Professional Documents
Culture Documents
Food Evaluation
Senior Editors
11. Food Constituents and Food Residues: Their Chromatographic Determination, edited by James F. Lawrence
editedbyLewis D. Stegink
12.Aspartame:PhysiologyandBiochemistry,
and L. J. Filer, Jr.
13.HandbookofVitamins:Nutritional,Biochemical,andClinicalAspects,
edited by LawrenceJ. Machlin
14. Starch Conversion Technology, edited by G. M. A. van Beynum and J.
A. Roels
15.FoodChemistry:SecondEdition,RevisedandExpanded,
edited by
Owen R. Fennema
16.SensoryEvaluationofFood:StatisticalMethodsandProcedures,
Michael O'Mahony
17. Alternative Sweeteners, edited by Lyn O'Brien Nabors and Robert
C.
Gelardi
18. Citrus Fruits and Their Products: Analysis and Technology,
S. V. Ting
and Russell L. Rouseff
19. Engineering Properties of Foods,edited by M.A. Rao and S. S. H. Rizvi
20. Umami: A Basic Taste, edited by Yojiro Kawamura and Morley R. Kare
21. Food Biotechnology, edited by Dietrich Knorr
22.FoodTexture:InstrumentalandSensoryMeasurement,
editedby
Howard R. Moskowitz
23. Seafoods and Fish Oils in Human Health and Disease,John E. Kinsella
24. Postharvest Physiology of Vegetables, edited by J. Weichmann
25. Handbook of Dietary Fiber: An Applied Approach,Mark L. Dreher
26. Food Toxicology, PartsA and B, Jose M. Concon
27. Modem Carbohydrate Chemistry, Roger W. Binkley
28. Trace Minerals in Foods, edited by Kenneth T. Smith
29. Protein Quality and the Effects of Processing, edited by R. DixonPhillips
and John W. Finley
30. Adulteration of Fruit Juice Beverages, edited by Steven Nagy, John A.
Attaway, and MarthaE. Rhodes
31. Foodborne Bacterial Pathogens, edited by Michael P. Doyle
32. Legumes: Chemistry, Technology, and Human Nutrition, edited by Ruth
H. Manhews
33.IndustrializationofIndigenousFermentedFoods,
editedbyKeith
H.
Steinkraus
34. International Food Regulation Handbook: Policy0 Science 0 Law, edited
by RogerD. Middlekauff and Philippe Shubik
35. Food Additives, edited by A. Lany Branen, P. Michael Davidson, and
Seppo Salminen
36. Safety of Irradiated Foods, J. F. Diehl
37. Omega3 Fatly Acids in Health and Disease, edited by Robert S. Lees
and Marcus Karel
38. FoodEmulsions:SecondEdition,RevisedandExpanded,
editedby
Kire Larsson and StigE. Friberg
39.Seafood:EffectsofTechnologyonNutrition,
George M. Pigonand
Barbee W. Tucker
40. Handbook of Vitamins: Second Edition, Revised and Expanded,
edited
by LawrenceJ. Machlin
41.HandbookofCerealScienceandTechnology,
Klaus J. Lorenzand
Karel Kulp
42. Food Processing Operations and Scale-up,
Kenneth J. Valentas, Leon
Levine, andJ. Peter Clark
43. Fish Quality Control by Computer Vision,
edited by L. F. Pau and R.
Olafsson
44. Volatile Compounds in Foods and Beverages,edited by Henk Maarse
45. Instrumental Methods for Quality Assurance in Foods, edited by Daniel
Y. C. Fung and RichardF. Matthews
46. Listeria, Listeriosis, and Food Safety,Elliot T. Ryser and Elmer H. Marth
47. Acesulfame-K, edited by D. G. Mayer andF. H. Kemper
48. Alternative Sweeteners: Second Edition, Revised and Expanded, edited
by Lyn O'Brien Nabors and Robert C. Gelardi
49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, ChiTang Ho, and MukundV. Karwe
50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee
51. Handbook of Food Engineering,edited by Dennis R. Heldman and Daryl
B. Lund
52. Food Analysis by HPLC, edited by Leo M. L. Nollet
53.FattyAcids in FoodsandTheirHealthImplications,
editedbyChing
Kuang Chow
54. Clostridium botulinum: Ecology and Controlin Foods, edited by Andreas
H. W. Hauschild and KarenL. Dodds
55. Cereals in Breadmaking: A Molecular Colloidal Approach,Ann-Charlotte
Eliasson andKire Larsson
56. Low-Calorie Foods Handbook,edited by Aaron M. Altschul
57. Antimicrobials in Foods: Second Edition, Revised and Expanded,edited
by P. Michael Davidson and Alfred Larry Branen
58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright
59. Rice Science and Technology,edited by Wayne E. Marshall and James
1. Wadsworth
60.FoodBiosensorAnalysis,
editedbyGabrieleWagnerandGeorgeG.
Guilbault
61. Principles of Enzymology for the Food Sciences: Second Edition, John
R. Whifaker
62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh
and Barry G. Swanson
63.
Engineering
Properties
of
Foods:
Second
Edition,
Revised
and
Expanded, edited by M. A. Rao and S. S. H. Rimi
64. Handbook of Brewing, edited by William A. Hardwick
65.AnalyzingFood
for NutritionLabelingandHazardousContaminants,
edited by IkeJ. Jeon and William G. lkins
66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G.
Gaonkar
67.FoodPolysaccharidesandTheirApplications,
editedby Alisfair M.
Stephen
68. Safety of Irradiated Foods: Second Edition, Revised and Expanded,
J.
F. Diehl
69. Nutrition Labeling Handbook, edited by Ralph Shapiro
Nondestructive
Food Evaluation
Techniques to Analyze Properties and Quality
edited by
Sundaram Gunasekaran
University of Wisconsin-Madison
Madison, Wisconsin
MARCEL
MARCEL
DEKKER,
1NC.
D E K K E R
N E WYORK BASEL
ISBN: 0-8247-0453-3
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above.
Neither this book nor any part may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopying, microfilming, and recording,
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Current printing (last digit):
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PRINTED IN THE UNITED STATES OF AMERICA
Preface
iv
Preface
Optical techniques are presented under four topical headings (Chapters1of electromagnetic spectrum: visible, IR, NIR, and
FTIR; computer vision; delayed light and fluorescence; and x-ray tomography.
Chapter 5 introduces the basic principles of nuclear magnetic resonance (NMR)
and magnetic resonance imaging (MRI). NMR and MRI are nondestructive techniques that can be used to probe the physical and chemical properties and anatomical structure of biological materials. Therefore, the quality parameters associated
with certain physical and chemical properties of foods can be evaluatedby NMR
and MRI. Use of NMR and MRI in analysis of water mobility, glass transition
in foods is described.
process, distributionof water and fat, and internal blemishes
Sound waves are transmitted through materials as compressions and rarefactions in their physical structure. Hence, it is often possible to relate the ultrasonic properties of a material to useful information about its macroscopic and
microscopic composition. In Chapter 6, the physics of high-frequency sound is
introduced, and applications of ultrasonic properties to monitor food quality are
described.
Mechanical methods of nondestructive food evaluation include low-intensity impact (tapping) and vibration testing and high-pressure air impingement
(Chapters 7 and 8). One of the most recent techniques used to evaluate food
texture is the small-amplitude oscillatory strain test, popularly known
as dynamic
5%) is used to study the material
testing. In this test, a very small strain (less than
structure-function relationships. Since food structure is the basis for its texture,
this method offers the advantageof obtaining fundamental information about the
eating quality of foods.
A taste panel traditionally measures many subjective food quality factors
such as aroma andtaste.Recent developments in providing objective, instrumented evaluations of such subjective factors are presented in Chapter 9, Biosensors in Food Quality Evaluation. A good example of such class of sensor
is the electronic nose, which mimics the human sense
of smell. The integration
of multiple gas sensors and artificial intelligence has led to a
new science of
machine olfaction. Biosensors offer the advantage
of rapid detection of bioactive
componentsthatcanbemeasured
and controlledtoensurefoodquality
and
safety. In food quality analysis and control, the data collected often are subjective
and ill-conditioned. To infer useful information out
of such data sets requires
methods other than those traditionally used. Chapter 10 describes some of these
data analysis procedures, such as neural networks, fuzzy logic, pattern recognition, and statistical process control.
I would like to thankallthe contributors and the Marcel Dekker, Inc.,
production staff for their enthusiastic and timely support in bringing this project
to fruition.
Sundaram Gunasekaran
Contents
Preface
Contributors
1.
2. Computer Vision
Suranjan Panigrahi and Sundaram Gunasekaran
3.
4.
...
111
vii
39
99
137
165
V
vi
Contents
6. Ultrasonics
John Coupland and David Julian McClernents
217
7. Firmness-MeasurementMethods
Yen-Con Hung, Stan Prussia, and Gabriel 0. I . Ezeike
243
8. LinearViscoelasticMethods
M . Mehrnet Ak and Sundararn Gunasekaran
287
9.Biosensors in FoodQualityEvaluation
Sudhir S. Deshpande
335
10. New Techniques for Food Quality Data Analysis and Control
Jinglu Tan
319
Index
417
Contributors
M. Mehmet Ak Department of Food Engineering, Istanbul Technical University, Maslak, Istanbul, Turkey
Paul L. Chen Department of Biosystems and Agricultural Engineering, University of Minnesota, St. Paul, Minnesota
John Coupland Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania
Sudhir S. Deshpande Signature Bioscience, Inc., Burlingame, California
Gabriel 0.I. Ezeike Center for Food Safetyand Quality Enhancement, Department of Food ScienceandTechnology,TheUniversity
of Georgia,Griffin,
Georgia
SundaramGunasekaran
Department of BiologicalSystemsEngineering,
University of Wisconsin-Madison, Madison, Wisconsin
Yen-Con Hung Center for Food Safety and Quality Enhancement, Department
of Food Science and Technology, The University of Georgia, Griffin, Georgia
vii
viii
Contributors
Joseph lrudayaraj
The Pennsylvania State University, University Park, Pennsylvania
1.
INTRODUCTION
Food quality may be defined as the compositeof those characteristics that differentiate individual units of a product and have significance
in determining the
degree of acceptability of that unit by the buyer (1). The qualityof many products
may be judged by the colors they display or fail to display. It is particularly
and vegetables,
critical in cases of food and biological products such as fruits
cereal grains, and processed foods. The primary goal
of quality control is to
maintain a consistent standard of quality at a reasonable cost and at levels and
tolerances acceptable to buyers.
Human evaluation has been the primary method of quality assessment for
operations such as grading and sorting of food products, but such evaluation can
hardly provide a general standardon a large scale and wide range of operations.
Factors such as eye fatigue, lackof color memory, variationsin color discriminain lightingconditions
tionbetweenindividuals,personalbias,andvariations
greatly influence an individuals decision when determining the qualityof a certain product. Moreover, the human eye is greatly limited by its perceptions in a
very narrow band of the vast electromagnetic spectrum. Some quality attributes,
external and internal defects, and compositional factors are more readily detect(UV) and infrared
able in the region outside the visible range, e.g., ultraviolet
(IR). This has led to considerable research in developing instruments sensitive
1
Optical Methods
Reflection
Specular
Radiation
Incident
Medium 1 , n,
n
Y
Medium 2, n2
Light Scattering
and Absorption
Diffuse kkmission\
\r
Regular Transmission
Fig. 2 Schematic representation of interaction of light with matter. 8, = angle of incidence, eR = angle of reflectance, OT = angle of transmittance, n,, n2 = refractive index
of medium 1 and 2, respectively.
A.
Color Specification
There are three characteristics of light by which a color may be specified: hue,
saturation, and brightness. Hueis an attribute associatedwith the dominant wavelength in a mixture of light waves, i.e., it represents the dominant color as perceived by an observer. Saturation refers to relative purity or the amount
of white
light mixed with a hue. Brightness is a subjective term, which embodies the
chromatic notion of intensity. Hue and saturation taken together are called chromaticity. Therefore, a colormay be characterized by brightness and chromaticity
(4).
B. CIE System
The Commission de Internationale de 1Eclairage (CIE) defined a system
of describing the color of an object based on three primary stimuli: red
(700 nm),
green (546.1 nm), and blue (435.8 nm). Because of the structure of the human
eye, all colors appear as different combinations of these. The amounts of red,
form any given color are called the tristimulus
green, and blue needed to
values, X, Y, and Z, respectively. Using theX, Y, and Z values, a color is represented by a set of chromaticity coordinates or trichromatic coefficients,x, y, and
z, as defined below:
Optical
x =
X
X + Y + Z
Y
y = x + Y + z
z =
X + Y + Z
(1)
111.
INTERACTION OF LIGHTWITHMATTER
A.
PhysicalLaws
IR
+ IT + IA
(2)
(3)
n2 sin 8, = n , sin 8,
where k is a constant and n is the number of molecules in the path of the beam.
of the sample and the thickness b
Since n is proportionaltoconcentrationc
through which the radiation passes, Eq. (4) is rewritten as:
(5)
lOg(IT/I) = abc
The ratio IT/Iis known as the transmittance T and is related to absorbance A as:
A = log( 1/T)
From Eqs. (5) and (6), absorbance A can also be written
A = abc
as:
(7)
Optical Methods
RII
+ R:
OO),
Rii =
where n , and n z are refractive index of the medium and object, respectively; and
8, is the incidence angle (Fig. 2).If the material is absorbing, the refractive index
is a complex number n( 1 - ik), where n is the real part of the complex number
and k is an absorption constant, and the regular reflectance is written as:
=
[I.?
(n?
nJ2+ ( n M ]
+ n,)? + (nzk)2
(1 1)
When the incident light is reflected from a surface evenly at all angles, the
object appears to have a flat or dull finish termed diffuse reflection. No rigor-
and
Gunasekaran
lrudayaraj
ous theory has been developed for diffuse reflectance, but several phenomenological theories have been proposed, the most popular being the Kubelka-Munk theory (12). The Kubelka-Munk model relates sample concentration to the intensity
way Beer-Lamberts
of the measured spectrum in a manner analogous to the
law relates band intensities to concentration for transmission measurements. The
Kubelka-Munk function f(R,) is generally expressed as:
f(RJ =
(1 - R-)2 - k
S
2R,
+ log(R) = a d s
(13)
(14) log(l/R)
(s/a)
c =k
where k = absorption coefficient. It should be noted that s is not a constant but
depends on a number of properties of the sample such as particle size (s is in-
Optical Methods
B. FactorsAffectingDiffuseReflectanceSpectralData
Diffuse reflectance spectroscopy offers exceptional versatilityin sample analysis.
This versatility results from both its sensitivity and optical characteristics. Classically, diffuse reflectance has been used to analyze powered solids in a nonabsorbing matrix of an alkali halide such as KBr. The sample is typically analyzed
at low concentrations, permitting quantitative presentation
of the datain KubelkaMunk units. This technique yields spectra that are qualitatively similar to those
produced by conventional transmittance or pellet methods. However,
they exhibit
higher sensitivity for quantificationand are less subject to scattering effects that
cause sloping baselines in pellet measurements.
Several factors determine band shape and relative/absolute intensity
in diffuse reflectance spectroscopy through their effect on the reflection/absorbance
phenomena specific to the sample. These include:
Refractive index of the sample
Particle size
Sample homogeneity
Concentration
10
1.
RefractiveIndex
Refractive index affects the results via specular reflectance contributions to diffuse reflectance spectra. With organic samples, the spectra display pronounced
changes in band shape and relative peak intensities, resulting in nonlinearity in
For some inorthe relationship between band intensity and sample concentration.
ganic samples, strong specular reflection contributions can even result in complete band inversions. This overlayof diffuse reflectance and specular reflectance
by diluting
spectra, as well as the resulting spectral distortions, can be minimized
the sample in a nonabsorbing matrix. In addition, accessory design can help reduce specular reflectance contributions.
2. ParticleSize
Particle size is a major consideration when performing diffuse reflectance measurements of solids. The bandwidth is decreased and relative intensities are dramatically altered as particle size decreases. These effects are even more pronounced in spectra of highly absorbing inorganic materials with high refractive
indices. For these samples, specular contributions can dominate thefinal spectra
if the particle size is too large. To acquire a true diffuse reflectance spectrum, it
is necessary to uniformly grind the sample and dilute it in a fine, nonabsorbing
matrix. Similar preparation must be applied to the nonabsorbing matrix material
in order to provide and ideal diffuse reflector for background analysis and as
a support matrix for the sample.
3. SampleHomogeneity
The Kubelka-Munk model for diffuse reflectance is derived for a homogeneous
sample of infinite thickness. However, some sample analysis methods, especially
of sample onto a powdered
those designed for liquid samples (e.g., deposition
supporting matrix), can result in a higher concentration of sample near the analysis surface. In these circumstances, variations in relative peak intensities may be
noticed. In particular, more weakly absorbing wavelengths tend to be attenuated
it is
at higher sample concentrations. To avoid these peak intensity variations,
necessary to distribute the analyte as uniformly as possible within the nonabsorbing background matrix.
4.
Concentration
One particularly important advantage of diffuse reflectance spectroscopy, especially in comparison to transmittance measures, is its extremely broad sampleanalyzing range. While it is theoretically possible to acquire usable diffusereflectance spectraon samples of wide-ranging concentrations, practical considerations
often complicate the analysis process. With high concentration samples, espe-
Optical Methods
11
cially those with a high refractive index, one can expect a dramatic increase in
the specular contribution to the spectral data. As a result, some sample data may
be uninterpretable without adequate sample dilution. Even when samples can be
measured satisfactorily at high concentrations, it is advisable to grind the sample
to a very uniform and
fine particle size to minimize both specular reflectance
and sample scattering effects, which adversely affect quantitative precision.
Alternative methods of sample analysis in diffuse reflectance include:
IV.
A.
Near-InfraredSpectroscopy
12
component
Chemical
Food
functionality
Wavelength
Water, carbohydrates
Unsaturated fat
Fats, proteins, carbohydrates
Fats
Pectin
Fatty acids, acetic acid
Water
Protein
Protein
Carbohydrates, fats
Source: Adapted from Ref. 19
of band
0 -H stretch
C -H of cis double bond
C-H
C=O, ester
C=O, ester
C =0, acidic
0 - H (bend)
C =0, amide I
N -H, amide I1
c-0, c-c
3600-3200
3030
3000-2700
1745
1725
1600- 1700
1640
1650
1550
1400-900 (complex)
Optical
13
B. FourierTransformInfraredSpectroscopy
Fourier transform infrared (FTIR) spectroscopy is based on the Michelson interferometer configuration designed a century ago (1 8,20,2I). It is used to detect
radiation in the mid-IR region. Fourier transform instruments obtain the data by
using interferometry while they calculate the spectrum by using Fourier transform
mathematics. The resultis increasing sensitivity of measurement.The interferometer consists of a fixed mirror, a movable mirror, and a beamsplitter (Fig. 3). The
beamsplitter transmits half the incident IR radiation to the movable mirror and
reflects the other half to the fixed mirror. The speed of the movable mirror is
monitored by a laser. The two mirrors reflect the two light beams back to the
beamsplitter and then the beams recombine. When the distance from the fixed
to the beammirror to the beamsplitter equals that from the movable mirror back
splitter, the amplitudes of all frequencies are in phase and recombine construca condition called zero
tively. There is no difference between the two beams,
retardation. As the movable mirror is moved away from the beamsplitter (retarded), the difference between the two beams
is generated because the two beams
travel different distances within the interferometer before recombining.A pattern
of constructive and destructive interference results, which depends
on the position
of the movable mirror and the frequency of the retardation. The intensity of the
Y
Source
Movable Mirror
14
1. AttenuatedTotalReflectionSpectroscopy
Attenuated total reflection (ATR) spectroscopy is one of the most powerful FTIR
methods for biological and liquid sample analysis. It is fast and yields a strong
signal even with small traces of the target molecule.
Reflection occurs when a beam of light passes from a dense to a less dense
medium. Total reflection occurs when the incident angle is greater than a critical
angle (Fig. 4). A light beam actually penetrates a small distance into the less
dense medium before reflection occurs(18). The depthof penetration varies from
a fraction of a wavelength up to several wavelengths. The depth of penetration
depends on the wavelength of the incident radiation, the index of refraction of
the two media, and the angle of the incident beam with respect to the interface.
Such penetration radiation is called the evanescent wave. When the less dense
medium absorbs the evanescent radiation, attenuation of a beam occurs at different wavelengths of the absorption bands. This is referred to as attenuated total
reflectance.
In the ATR method, the sampleis placed in contact against a special optical
crystal, which is called an internal reflectance element. An IR beam from the
spectrometer focused onto the beveled edgeof a setof mirrors is reflected through
the crystal, usually numerous times, and then is directed to the detector. Penetration d, is calculated as (26):
15
Optical Methods
"2
critical
Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) is a sampling technique developed forIR analysis of powder materials and turbid liquids.
This technique has been applied for the analysisof pharmaceutical (30) and food
(3 1 ) samples. DRIFTS is also utilized for quantitative (32) and qualitative(33,34)
determination of the composition of samples (forages and coffee, respectively)
with an accuracy equal to or better than that found using NIR spectra.
DRIFTS has been used primarily in UV-visible spectroscopy studies where
the beam energy is high enough for proper detection. Diffuse reflectance studies
found little use in the caseof classical scanned IR beam. The advantages brought
16
IR beam
source
Optical
detector
V.
QUALITYEVALUATIONOFFOODPRODUCTS
A.
MaturityandRipenessEvaluation
The ripening of fruits is associated with changes in color, flavor, and texture that
lead to the state at which the fruit is acceptable to eat. Readily apparent phenom-
Optical
17
ena associated with the ripening of most fruits, among others, include changesin
color, which involve loss of chlorophyll leading to the unmaskingof underlying
pigments and the synthesis of new pigments. Ripening is regarded as an indication of senescence accompanied by several physiological and chemical changes
(12,13). It may also represent a process requiring synthesisof specific enzymes.
Most fruits exhibit an increased reflectance and reduced absorbance in the 670
nm region because of the loss of chlorophyll. This hasbeenthe single most
(35). The mechanismof color
important criterionin optically judging fruit quality
in Hutchings
changes in fruits and vegetables during ripening is further discussed
(3).
Determining degree of maturity by surface color evaluation, however, has
its limitations. In many fruits, external color changes donot reflect internal ripeness. Similarly, external color evaluation cannot truly differentiate between immature and mature green fruits. Such a distinction
is important because mature
green fruits eventually ripen while immature fruits
will not. Where skin color
does not truly represent fruit quality or does not sufficiently indicate the stage
of maturation, internal flesh color can serve as an index of quality (36).
The success of establishing a valid index of quality largely depends on
the appropriateness of the optical property measured. Basic requirements for a
successful optical measurement are that the magnitude of relative measurement
should vary as greatly as possible over the full range
of maturation, andthe
change in the measurement between consecutive stages of maturity should be
great enough to permit precise color differentiation.
In addition, the nature of
the measurement criterion should also be formulated judiciously so as to permit
measurements insensitive to variations in the product and the measurement system. Over the years, the measurement criterion has taken various forms, which
can be broadly grouped under the following four classes:
1.
18
Optical
19
Maturity and other quality indices for several other fruits and vegetables have
been developed following a similar pattern. A detailed list is available in Gunasekaran et al. (42).
IR and NIR methods can beused to determine the sugarsin fruits that have
a thin skin, such as apples, peaches, prunes, or cherries, which, in turn, can be
correlated to their stateof ripeness (43). Sensors basedon this principle have been
developed. However, this method is ineffective for thick-skinned fruits (44,45).
Optical properties have been used to evaluate maturity in peanuts. Kramer
et al. (46) investigated light absorption properties
of Virginia-type cured, un-
20
roastedpeanuthalveswithoutskins.AOD(480-510)wasfoundusefulasan
indicator of peanut maturity. Due to the absorption band at 445 and 470 nm of
the pigment xanthophyll in immature peanuts, the above measurement values
also found
werehigherforimmaturepeanutsthanformaturepeanuts.They
the tastepanelratingof
offgoodcorrelationbetweenAOD(480-587)and
flavor in peanuts. Beasley and Dickens (47) indicated that
oil extracted from
as amaturityindex.Theyreportedthatoilfrom
peanutscouldalsobeused
mature peanuts generally transmitted more light at about 425, 456, and 480 nm
than oil from immature peanuts. Apart from xanthophyll, p-carotene and lubein
to
were also suggested as influencing the spectral properties, which are related
maturity.
Gloss characteristics of a number of fruits and vegetables have been determined. Unwaxed oranges, bananas, and onions have significantly lower gloss
than eggplant, green pepper,and tomato (48). Commercially mature eggplants are
glossier than green tomatoes and apples. This
is partly explained by differencesin
epicuticular wax structure. The lamellae-type wax covering the eggplant reflects
light more efficiently than the amorphous wax layer covering the tomato and the
as they
large overlapping platelets of the apple (49). Bananas also lose gloss
mature. This is probably due to epicuticular wax from the surface and possibly
also due to an increase in surface roughness caused by water transfer by osmosis
to shriveling, separationof epidermal cells,
from the skin to the pulp, which leads
and the appearance of longitudinal cracks (50).
Optical Methods
21
detection device to sort out bruised fruit beganin the early 1970s. While studying
the rate of discoloration for impact injuries versus the changesin selected phenolic compoundsin bruised apple pulp, Ingle and Hyde( 5 l ) observed a consistently
(52j reported a
lower reflectance at 600 nm for bruised apple pulp. Woolley
decrease in the NIR diffuse reflectance when water was used to replace air in the
intercellular air spacesin plant tissues. Since an apple bruise primarily consists
of
crushed cells surroundedby free liquid, measuring NIR reflectance seems promis(53)
ing to detect bruising in apples. Using a similar technique, Brown et al.
extensivelystudiedtheNIRreflectance
of threevarieties of freshandstored
apples. At all wavelengths between 700 and 2200 nm, bruised apple skin exhibited a less average reflectance than unbruised skin. It is thought that reflectance
a combifor a bruised surface is less than that for an unbruised surface because
of
nation of cell destruction in the bruise (fewer rigid cell walls to scatter light), an
in
unaltered water-air relationship in the tissue, and a gradual chemical change
the cell material. The differences
in reflectance at 800, 1200, and 1700 nm or
the ratio of reflectance (unbruised to bruised) at some wavelength between 1400
and 2000 nm may be useful in optical bruise detection (53). Reflectance properties of apple tissue cannot reliably predict bruise depth, but two-wavelength derivative models distinguish between good and bruised tissue better than nonderiva( 5 5 ) alsoinvestigatedtypicalreflectanceproperties
of
tivemodels(54).Reid
three varieties of apples for use in automatic trimming operations. He determined
that a detector sensitive in the wavelength range from 400 to 450 nm could be
used for bruise detection, and one sensitive in the 725-800 nm region could be
used for stem and calyx detection.
in
Reflectance properties have also been used to detect water core defect
apples. a heat-initiated disorder. In affected tissue, intercellular spaces are filled
with liquid or cells become swollen so as to eliminate air spaces, resulting in a
translucent or water-soaked appearance. Moderately water-cored apples are difficult to sort out visually from sound apples. The inability
to detect such disorders
not only causes a marketing problem butalso prevents investigation of the development of the disorders in intact fruits. Olsen et al. (56) were among the first to
use the differencein optical density at wavelengthsof 760 and8 15 nm to measure
water core concentration in apples. Birth and Olsen (37) made a more definitive
study of this technique to detect water core in Delicious apples. This technique
takes advantageof the physical changesthat occur in apple tissue that affect lightscattering properties. Since the air spaces are eliminated in water-cored tissue. it
scatterslesslightthannormaltissue.Water-coredtissuethustransmitsmuch
more energy. The relative optical density values indicated a water absorption
band at 760 nm. Also, there wererelatively few substances in normal apples that
absorbed energy at about 800 nm, so it was considered the best wavelength rein
gion. An optical density difference of AOD (760-840) gave the best results
identifying water-cored apples.
22
Felsenstein and Manor (57) studied certain surface defects of oranges that
are characterized by color change. They reported that within the vicinity of 667
nm, there was a difference of at least 17% in the intensity
of light reflectance
from the surface of a good orange and one with a blemish. Gaffney (58) investias plug, oleocellosis, rots,
gated fruit grade color and several surface defects such
molds, windscar, scabs, thorn scratches, etc. of oranges and grapefruit. Several
of these defects affect the keeping quality of fruits, whereas others detract from
to be sorted out. The
surface appearance and lower the grade. Such fruits have
Hamlin, Pineapple, and Valencia varieties of oranges and Marsh and Thompson
varieties of grapefruit exhibited definite differences in reflectance characteristics
in the wavelength band of 650-700 nm according to surface color. The wavelength band 550-610 nm was sensitive to changes in surface color or nature due
to various defects.
A color index has been developed to evaluate the quality of orange juice.
The color number (CN) is defined in terms of the tristimulus values X, Y, and
Z as (59):
X
Z
CN = 56.5 - - 18.4 Y
Y
+ 48.2 I
- 8.57
(18)
Optical Methods
23
2. Food Grains
Separating foreign material such weed
as seeds from thatof other grain cropsis an
important operation in the grain industry. Hawk et al. (70) studied the reflectance
characteristics of 12 grains. Their results indicated that the difference in reflectance between grains in the IR region is small and the greatest differences occur
between 450 and 750 nm. The different reflectance properties were
used in evaluating grain samples for admixture grain, i.e., grains other than the primary one.
1 ) used light reflectance measurementsto detect exterGunasekaran et al. (7
nal cracks in individual kernels of corn. Using a laser light (632.8 nm), they
could detect cracks smaller than 1 mm. The defect detection accuracy was 100%
for broken, chipped, and starch-cracked kernels and 80% for surface-split kernels.
24
Optical
25
hemoglobin selectively absorb light, while structural and myofibrillar protein absorb relatively less light but cause more scattering. This suggests that the general
optical quality standards for meat products should take factors other than pigments into consideration.
Davis et al. (79) reported that the interaction between light and muscle
pigments could provide a nondestructive means of evaluating pork muscle quality. They investigated the reflectance spectra of longissimus muscle from pork
loins of the qualities PSE, normal, and dark, firm, and dry (DFD). A pork quality
at 633 and
index (PQI) was suggested based on light reflectance measurement
627 nm as follows:
PQI
- 1.67 - 254log
(RI)
(RI,)
+ 258log
This yielded a correlation of 0.8 with visual rating of quality and 0.86 with the
that the measurements involving
OD of Hart extract. Davis et al. (79) commented
pigments might be affected by the chemical reactions involving porcine myoglobin and by other external factors such as bacterial growth and oxidation. The
light-scattering property of muscle, independent of the above factors, was suget (36) reported
gested as a desirable factor in evaluating muscle quality. Birth al.
a high correlation between the scatter coefficient at 632 nm and the OD of Hart
extract.
The presence of blood and meat spots is one of the most common defects
found in eggs. Its incidence may range from less than 1% to nearly 100% (80).
One of the earliest attempts to develop a spectrophotometric technique to detect
blood in eggs is credited to Dooley(SI), who developed a device to automatically
detect eggs containing blood using radiation
in the region of 1260-1400 nm.
However, Brant et al. (80) identified three absorption bands
for blood at 415,
541, and 575 nm in the visible region. Their method of detecting blood spots i n
white shell eggs was based on the relative transmittance measurement between
555 and 565 nm. Although a success rate of 97.5% was reported, this measurement was specific to the color of the eggshell. Norris and Rowan (82) applied a
similar technique to detect blood spots regardless of shell color. Based on the
relative absorbance measurements at 577 and 600 nm, they could detect 70% of
all eggs having blood spots from 3 to 6 mnl in diameter and 100% having spots
larger than 6 mm in diameter.
C. Composition Analyses
Composition analyses of food materials are very important as quality indices for
a variety of food materials. Such evaluation is normally performed by NIR and
FTIR spectroscopy.
26
7.
MoistureContent
Goulden (88) first used IR radiation to measure fat, protein, and lactose in milk.
Fat measurement was based on absorbance at 1724 cm" (fatA) by ester carbonyl
groups of fat molecules. Protein measurement was based on absorbance at 1538
cm" by peptide bonds of protein molecules, and lactose measurement was based
on absorbance at 1042 cm" by hydroxyl groups of lactose molecules. NIR spectroscopy can be used to analyze moisture, fat, protein, and total solids in cheese
(89,90). Rodriguez-Otero et al. (90)
used NIR reflectance spectroscopyto analyze
fat, protein, and total solids in cheese without any sample treatment.
Norris (91) studied light absorption characteristics of ground beef samples.
Of the observed absorption bands at 540, 575, 640, and 760 nm, he selected the
of ground beef. The
one at 760 nm as a criterion to estimate the fat content
other absorption bands were rejected because they were closely related
to light
absorption by blood. A fat absorption band
at 928 nm was also reportedby Massie
50
Advantages
Standard conventional method
Convenient
Relative speed and precision
Accommodates more samples
Attains desired temperature more rapidly
One of the standard methods
More accurate and precise than other methods
Useful for determining water in oils and fats by preventing samples from oxidizing
Very rapid once apparatus is set up (within minutes)
IR absorption
NIR reflectance
Rapid
Precise
Nondestructive
No extraction required
Minimal sample preparation
High sensitivity due to large dielectric constant of water
Convenient to industrial operations with the continuous
measurement system
Universal aplicability
Dielectric capacitance
Source: Ref. 83
Disadvantages
5
0
Q
u)
28
(92).Fromthespectralreflectancedata,
formula:
heproposedthefollowingempirical
where A andBareconstants.Comparisons
of fatcontentestimated bythis
methodwiththevaluesobtained
by chemicalanalysis(Soxhletprocedure)
-+ 1.98% fat.
yielded a correlation of 0.82, and the measurements are within
NIR spectroscopy has been used
to determine the sum of dimer and polymer
triglycerides and acid value to evaluate frying oils (93). This
is a rapid, lowcost technique to assess whether a sample complies with food legislation. FTIR
spectroscopy is another interesting approach to authenticate extra virgin olive oil
(94). The problems associated with identifying adulterated oils are complicated
by the ever-changing nature of adulteration techniques and the numberof procedures that can pass undetected through official quality control.
Some oils can be added to other oils without being detected
by routine
physical and chemical characteristics due to their fatty acid composition. In such
cases, gas-liquid chromatography (GLC) analysis of fatty acids proves useful. IR
spectra between 300 and 357 cm-l and 770to 1175 cm" can distinguish between
oils of peanut, sesame, sunflower, etc. Peanutoil has a characteristic band at 9 13
cm",sunfloweroilat847,andsesameoilat812and
913 cm". IR spectra
oil and its
between 4000 and 850 c1n-l have shown differences between olive
adulterant, rapeseed oil. Differences have been noted at 3100 and 1750 cm" and
of the differential spectrum
from 1400 to 1300 cm", the most striking feature
being the negative peaks at 1130 and 1080 cm" with a characteristic contour
from 900 to 1200 cm". Thesecharacteristicspersist in a mixture containing
in
as little as 10% rapeseed oil. These differences are attributed to differences
unsaturated fatty acids, particularly oleic and linoleic acids (95).
In contrast to NIR, FTIR has much to offer the analyst because specific
bands may be assigned to specific chemical entities. Statistical correlation methods are not always necessary, but they are not excluded and may be required
in
very complicated mixtures (63). This techniques has been widely used to deter(96), meat (97), sweetened condensed
mine fat, moisture, and protein in butter
milk (98), and other high-fat products (99). It has also been used to monitor the
oxidation of edible oils (100) and to determine the level of tram-unsaturation in
fat (101).
By combining attenuated total reflectance and mid-IR spectroscopy with
statistical multidimensional techniques, Safer et al. (102) obtained relevant information from mid-IR spectraof lipid-rich food products. Wavelength assignments
for typical functional groups in fatty acids were made for standard fatty acids.
Optical Methods
29
Absorption bands around 1745 cm" due to carbonyl group, 2853 and 2954 cm"
due to C-H stretch, 3005 and 960 cm" due to
C = C bonds, 1160 cm" due
to C - 0 bonds, and 3450 and 1640 cm" due to 0 - H bonds were observed.
Water strongly absorbs in the region of 3600-3000 cm-l and at 1650 cm" in
butter and margarine, allowing one to rapidly differentiate them as a function of
their water content. Principal component analysis was used
to emphasize the
difference between spectra and to rapidly classify
27 commercial samples of oils,
butter, and margarine.
Belton et al. (103) studied the components of fat, protein, and sugar in
confectionery products usingFTIR spectroscopy coupled with photoacoustic and
attenuated totalreflectancedetectionmethods.Theyconcluded
that peaks at
1744, 1477- 1400, 1240, and 1 195- 1 129 cm" could be from an ester carbonyl
group, C-H bond, and C - 0 stretching of fat, respectively. Peaks at 1650 and
1540 cm" are from protein,
and those at 1128 to 952 cm" are from sugars.
3000 cm" and at 1650 cm" in
Water is strongly absorbed between 3600 and
fat-rich foods (96,103). Principal component analysiswas used to emphasize the
differences between spectra and to rapidly classify each sample (96).
Usually wavelength assignments for typical functional groupsin fatty acids
A) forestercarbonylgroups
areabsorptionbandsaround1745cm"(fat
in methylene
(R(CO)OR/OH), 2930 and 2853 cm"' (fat B) for C-H stretch
groups, and 1 160 cm" for C - 0 bonds of lipid (104).
IR spectroscopy hasalso been used to detect adulterationof fat with lowerquality/cost oil. Attempts have been made to detect concentrations of less than
10% of vegetable or animal fat
in butter fat by GLC in conjunction with IR
spectroscopy (percentage transmission at 967 and 948 cm", denoted as T967
and T948 and assigned to isolated trans and conjugated cis-trans isomers) can
reliably distinguish butter from its adulterant substitute fats (95).
3. ProteinContent
Proteins have three characteristic absorbance bands
in the mid-IR spectrum (104).
Two of these, amide I (about 1600- 1700 cm") and amide 111 (about 1200- 1400
cm"), are sensitive to polypeptide backbone conformation and might be able to
I band is moreintense, but it
distinguishbetweenproteins(105).Theamide
overlaps with an intense water deformation band at 1645 cm". The amide 111
band, although less intense, is not overlapped by water absorptions. This band
has been used as a tool to detect adulterations of NDM with SPC (106).
Wheat protein content and grain hardness can be rapidly determined
by
IR and NIR spectroscopy (107). The NIR and Brabender hardness tester results
correlate significantly with percentage of dark hard and vitreous grains as shown
by commercial red winter wheats which have similar protein contents. NIR spec-
30
troscopy has also been used to differentiate between hard red winter and hard
red spring wheat. Examination of the principal component factors has indicated
that hardness, protein level, and the interaction of water with protein and other
constituents are responsible for correct classification based on NIR (108).
IR absorption can be used to determine the protein content in whole milk
at 6460 nm, which is the absorption maximum for the peptide bond. Other components of milk such as lactose and fat can be simultaneously measured at their
respective absorption bands at 5730 and 9597 nm. Water absorbs significantly
at 1000-5000 cm. Therefore, it interferes excessively with protein absorption
bands in the IR spectrum. For protein determination in milk, this couldbe alleviated by using a double-beam spectrophotometer with water in the reference cell
and milk in the sample cell (109).
NIR spectroscopy is a popular method for determining protein
in cereal
products primarily due to its speed, simplicity of operation, safety, and low operating cost. To avoid excessive interference by starch, fat, and water, a wavelength of 4590 cm corresponding to a combined vibration of amide groups is
chosen to quantify protein components ( 1 IO).
VI.
Optical propertiesof food and biological materials vary widely and are dependent
upon many factors. Quality evaluation based
on these properties requires accurate
optical property values. Undera given setof conditions, precise values can probably be obtained for any particular substance by careful measurements. However,
good estimates can be made for many materials on the basis of the investigations
already reported. Such optical property estimates will permit a quick evaluation
of various properties that have servedas quality indices and help in the selection
of those most suitable for any particular application.
Generally, nondestructive quality evaluationof food and biological materials focuses on three major aspects: maturity and/or ripeness evaluation, internal
For maturity evaluation,
and external defect detection, and composition analysis.
the interaction of various pigments and the changes associated with them during
maturation are taken as the prime indicator. The importance of chlorophyll in
this regard has been adequately established. Both external and internal defects
have been found to affect the normal propertiesof interaction of light with products in consistent ways. Hence, defect detection is accomplished by comparing
optical propertiesof a normal product with thoseof the defective ones. Moisture,
protein, fat or oil content, and other compositions have been analyzed based on
certain absorption bands in the electromagnetic spectrum.
The wide variety of sizes, shapes, and textures of food products makes
most commercial instruments difficult to use for measuring optical characteristics
Optical
31
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Computer Vision
Suranjan Panigrahi
North Dakota State University, Fargo, North Dakota
Sundaram Gunasekaran
University of Wisconsin-Madison, Madison, Wisconsin
1.
INTRODUCTION
39
40 Gunasekaran
11.
and
Panigrahi
COMPUTERVISION SYSTEMS
Computer vision is the science that develops the theoretical and algorithmic basis
by which useful information about an object or scene can be automatically extracted and analyzed from
an observed image, image set,
or image sequence.
Computer vision is also known as machine vision or computer imaging. It is a
branch of artificial intelligence technique and deals with simulating human vision.
1):
The essential components of a typical computer vision system are (Fig.
Computer (analogous to the human brain)
Sensor or camera (analogous to the human eyes)
Illumination system (light source and illumination chamber, etc.) to facilitate image capture
Frame grabber/digitizer to digitize the image information from the camera,
monitor(s)
In todays modern computer imaging system, the camera and frame grabber
are joined together to form the digital camera. The use of digital cameras thus
eliminates theneed to use a framegrabber in the computer. The imageis digitized
in the camera and sent to the computer for further processing. More modern
computer imaging systems do not need a separate monitor either. Images can be
displayed directly on a high-resolution computer monitor. For special applications, many users still use a high-resolution display monitorto separately display
images.
Recognizing and extracting useful object features from image data are complex tasks involving a series of steps that can be grouped into three major parts
Frame Grabber/
Computer
Sensor/
Camera
Light source
Illurnination
Chamber
Display
Monitor
Computer Vision
41
(Fig. 2): image acquisition, image processing, and image understanding. Image
acquisition deals with such issueskomponents as illumination, camera, digitizer,
etc. The image processing step encompasses preprocessing, segmentation, and
feature extraction. The image understanding part consists of image recognition
so as to make
and interpretation. Eachof these steps must be carefully performed
each subsequent step progressively easier and result inan improved end result.
For example, a poorly formed or acquired image cannot provide good results
with any amount of further processing.
All the steps are closely linked
with
knowledge base available about the system studied and the featuresof interest.
111.
IMAGE ACQUISITION
A.
Digital Images
42
5 12 X 480 or 640 X 480. For best results it is important to match the spatial
resolution of the camera to that of the frame grabber.
The intensity of the monochrome image is known as the gray level. The
limit on gray level is that it is positive and finite. The gray level interval (from
low to high) is called a gray scale. A common practice is
to shift this interval
numerically to the interval (0,L) where the lowest value 0 represents pure black
All intermediate values are
and the maximum value L represents pure white.
shades of gray varying continuously from black to white. For example, when an
8-bit integer is used to store each pixel value, gray levels range from
0 to 255
(i.e., 2" - I to 2' - I).
Inferring an object's size, shape, position, orientation, and other attributes
from the spatial distribution of gray levels requires the capability to infer which
pixels belong to the object and which do not. Then, from the pixels that belong
to the object, it requires the capability to identify the object features of interest.
Algorithms have been developed to translate the gray levels of a pixel in a way
that accentuates the desired information.
In the case of color images, the image intensity
is represented by three
components representing red, green, and blue (RGB system) or hue, saturation,
and intensity(HSI system). Further detailsof the color digital image are presented
in a following section.
B. illumination
The prerequisite for any vision application is that the features to be examined
can be seen in the image. Therefore, despite all the progress in image analysis/
processing algorithms, the performance of the camera and illumination subsystem
can greatly affect the reliabilityof a machine vision application. A well-designed
lighting and illumination system can assist in the accuracy and success of image
analysis by enhancing image contrast. Good lighting will improve feature discrimination,reduceprocessingtime,andreduceprocessinghardwarerequirements. Thus, it is almost always cheaper to improve lighting than image processing (2). Food materials are nonhomogeneous and randomly oriented; the raw
materials may be dirty. Singulation of objects for examination is often difficult,
so we have to cope with objects that touch and/or overlap, which
may cause
shading during image acquisition. Therefore, vision applications in the food industry present unusual challenges when designing proper illumination systems.
Illumination consists of selecting appropriate light sources and identifying
suitable configurations for the light sources so as to obtain the highest quality
images. The geometryof the imaging system should be well known. This requirement is especially important for dimension measurements. When the viewing
geometry is more complicated, either becauseof the nonplanar image surface or
nonperpendicular imaging angle, measurements are more
difficult and require
Computer Vision
43
determining the geometryof the imaging system. Elaborate discussions of different types of illumination techniques for general machine vision applications have
been presented by other authors (3,4). Most lighting arrangements can be grouped
as either front-lighting or back-lighting. The front-lighting option is best suited
for obtaining surface characteristics of an object, while back-lighting is best for
(5)
subsurfacefeatures. For example, using back-lighting, Gunasekaran et al.
examined internal stress cracks
in corn kernels and Upchurch and Throop
(6)
detected watercore in apples. The appropriatenessof a well-designed illumination
system can be evaluated by the suitability of acquired images for successful further processing. The most commonly used illuminations system configurations
are summarizedin Fig. 3. Associated advantages and disadvantages of these techniques are compared in Table 1.
A wide variety of light sources and lighting arrangements are available
(2).
Most general computer vision applications are implemented using either incandescent or florescent lighting. However, use of polarizers and polarized light can
improve the light intensity contrast, eliminate unwanted glare, and minimize diffuse reflectance (7). This is especially suitable for transparent and translucent
objects. Since an objects color dependson illumination, color measurements are
easily affected by changesin the color temperatureof an incandescent bulb. Thus,
or colorvalues,requiresa
measuringbrightnessinformation,suchasdensity
very stable illumination source and sensor. Bright specular reflections may cause
saturation, blooming, or shifts in image magnification. Sometimes the color of
two objects will appear similar under one light source but much different under
another. So, a number of light sources of different spectral responses must sometimes be tried when attempting to maximize image contrastfor the best possible
results. For multiple or brightly colored fruits and vegetables, a multiple spectral
lighting system is needed to assure accuracy over a large spectral range. Spectral
reflectance properties of products should be considered when developing an appropriateilluminationsystem(lightingandviewinggeometries,lightsources,
(8). The specand sensor components) to obtain maximum discrimination power
tral output of different light sources can be obtained from respective manufacturers.
For on-line evaluations where speed of operation becomes an important
look the same
criterion, global uniformity (i.e., the same type of feature should
wherever it appears in the image) is essential. This means that brightness and
color values are the same and
thus it requires uniform, consistent image illumination (9). Furthermore, the optomechanical constructionof a camera and illuminator should withstand environmental conditions suchas mechanical vibrations and
dust common in industrial applications. Strobe lightingis useful for on-line applications to virtually arrest themotion to aid in acquiring images without worrying
about image blur due to image motion.
The strobe repetition rate should be
selected to match the speed of object motion. A strobe unit designed for machine
44
Light
Sources
Light
Sources
Diffuser
Diffuser
kpiece
Fig. 3 Different configurations of illumination system for computer vision. (A) Diffuse
front illumination, used for general top lighting. (B) Directional front illumination creates
shadows and will not reflect into the camera if surface
is highly reflective. (C) Light tent
(cloudy day) is nondirectional, totally diffuse top lighting that produces illumination like
that on a cloudy day (good for metal parts and electronic components).
(D) Back-lighting
through a collimating lens
so that the light rays
are pseudo parallel. (E) Dark field illumination in which incident light reflects away from the camera and illumination
is created from
specular reflections. (F) Diffuse back-lighting, in which light is on the opposite side of
the part as the camera and goes through a diffusing material suchas lexan or opal glass.
(G) Low-angle illumination, in which incident lighting is almost parallel to the surface
of the part. (H) Polarized front illumination, involving front-lighting with a polarizer on
the light and a cross-polarizer on the lens.
(I) Polarized back-lighting, in which a polarizer
and a cross-polarizer are on opposite sides of the part over some form of back-lighting.
(J) Stmbed illumination, in which microsecond duration lighting
is used to freeze the
motion of moving parts. (K) Structured light, in which a plane of light generated via
structured white light with focusing optics,
or laser line converter,is used to show contour/
3-D information of the part. (L) Coaxial lighting, in which the illumination is along the
same path as the cameras viewing path. (Courtesy of Machine Vision Association, Society
of Manufacturing Engineers, Dearborn, MI.)
vision use must be able to withstand continuous operation with repetition rates
45
Computer Vision
L i g h t Soorcc
W orkpiece
(b)
46
W orkpiece
Light
Sources
(r)
Fig. 3 Continued
47
Computer Vision
r s
\I
Light
Source
Light
Source
Cross-Polarizer
Translucent Part
1Polarizer
Light
Source
(i)
(1)
Fig. 3 Continued
49
Computer Vision
Workpiece
(1)
ul
0
Light tent
Advantages
Soft, fairly nondirectional
Reduces glare on metallic surfaces
Relatively easy to implement
Easy to implement
Good for casting shadows
Fiber optic delivery in many configurations
Eliminates glare
Eliminates shadows
Illuminates defects
Provides a high contrast image in some applications
Easy to implement
Creates silhouette of part
Very high contrast image
Low cost
Diffuse backlighting
Disadvantages
Edges of parts may be fuzzy
Low contrast on monocolor parts
May create unwanted shadows
Illumination is uneven
Must surround workpiece
Can be costly
Size can be a problem
Difficult to implement if material handling interferes
May be too bright for camera without neutral density
filters
Does not illuminate flat smooth surfaces
Eliminates glare
Highlights certain types of features or defects
in translucent materials
Relatively easy to implement
Strobed illumination
Structured light
Coaxial lighting
Complicated to implement
Harsh illumination for shiny surfaces
Source: Courtesy of Machine Vision Association. Society of Manufacturing Engineers. Dearborn. Michigan.
52 Gunasekaran
and
Panigrahi
advancements, fiber optics has been used for sensors as well as computer vision
applications. For computer vision applications,fiber optics has been used mostly
of fiber optics
for illumination and image transmission. It is expected that the use
as part of an image illumination subsystem of the computer vision system will
dramatically increase in the future.
In many computer vision applications, becauseof space and environmental
or to
constraints, it is necessary to provide illumination from remote locations
transmit image information to a remotely located computer system. Under these
conditions, the use of fiber optics is justified ( 1 1).
Fiber optics is highly desirable under the following conditions ( 1 1 ) :
Space for positioning the light source is restrictive.
The application requires camera movement along with movement
of the
illumination.
Multiple lighting with varied angle of incidence is required.
Maintenance of temperature profile of heat sensitive objects is critical.
Insertion of light through a small opening is required.
The light source could be hazardous in an explosive environment.
The application requires examination at micro or macro levels.
Optical fibers used for light transmission, illumination, and image transmission can be of the high-loss type, made up of optical glass or plastics. High-loss
type fibers are also cost-effective as compared to low-loss type fibers, which are
used primarily for data transmission, communications networks and other sensing
applications (12,13).
Fiber optics operates on the principle of total internal reflection. Optical
fibers exploit this phenomenonby encasing a cylindricalfiber in another cylindrical casing of lower refractive index (12,124). The outer casing is called clad,
and the inner fiber is called core (Fig. 4).
When light enters the fiber end at an angle A (incident angle), it undergoes
refraction at the interface of the core and its surrounding. Generally, the core has
a higher index of refraction (n,) than that of clad (11~). Under this condition, if
the angle of incidence (A) is greater than or equal to A, (critical angle), the light
will undergo total internal reflection (multiple times) at the core-clad interface.
Finally, it will leave the fiber through the outer end ( 1 19).
Using Snellslaw for optical fiber, its numerical aperture(NA) can be determined from the refractive indices of the core (n,) and cladding (n?), respectively
( I 19):
NA
[(nf - n;)]
NA
n sin (A,)
53
Computer Vision
of incidentlight
inan
opticalfiber.
where n is the refractive index of the surrounding medium. Since for air n = I
(for many cases, air is usedassurroundingenvironment),
N A = sin A,. N A
represents the ability of the fiber to accept light. Thus, the higher the NA, the
greater is the amount of light entering into the fiber. Therefore, to allow more
light through thefiber, consideration should be givento choose larger fiber (core)
and N A ( 1 19).
For both illumination and transmission applications, several criteria need to
be considered before selecting the appropriate optical fiber for a given computer
imaging system ( 120).
54
Chalcogenide
Fluoride
Glass
Enhanced
Glass
Silica
I
0.1
0.2
0.3
Ultraviolet
I
0.5
0.7
Visible
I l l
1
I
2
3
Wavelength
- (wm)
..
.
IIIII
7
10
20
Near-infrared
Fig. 5 Opticalfibermaterialsandtheirspectraltransmission(Adaptedwithpermission
fromRef. 12.)
Computer Vision
55
Camera
C.
The camera is the sensor of a computer imaging system used to capture image
information. It functions similar to the eyes in human vision. Charged coupled
device (CCD) cameras have been used for nearly all computer imaging applications since their introduction almost 25 years ago (14). There have been many
developments basedonthe CCD technology.Recentlycomplementarymetaloxidesemiconductor(CMOS)technologyhasbeenintroduced(14,17).Many
varieties of black-and-white and color cameras are commercially available. A
black-and-white camera will suffice for food quality evaluationif attributes unrelated to color (dimensions, shape, other geometrical attributes) are to be evaluated. To evaluate color or color-related attributes, an appropriate color camera
should be selected. Color cameras range in cost from several hundred to several
thousand dollars. For higher-quality color images, three CCD color cameras perform better than one CCD color camera.
Though most cameras available are of area-array types, line-scan cameras
arealsoavailable in bothcolorandmonochromemodes.Area-arraycameras
are useful for imaging 2-D scenes, while line-scan cameras offer high positional
accuracy, rapid frame rate, and a wide dynamic range (14). They are available
in resolutions that include 128, 256, 5 12, 1024, 2048, 4096, and 8 196 pixels per
line. Many line-scan cameras have square pixels also (14).
For some food product evaluation, the nature of the inspection might re-
56
quire the camera to deal with low-light level images. This requirement can be
fulfilled by using another variationof line-scan camera, time-delay integration
(14). These cameras typically have 1024 lines and a number
of stages or rows
of sensors positioned side by side horizontally. Because these sensors also incorporate rows of photo elements, multiple views of objects can be captured. The
electrical charge from each rowis transferred from row to rowin synchrony with
the moving object to eliminate blurring. These cameras provide high signal-tonoise ratios as compared to their line-scan camera counterparts (14).
For imaging 2-D scenes, the most commonly used cameras are either frame
transfer or interline transfer CCD types. A frame transfer type camera has both
a sensing array and a storage array.
The storage array is positioned above the
sensing array. At the end of a field period (1/60 s, RS-170 signal), the data are
rapidly shifted vertically from the sensing array into the storage array. After the
data have been shifted into the storage array, they are shiftedto a horizontal shift
register, two lines at a time. The advantage of the frame transfer device is that
is
the entire area of the sensing array is sensitive to light. Their disadvantage
that the transfer process takes longer
and, thus, under certain conditions where
objects move, error in image data may be introduced (1 5).
Interline CCD type chips are used for most CCD cameras in the market.
of their low cost and their
They are attractive for many applications because
ability to handle bright localized
light overloads without streaking (15). They
can image moving objects without blurring, provided the scene has sufficient
light (1 5 ) .
Recently, progressive scan technology based on
CCD chips has created
progressive scan cameras that can be used for imaging fast-moving objects (14).
The emergence of these cameras has increased the potential of integrating computer-imaging techniques for high-speed, on-line applications in a cost-effective
manner. Progressive scan means noninterlaced or sequential line-by-line scanning
of the image information out of CCD as opposed to traditional interlaced fields
(16). Although this technology has been available for some time, especially for
frame-transfer type cameras, the recent introduction
of cameras basedon interline
transfer-based progressive scan devices has created new application advantages,
especially for high-speed imaging (16).
In these cameras, technology depends
heavily on effective shuttering (16). The technological advantages of interline
progressive scan ( 1 6) now can be applied to all types of high-speed and precision
imaging applications related to food quality evaluation.
Cameras for computer imaging that use charge injection devices (CIDs)
are also available. These sensors differ significantly from typical
CCD sensors.
Although the sensing pixels are constructed of metal oxide, semi-conductor capacitor integrating sites, the collected photon charge is read out differently ( I 5).
Major advantagesof CIDs includerandom access, radiation tolerance, nonbloom-
Computer Vision
57
D.
FrameGrabber
58 Gunasekaran
1.
and
Panigrahi
Video Input
It is critical to ensure that the output of an image source (analog camera, digital
camera, VCR, etc.) matches the input of the frame grabber. The camera can be
monochrome (black-and-white) or color providing standard outputs as RS-170
monochrome or National Television System Committee (NTSC) color signals
(60 Hz video signals are used throughout North America and Japan) ( 1 18). The
camera can also provide Comite Consultatif International Radio (CCIR, an international organization) monochrome or phase alteration line color signals (50 Hz
or progressive
video standard)( 1 18). Nowadays, other cameras, such as line-scan
scan, are also available with nonstandard video outputs.
It is also possible to take image information from a video cassette recorder
(VCR) or digital camera.
It is often thought that digital cameras
do not need frame
grabbers. However, most frame grabbers need
an interface with a computer. It
is recommended to verify the proper required interface of the digital camera.
or of low
Some video signals, including those from VCRs, can be noisy
quality, resulting in blurry image acquisition due to missing or extraneous sync
(synchronization)signals.Thus,specialtimingcircuitry
is requiredonframe
( 1 18).
grabbers to correct for missing, extraneous, or low-level sync pulses
It is sometimes necessary to acquire monochrome images from color signals. The color (chrominance) content of these signals can cause interference
patterns, which degrade the quality of the image. Thus, frame grabbers are provided with a hardware/software selectable chrominancefilter that produces better
images ( 1 18).
2. Analog/DigitalCapability
col-
h. BrigktrwssResolution.
Thisrepresentsthemaximumdiscrimination
of a given
capability of digital pixel value representing the brightness or color
pixel. It is expressed by the resolution of the AID converter, which is expressed
in number of bits (1 18). For a resolution of 12 bits, the resulting number of gray
levels is 2.
For color images, three A/D converters simultaneously convert red, green,
and blue information. For color frame grabbers, brightness resolution
is determined by the sum of all the A/D converters resolutions, i.e., 3 X 11 bits. For
example, a color frame grabber with
an 8-bit A/D converter has a brightness
resolution of 3 X 8 or 24 bits. The total number of colors is 2 or 16,777,2 I6
(25,43,118).
Computer Vision
59
I O MHz
3. SignalHandling/Conditioning
The quality of an incoming video signal directly affects the qualityof a digitized
image. Poor lighting conditions, signal loss due to long cables, irregular sync
of camera are some parametersthat can cause
signals, and gray scale nonlinearity
poor quality in an incoming video signal. Well-designed frame grabbers can compensate for many of these problems by having different in-built capabilities (20).
Gain control adjustments, offset adjustments, and sync timing are three very desirable capabilities for frame grabbers (20).
(1.
Guin Adjrrsftnents. Gain adjustments can help in many situations such
as when adjustment of illumination is not possible and when the application depends on natural lighting (20). Other applications which are inherently of low
contrast (typically food/biological applications) or those which deal with passive
60
infrared imaging can also benefit from this feature. Thus, a gain control
frame grabber provides more versatility (20).
in the
b.OnsetAdjustments.
Offsetadjustment is verycritical,especially
if
the camera does not compensate for low or high lighting conditions. For best
results, a frame grabber should have the capability to offset a video signal by
2 100% in small, precise increments (20).
C.
ProgrammableGainand
Offset Controls. Becausegainandoffset
adjustments are very complementary, it is desirable that a frame grabber have
the capability for programmable control for gain and offset (20,118). The frame
grabber that is to be used with resettable cameras should detect each horizontal
sync pulse and instantly resynchronize its pixel timing. (Resettable cameras are
mostly used for industrial applications such as inspecting parts on conveyer
belts.)
For such applications, a desirable frame grabber is one with proper sync-timing
capability such as a crystal-controlled digital clock that can resynchronize instantly (20).
To digitize video
Another parameter to be considered is pixel jittering.
images, frame grabbers sample analog video at uniform intervals on each line to
determine the gray scale levelof each pixel (20). However, the inability
of frame
grabbers to precisely adjust the sampling points relative
to horizontal sync causes
pixel jitter (20). Phase locked loop (PLL) is the traditional timing mechanism
used for sync timing. PLL creates a clock from reference frequency.Pixel jitter,
in other words, is the timing accuracy of this clock and is expressed in nanoseconds. Pixel jitter of PLL varies greatly dependingon design and implementation
(21). For example, a frame grabber with 2 5 ns implies a pixel positional error
of 12.5% (20). Though frame grabbers are available that use both digital clock
circulating and modified PLL for precision applications with pixel jitter of *2
ns, pixel jitter of ? 10 ns is reasonable. The selection of acceptable pixel jitter
depends on the application (21).
Computer Vision
61
62
IV.
IMAGE PROCESSING
A.
Basic Steps
The basic steps in image processing are image preprocessing, segmentation, and
feature extraction (Fig. 2). The purpose of image preprocessing or image conditioning is to enhance the quality of the acquired image, which is often degraded
by distortion and noise in the optical and electronic systems of the input device.
(24): noise reducImage preprocessing steps include one or more of the following
tion, geometrical correction, gray level correction, and correction of defocusing.
These steps are typically applied uniformly and are context independent.
As thenameimplies,imagesegmentationrefers
to theprocessofsegorobjects.
menting or partitioningacompositeimageintocomponentparts
Proper segmentation is very critical. Often, the first step in assuring successful
segmentation is control of background uniformity. For monochrome images, segmentation normally is performed by examining the gray scale histogran-a bar
chart of the number of pixels in the image at different gray levels. Segmentation
algorithms are based on discontinuity or similarity of the gray level values. Discontinuities in image gray scale indicate sharp changesin image brightness such
as background and object.
In general, autonomous segmentation is one of the most difficult tasks in
image processing (25). Macaire and Postaire (26) described a real-time adaptive
thresholding to be used for on-line evaluation with line-scan cameras.
Segmented image data constitute raw pixel data of the image boundary or
or region
a region of interest in the image. The image representation as boundary
should be selected based on the intended application. For example, boundary
representation is appropriate for image size and shape characterization.
The region representation is suitable for evaluating image texture and defects.
The feature extraction step is thekey in deciphering the require image data
form the composite image information.The successof the feature extraction step
of the previous steps, including image
depends largely on the appropriateness
acquisition. The knowledge of the feature under consideration is also critical
at this stage in designing appropriate algorithms to extract information pertaining
to the desired feature(s).
Featureextractionfacilitatesobtainingsomequantitativeinformation
of
interest, which is then processed in conjunction with the knowledge base available for the feature studied.
6 . KnowledgeBase
At all steps during image processing, interaction with the knowledge base enables
more precise decision making. Thus, knowledge about the system being studied
of an image-processingsystem.Withoutan
shouldbeanintegralcomponent
Computer Vision
63
appropriate knowledge base, the vision system cannot think and make intelligent decisions (27). This problemis further complicated by the fact that the output
of a vision sensor is a complex combination of many parameters: size, shape,
texture, color, etc. Some requirements for intelligent decision making are (a) the
ability to extract pertinent information from a background of irrelevant details,
(b) the ability to learn from examples and generalize this knowledge and applyit
in different circumstances, and (c) the ability to make inferences from incomplete
information.
of
Expertsystems,neuralnetworks,andfuzzylogicaresomemethods
building knowledge bases into computer memories, enabling them to recognize
and interpret image data and to provide on-line control capabilities. The image
understanding part of the computer vision systemis inherently tied with the completeness and accuracy of the valid knowledge base available for the product(s)
and the feature(s) being studied. The successful image understanding step will
lead to the ultimate goal-translating image analysis data into information useful
for further action such as process/machine control. Applying neural networks
and/or fuzzy logic in conjunction with computer vision systems is rapidly growfor quality sorting of fruits and vegetaing, and commercial systems are available
bles (28).
C. PatternRecognition
Pattern recognition at some level is fundamental to image analysis. A pattern is
ina quantitative or structural description of an object or some other entity of
is formed by one or more descriptors
terest in an image. In general, a pattern
(features). Pattern recognition by machine involves techniques for assigning patterns to their respective classes automatically and with as little human intervention as possible.
In machine recognition of image patterns and shapes, generally two approaches are used: a statistical or decision-theory approach, in which features are
extracted and subjectto statistical analysis, and a syntacticor structural approach,
in which image primitives are selected and subjected
to syntax analysis.
The statistical or decision-theory approach is the traditional approach to
1960s. The system (Fig. 6)
pattern recognition that has been studied since the
consists of two parts: analysis and recognition. In the analysis part, a setof image
features that are judged to be nonoverlapping (or as widely apart as possible) in
the feature space is chosen (29). A statistical classifier (e.g., based on a fuzzy
logic or neural network system) is designed and trained with the chosen set
of
features to obtain the appropriate classifier parameters. In the recognition part,
an unknown image is filtered or enhanced in the preprocessing stage, followed
by feature detection and classification. This approach, however, does not describe
or represent structural informationin a pattern that is often desirable or necessary
64
Classification
t """_
RECOGNITION
-------------"""""
ANALYSIS
t "_
I
Sample Pattern
I
Input
Image Pattern
rL
Decomposition
,.
FPrimitive
& Relation
Recognition
RECOGNITION
"--""""""""_
ANALYSIS
"1
Training
Selection
Syntax or
Structural Analysis
'
"
"
"
~
"
"
"
"
"
"
"
"
"
"
~
il
Relation Selection
Grammatical or
Structural lnferena3
b,
(8)
Fig. 6 Pattern recognitionsystems: (A) statistical and (B) syntactic. (Adaptedfrom
Ref. 29.)
D. Image Morphology
Image morphology refers to the geometric structure within an image, which inA general
cludes size, shape, particle distribution, and texture characteristics.
Computer Vision
65
approach in analyzing image morphology is to transform the given image to another where the information represented in the transformed image is more easily
understood. For example, investigationsof the shape of objectsin a binary image
a minimal set of pixels
often use thinning algorithms. Reducing an object to
representing an invariant of the objects geometrical shape is called thinning. A
skeleton is a line-thinned caricature of the binary image that summarizes the
shape and conveys information about its size, orientation, and connectivity (25).
An image resulting from the thinning process has many fewer black pixels representing the object and is, therefore, easier to manipulate.
If themaingoal of
thinning is data reduction and exact reconstruction of the original image is not
essential, many techniques are available that yield acceptable skeleton representations. However, if close or exact reconstruction is desired, care must be taken in
choosing an appropriate algorithm.
Morphological image-processing algorithms (thinning,
region filling, thickening, pruning,etc.) remain a useful tool in image processing and computer vision.
Some of the requirements for image thinning are (30):
Connected image regions must thin to connected line structures.
Approximate end-line locations should be maintained.
Thinning result should approximate the medial lines.
Extraneous spurs caused by thinning should be minimized.
The morphological approach has been successfully applied to a wide variety of problems. The power and usefulness of some basic morphological processing algorithms have been illustrated by McDonald and Chen (31). Morphologicalprocessingforisolated,nontouchingobjectsiseasilydoneusing
commercialpackages, which canperformobjectcounting
and dimensional
measurements, etc. However, touching and overlapping objects pose problems
unique tothe products being examined. Thus. separate algorithms and procedures
need to be developed. McDonaldand Chen (31) developeda morphological algorithm to separate connected muscle tissues in an image of beef ribeyes.
Recently, Ni and Gunasekaran ( 3 2 ) used imagethinning in conjunction
with a syntactic approach to evaluate the morphology and integrity of touching
and overlapping cheese shreds (Fig. 7). The algorithm performed very well with
less than 10% error in individual shred length measurements.
Evaluation of an image skeleton was also used to characterize granular
foods that may agglomerate (33). Smolarz et al. (34) used morphological image
processing to define structural elements
of extruded biscuits and then to discriminate biscuit type.
E. ShapeFeatureExtraction
The statistical or decision-theory approach has been widely used for food shape
it isoften
featureextraction.Foodmaterialshapeisveryimportantbecause
66
Fig. 7 Morphological image processing for evaluating integrity of cheese shreds: (A)
cheese shreds, (B) binarized image, (C) after image thinning. (Adapted from Ref. 32.)
closely related to quality. Due to the demands of high quality, automated food
shape inspection has become an important need for the food industry. Due to
large inhomogeneities of food materials, however, such invariant shape features
cannot be used to detect local defects.
In many cases, therefore, the invariant
feature extraction methods cannot accurately distinguish between damaged and
undamaged categories. Panigrahi et al. (35) evaluated invariant moments and
fractal geometry for shape classificationof corn. Recently, variant shape extraction methods (position, orientation and scale) are gaining popularity for food
material shape inspection (36).
In the variant method, the edge contour of the inspected object is transformed to a given position, orientation, and scale. Then
the shape features are
extracted from every local edge point. Ding et
al. (37) presented a statistical
model-based variant feature extraction method for shape inspection
of corn kernels. This was based on a reference shape, a transformed average shape
of some
undamaged corn kernels. After the reference shape was obtained, the shape
of
kernels being inspected was compared with the reference shape.
(38) proposed a new algorithm with
More recently, Ding and Gunasekaran
improved ability to adjust object location, orientation, and scale to determine the
edge contourof a numberof food materials for shape evaluation. This multi-index
active model-based feature extractor is based on a reference shape comparison
Computer Vision
67
F. Image Texture
Texture is characterized by the spatial distribution of gray levels in a neighborhood. For most image-processing purposes, texture is defined as repeating patterns of local variations in image intensity, which are too fine to be distinguished
(30). Thus, a connected setof pixels
as separate objects at the observed resolution
Fig. 8 Food shape evaluationto detectdamaged products by comparingobject and reference edge contours: (A) corn kernel, (B) animal cracker. Object edge contour has been
converted into Transformed edge contour and comparedwith Average of transformed
good kernel edge contours. (Adapted from Refs. 27 and 38.)
68 Gunasekaran
and
Panigrahi
V.
COLORIMAGE PROCESSING
A.
ColorCoordinates
Computer Vision
69
ever, RGB and HSI havebeen extensively used for color image processing applications. Each color coordinate has three components. The combination of these
components produces a color image.
1. RGB
In the RGB color coordinate system, the three color components are the three
primary or fundamental colors red, green, and blue. Different combinations
of
these primary colors produce various secondary colors. The Commission Internationale de 1Eclairage (CIE) uses the spectral primary system RGB to define any
color by combining red, green, and blue (light generated by a monochromatic
light source at 700, 435.8, and 546.1 nm, respectively) (43). The chomaticities
r, g, and b (normalized red, green, and blue) are defined as:
r=R/R+G+B
g=G/R+G+B
b=B/R+G+B
The RGB color coordinate system is commonly used in television, color
cameras, and color display monitors. In the television industry, RGB signals are
encoded into luminance (Y) and chrominance (I and Q) to minimize bandwidth
for facilitating broadcast (43). RGB cameras and monitors have
been used for
computer graphics because RGB isa good system for generating and displaying
images. Similarly, digitizers use three A/D converters to digitize the RGB signal.
The digitized color imageis stored in three color components/buffers, which are
mixed together only during display to show one composite color image.
According to Travis (42), RGB technology is fine for grabbing, storing,
in RGB space is computationand displaying the image, but processing the image
ally intensive and algorithmic implementation is complex. Moreover, RGB is a
poor way to represent images basedon human vision because people do
not think
of color in terms of combinations of red, green, and blue.For example, it would
be difficult to look at a yellow cake and specify the percentages of red, green,
and blue that combine to form the color of the cake.
2. HSI
The HSI color coordinate system is an alternative to the RGB system. Hue (H)
is defined as the attribute of color perception by means of which an object is
judged to be red, yellow, green, or any other color. Intensity (I) represents the
attribute of color perception by means of which an object is judged to reflect
more or less light than another object. Saturation (S) represents the attribute of
70
color perception that expresses the degree of departure from gray of the same
lightness.
Understanding and manipulating color images in HSI is much easier and
less complicated than in RGB because the HSI system resembles the way we
perceive color. Individual values of H, S, and I contain information that is meaningful for human color perception and can be analyzed independently. Thus, the
algorithmdevelopment is lesscomplicated(43,122).Thefollowingempirical
relationships between RGB and HSI color coordinates make it possible to switch
between the two (25):
I =
S
R + G + B
3
= 1
(6)
3 .
-[mm(R,G,B)]
I
H = cos
[(R
(7)
[(R - G) + (R - B)]/2
G) + (R - B)(G - B)].5
Computer Vision
71
source ( I 23). On a CRI scaleof 0- 100, 100 represents a source with the renderof 0 (zero) implies an illumination source
ing capabilities of daylight (45). A CRI
incapable of rendering color. Thus, the higher the CRI, the more vibrant or brilliant the color is. Light sources with a CRI of 80 or higher have excellent color
rendering properties. A CRI of 70-80 implies a good color rendering property
(45,123).
Color temperature is the absolute temperature of a black body radiator
having a chromaticity equal to that of the light source (123). It is expressed in
of 3000 K (low) corresponds to warm or
degrees Kelvin. A color temperature
red-yellow appearances. Light sources at 3500 K provide neutral white light and
those at 4100 K provide cool bluish light. Light sources witha color temperature
of 5000 K give off daylight (45).
Both CRI and the color temperature of a light source can be obtained from
the manufacturer. For developing a color computer imaging system, i t is always
recommended to select a light source with CRI above 85 and a color temperature
close to 5000 K (123). Another secondary but important parameter
is the stability
of the color temperature of the light source over time. The color temperature of
many light sources changes with time. Thus, it is recommended to select a light
source whose color temperature does not change with time.
C.ColorCalibration
Calibration of a color computer imaging system impliesthat all the critical comto
ponents (i.e., camera, frame grabber, display monitor) should be calibrated
handle, process, or display color information. Thus, calibration of a color computer imaging system includes the calibration of its components, such as a color
camera, frame grabber, and display monitor.
Many end users or developers, unfortunately, have not practiced color calibration. Often it is assumed that all components are working satisfactorily. In
many cases, however, a small deviation
of calibration of one component can
introduce errors in the final result provided by the color computer imaging. For
example, if a color camera looking at an orange puts out the color information
as yellow or red instead of orange, then error is introduced.
1. DisplayMonitorCalibration
Video test generators or test pattern generators available commercially can be
The generatorsendsdifferentsingle-color
connected to thedisplaymonitor.
charts such as red, green, blue, etc. and white, black, or multiple color charts to
the monitor to be displayed on thefull screen. The user thencan adjust the monitor for proper calibration for a wide range of color conditions or for user-desired
specific color condition. At present, computer add-in boards are also available
72
that can be used in place of stand-alone video test or pattern generators. A list
of manufacturers of video test generators are listed in Ref. 46.
2. CameraCalibration
A standard method to calibrate a cameras output uses two pieces of test equipment: an NTSC vectroscope and a waveform monitor. Vectroscopes and waveform monitors are extensively used by the television industry, and they complement each other. Waveform monitors display the video signal to allow the user
to measure its amplitude and time parameters. At the same time, the vectroscope
can show the relationship between a chrominance signal and its reference burst
(or phase) and gain distortion (46).
or
Under a given lighting condition, the camera can look at standard, single,
multiple color bar charts. The camera output is sent simultaneously to a waveform
monitor and an NTSC vectroscope. The waveform monitor measures the amplitude of a video signal, and the vectroscope shows the relationship between color
information (46). Necessary adjustments canthen be madein the cameras control
unit to calibrate the camera.
Note that an NTSC vectroscopeonly accepts NTSC composite input,
which
most analog cameras provide. If the camera does not have an NTSC output,
individualRGBoutputscanbeinterfacedwithRGBinputs
in any calibrated
video display monitor. In this case, the NTSC vectroscope is not used. Using
visual observation of the displayed color on a calibrated video monitor, proper
adjustments can be made in the color camera.
3. FrameGrabberCalibration
After the camera and display monitor are calibrated, the frame grabber can be
calibrated. Standard single-color bar charts typically havea 94-96% reflectance
value. Images can be acquired separately using single red, green, blue, white,
and black charts. For red, green, and blue conditions, the average pixel values
of the respective buffer for the digitized images should be about 252-255. Any
deviation can be addressed by changing the gain and offsets of respective A/D
converters of the frame grabber. Note that the frame grabber has three A/D converters for each of the red, green, and blue channels. The frame grabber should
have programmable gain and offset adjustment capabilities. Similarly, the frame
grabber can be adjusted under white (presence of all color) and black (absence
of all color) conditions. Different researchers have related incorporationof calibration of different color imaging components or systems. Panigrahi (47) described calibration of a color imaging system for corn quality evaluation, and
Hetzroni and Miles (48) described color calibration of RGB video images. Several researchers have reported additional procedural
and mathematical techniques
for calibrating cameras, even in on-line conditions (49-51).
Computer Vision
73
D. ColorImageProcessingApplications
In recent years, color computer imaging technology has been extensively applied
to numerous food-related applications, which can be broadly grouped into color
evaluation, defect detection, and texture evaluation
of different food products,
including dairy, meat,fish, fruit, vegetables, and others(52). This variety of applications, however, presents challenging color image processing issues. These applications can be discussed under two broad image processing categories: color
segmentation and image analysis and understanding.
1. Color Segmentation
In processing color images, segmentation refers to isolating or separating a homogenous or desirable region of interest in the image. Removal of a background
from an image is a simple example. Segmentation is also used to identify and
quantify all sorts of defects, diseases, and other abnormalities. The segmentation
problem in color image processing is more complex than in gray scale applications. A color imageis comparable to three gray level images having color information contained in three color components, e.g., red, green, and blue. Thus,
segmentation becomes more time-consuming and involved for color images. Of
course, the level of complexity depends significantly on a given application. The
inherent random variability of quality attributes of raw materials (agricultural
products) for food products further adds to the complexity
of segmentation of
color images of many food products.
Thresholdingbasedonhistogram
is usedforapplicationsthatseparate
background from the object or separate two
or three dissimilar contrasting regions
in the image. One requirement is that there should be a good amount
of color
difference among the regionsto be segmented. Sometimes investigationis neces(54).
sary to choose the appropriate color coordinate for performing segmentation
To evaluate the color
of French fries, for example, removing background information from the French fries was required. Finding the threshold based on histogram
on the RGB color component was difficult and time-consuming. Using the HSI
coordinate, a histogram was obtained on the I (intensity) component. Finding a
threshold in the intensity image alone was easier than with the RGB image. Using
the determined threshold, the background was isolated from the image. All backgroundpixelswerelabeled
in intensityimageandsubsequentlymappedinto
saturation and hue images. Thus, for color evaluation, the hue and saturation
information of background pixels were not considered (54).
Use of adaptive thresholding techniques for a histogram-based segmentation is also recommended for food images. They promise higher accuracy and
robustness than a fixed (global) threshold. Adaptive thresholding techniques can
adapt to changes in lighting and spectral characteristics of an object as well as
the background. Therefore, they are well suited for real-world applications and
74 Gunasekaran
and
Panigrahi
2. ImageAnalysisandUnderstanding
In analyzing color images of food, the exact techniques for image analysis and
understanding differ from applicationto application. For example, an image analof corn might
ysis and understanding algorithm developed for color classification
not work fully with high accuracy for potatoes or potato products. This provides
additional challenges and requires investigation for developing successful applications.
Selecting appropriate color coordinates for analyzing a given food color
image is critical. An accurate answer to the question, Which color coordinate
do I choose? can be obtained by experimentation only for a given application.
For color evaluation of edible beans, Panigrahi et al. (63) evaluated both r-g-b
(normalized RGB) as well as hue h-s-i (normalized HSI) coordinates. Both sets
of coordinates provided accuracy up to 100% in classifying beans in three color
groups (63). To identify and quantify fat in meat images, RGB color space was
usedwitharectangularprismand
Mahalano bois distance criteria (64). RGB
color coordinates were used for locating citrus fruits for harvesting (65) and RG
color space wasutilized along with Bayes decision theory for image partitioning
and subsequent color classification of stone fruits (66).
Recently, the potential of artificial intelligence technologies, such as neural
networks and fuzzy logic, has also been explored for image classification and
understanding purposes. Both neural network and fuzzy logic techniques are intelligent, adaptive, and fault-tolerant(57),and they complement each other. Neu-
Computer Vision
75
VI.
THREE-DIMENSIONALCOMPUTERIMAGING
Most of the food materials are three-dimensional (3-D) objects, and hence for a
complete and thorough evaluation of food quality, 3-D information is necessary.
Therefore, there is an increasing need for extracting 3-D information, which will
76
A.
1.
Time of Fhght
Fig. 9 Differenttechniquesformeasuring3-Dinformation.(Adaptedwithpermission
fromRef. 77. 0 1988 IEEE.)
Computer Vision
77
Target Pomt
I\
Beam
Transmitted
Transmitter
Laser
* Pulsed Time Delay
* Modulated (Frequency
Amplitude)
or
Ltght
\Recewed
Light
Recewer
Range
Intensity
Fig. 10 Configuration for pulsed time delay and phase shift laser range sensing systems.
(Adapted with permission from Ref. 77. 0 IEEE 1988.)
78 Gunasekaran
and
Panigrahi
mine the range based on the measured time of flight of the transmitted signal
(76,77). In a phase shift system, a frequency or amplitude modulated laser sends
the signal. The measured phase shift between the transmitted and the received
signal determines the range (77).
Laser-based range sensors could provide better resolution than ultrasonic
range sensors, but they are relatively costly (76,77). Moreover, slow measurements and ambiguity (when phase shift is greater than 360) issues create additional problems for laser range sensors (77). Nevertheless, the rapid reductionin
the cost of the laser systems, along with their increased capabilities, could eliminate some of these problems.
2.
Triangulation
Epipolar Plane
Projector
(StructuredLtght)
Fig. 11 Illustrationofthetriangulationprocess.(Adaptedwithpermission
77. 0 1988 IEEE.)
from Ref.
Computer Vision
79
Baseline
80 Gunasekaran
and
Panigrahi
Object
Detector
(A)
(B)
(C)
Fig. 13 Different structured lighting systems:(A) simple system,(B) light stripe system,
(C) multiple stripe system. (Adapted with permission from Ref. 78. 0 Kluwer Academic
publishers.)
The width of light stripe defines the spatial resolution (84). If the object is moving, the vision system will needa few or no moving parts. However, the direction
of travel should be perpendicular to the light stripe (78).
The third technique uses multiple stripesof light simultaneously (Fig. 13C)
(78,82) and is very similar to the second arrangement (Fig. 13B). One disadvantage with this approach is that multiple stripes can cause ambiguity during the
process of image recognition. However, the use of several gray-coded light stripes
might solve this problem (82,83). The gray-coded light stripe method provides
computational advantages too. For example, if N ordinary light strips (N scans)
were required to completely scan an object, only log, N gray-coded light stripes
(log? N scans) would cover the entire object (81-83).
Bayer and Kak(1 16) reported the integration of color with structured lighting. This method of color-encoded structured lighting shows a few benefits, i.e.
increased speed and better accuracy. In this technique, they ( 1 16) used a single
encoded grid of colored light stripes to obtain range information. Grid to grid
alignment problems generally found in multiple stripe technique, were overcome
with this method (1 16). However, problems were encountered in dealing with
objects having deeply saturated colors. Thus, the applicability of this technique
might be limited to applications where the objects are of neutral color (1 16).
Many benefits are associated with structured lighting. Its inherent simplicity
to acquire 3-D information.
has madeit one of the most commonly used technique
it. Objects with high
Nevertheless, several difficulties are still associated with
specular surface characteristics could provide incorrect
or sometime very little
range information (76,77,82,83). Though structured lighting technique sometimes
could be slow in acquiring image information (77,82,83), recent developments
in high speed processors and detectors might eliminate this problem.
In cases
where triangulation techniques are used with structured lighting, sometimes hidin acquiring quality
den surface or edges
of the object might cause problems
images or processing acquired images (77,8 1,83,84).
Computer Vision
81
Fig. 14 A typical stereo configuration for 3-D imaging. (Adapted with permission from
Ref. 117. 0 SME)
Structured lighting has been used for several agricultural applications, some
of which dealt with production and animal agriculture. Structured lighting with
two-constraint propagation was used to measure 3-D surface featureson potatoes
(85). Structured lighting was also used to find stalk and calyx of apples during
high-speed detection of blemishes on apples (86).
Another 3-D measurement system using structured lighting was developed
to determine the shape of soybean seed, its axial length, surface area, volume,
particle density, compactness, and sphericity by Sakai and Yonekawa (87). In
at the center
their study. A soybean sample was mounted on a needle located
of a supporting table. A camera was placed over the sampleat a known distance.
to the
A vertical plane of light struck the sample at an oblique angle relative
cameras horizontal axis. A helium-neon laser light source was used, which emitted structured light vertically deflected by a polygon mirror at 10,000 rpm and
was narrowed by a biconvex lens. The systems performance was satisfactory
(87). However, more work is necessary to further develop the system.
82 Gunasekaran
and
Panigrahi
Camera modeling and calibration are important procedures for 3-D information acquisition using stereo (79,117). According to Bachnak ( 1 17), Camera
calibration is the procedure for determining intrinsic and extrinsic parameters of
the camera. Such parameters include the focal length of the camera, the scaling
factor of the system, the transformation relationship between the 3-D scene or
object, and the image plane. Detailed description of camera calibration is mentioned by Faugeras (79).
Extraction of depthor3-Dinformationusingstereoinvolvesdisparity,
cameraparameters,andimagecorrespondence(76,88,117).Imagecorrespondence implies matching corresponding points in the stereo pair images (76). For
determiningcorrespondencefromimages,Jarvis(76)emphasizedthatthere
must be sufficient visual informationat the matching points to establish a unique
pairing. Two basic problems arise with this process.
The first occurs at parts
where uniformity of intensity or color makes matching impossible. The second
happens when the image of some part of the scene or object appearsin only one
view of the stereo pair because of occlusion effects or limited field of view captured in the images.
According to Bachnak (1 17), two regularly used approaches to matching
or correspondence are area-based and feature-based matching. Area-based approaches result in reasonably accurate disparity maps, but they are sensitive to
changes in contrast and depth. Feature-based methods, on the other hand, focus
on easily distinguished properties i n the images such as lines, corners, etc. The
result is normally an accurate sparse disparity map. Further discussion on tackling correspondence problems are mentioned by Yakimovosky and Cunningham
(88).
Another critical step to be emphasized for using the stereo techniqueis the
optimization of baseline or distance between two cameras (76,77,88,117) (Fig.
1 I). If the baseline is smaller than optimum, accuracy in 3-D measurement could
be affected. The largerthe distance than optimum between cameras, on the other
hand, could increase the accuracy of depth measurement. However, the problem
of hiddedmissing surfaces could occur (76,77,117). A stereo system developed
at NASAs jet propulsion laboratory for guiding a robotic vehicle
is described
by Matthies and Anderson (89).
B. 3-D Microscopy
Study of microstructure is one of the most fundamental ways of evaluating food
quality because the macroscopic textural properties are, in fact, a manifestation
of the microstructural arrangement of constituents of a complex food material.
Stanley and Tung (90)
defined microstructure as a complex organizationof chemical componentsundertheinfluence
of externalandinternalphysicalforces.
Foods having similar structures can be loosely grouped together as foods that
Computer Vision
83
84 Gunasekaran
and
Panigrahi
Oneprincipaladvantage
of opticalsectioningoverserialsectioning
is
avoiding physical specimen distortion dueto cutting and having image alignment
from the various image planes. Another advantage is depth resolution; while inferior to the lateral resolution in each plane by a factor of about 2-3,
it is still
useful for many applications (9). The minimum separation between observation
planes is 0.05 pm (94), a difficult resolution to achieve using regular light microscopy. Maximum observation depths of 10-100 pm can be achieved, depending
on a specimens opacity and absorption characteristics.
The CLSM has been a proven technique for a number
of biomedical applications. However, it is still in its infancy for food quality evaluation (3). Its application for studying food materials is expected to increase within next few years
due to the ability of CLSM to:
Penetrate deeply but noninvasively into the specimen
Obtain large numbers of sequential, thin optical sections that may be reassembled by a computer to produce 3-D images or stereo pairs
to threeorfourseparatechemicalcomponents
Identifyandlocalizeup
(depending on the number of laser lines available on the instrument) by
using specific fluorochrome labeling techniques (95)
In addition, specimen observations can be made within a plane both transverse to and along the optical axis, as compared with conventional light microscopy, which can only make images transverse to the optical axis. Sample preparation for CLSM involves staining the lipid or aqueous
protein phase of cheese with
fluorescent dyes and subsequent observation after laser excitation (97). However,
CLSM is limited by the maximum possible magnification, which
is about 400.
Vodovotz et al. (96) provide details of CLSM functioning.
Brooker (97) presented some figures of mozzarella cheese microstructure
obtained using CLSM.Hassan et al.(98) used CLSM to observe coagulum formation resulting from milk acidification. Vodovotz et al. (96) and Blonk and van
( 1 00)
Alast (90) reviewed many other CLSM applications. Ding and Gunasekaran
have developed a 3-D image reconstruction procedure to build a 3-D network of
fat globules in cheese. The 3-D view of fat globules in Cheddar cheese is presented in Fig. 15. Throughthisreconstruction,informationabouttheglobule
volume and related properties were measured and related to cheese-making procedures, which were not otherwise possible (101).
Computer Vision
85
Layer 16
86 Gunasekaran
and
Panigrahi
VII.
NONVISIBLECOMPUTERIMAGING
Although the majorityof computer imaging technology uses the visible spectrum
(380-700 nm), the nonvisible electromagnetic spectrum also has potential for
(UV),
use in computer imaging. These nonvisible bands include x-ray, ultraviolet
near-infrared (NIR), and infrared (IR). Advances in semiconductor-based detector technology and declining component prices have triggered the integration of
nonvisible imaging techniques for food quality evaluation.
A.
FluorescentImaging
Most food products or raw materials of food products can use fluorescent imin the next chapter. Formost cases
aging. Principles of fluorescence are described
of fluorescent imaging, the wavelengths used range from the far end ofUV (300
Computer Vision
a7
nm) to the far end of VIS (700 nm). Intensified CCD cameras have been used
for this typeof application. Because of the low amount of signals available, these
intensified CCD cameraswork better than a conventionalCCD camera. Recently,
however, the introduction of DSPs with conventional CCD cameras has made it
possible to create low-light cameras for acquiring fluorescent images without
using intensified CCD. These low-light cameras have the ability
to vary the time
1/60 or 1 / 130 second to several minutes.
of integration of image information from
By integrating a weak fluorescent signal for a longer time, a quality fluorescent
image is obtained.
The introduction of BCCD has also generated another viable option for
acquiring quality fluorescent and NIR images.
The spectral sensitivityof a BCCD
camera is significantly higher than that of intensified
CCD and conventional CCD
cameras, especially at the UV and NIR ends of the visible spectrum (19).
B.
NIR Imaging
NIR images can be very valuable for food quality evaluation. For imaging pur700-1 100 nm and
poses, the NIR waveband can be divided into two groups:
> 1100 nm.
Because of the higher sensitivity of BCCD cameras in the lower NIR region, they can be used for NIR imaging of food products. Similarly, some monochrome CCD cameras have relatively high sensitivity in the lower NIR region.
Although the sensitivity of monochrome CCDs in the 900- 1 100 nm zone is not
as high as that of a BCCD, there is a big difference in cost. Thus, which camera
one chooses to use depends on the application. Note that before using a monochrome CCD camera for NIR imaging, the IR filter in front of the CCD sensor
head must be removed. It is also highly recommended that the sensitivity curve
of the camera be obtained from the manufacturer
to verify that the camera is
appropriate for the application.
NIR imaging can also be achieved by using a liquid crystal tunable
filter.
to a standard CCD detector to produce
Tunable filters can be easily coupled
digital images at any wavelength within 400- I 100 nm (102). It has no moving
parts. Since it is capable of acquiring images at many wavelengths,it can be used
to generate multispectral images (102). Note that the quality of the image still
depends on the sensitivity of the CCD detector used.
NIR images based on 700-1 100 nm can be used for detecting defects and
formappingmoisture (970 nm)andprotein (1020 nm) in foodproducts. An
example is detecting defects in peaches. A monochrome CCD camera witha
band pass filter centered at 750 nm (with a bandwidth of 40 nm) produced the
images. The images were further analyzed for placing a peach into one of eight
classes based on different defects. The classification error based on NIR images
was 3 1 % compared to 40% obtained with color images (103).
88 Gunasekaran
and
Panigrahi
C.InfraredImaging
Focal plane array thermal infrared cameras without liquid nitrogen cooling (using
a sterling cycle-based cooler instead) are now available from commercial sources.
Some of them are compact and easy to use and provide better spatial resolution
and thermal sensitivity. They are sensitive to the thermal infrared band (3-5 p n )
and can capture images at 30 frames/s with 12-bit dynamic ranges. With emissivity and atmospheric correction capabilities, they can create thermal images
of
food products. 1R cameras can also measure temperatures from - I O to 1500C.
Thus, IR cameras promise another rapid and nondestructive technique for food
quality evaluation. especially for characterizing thermal properties, thermal mapping, and moisture-related studies. 1R imaging was used to estimate the internal
temperature of chicken meat after cooking (106).
Computer Vision
89
D. X-Ray Imaging
X-rays are another component
of the electromagnetic spectrum. They contain
high energy and can be used for nondestructive imaging. Recently, the development of filmless and low-energy x-ray detectors has created expanded possibilities for x-ray imaging for food and agricultural applications.
X-ray line-scan imaging was used to classify apples based on water core
features (107). X-ray technology was used to predict grain yield (108). Although
the integration of x-ray technology might find some obstacles for food quality
evaluation from consumers, low-energy x-ray devices might gain acceptance
in
the future for nondestructive evaluation where other imaging techniques will not
work.
90 Gunasekaran
and
Panigrahi
Computer Vision
91
General requirements for on-line applications are throughput (speed), accuracy, consistency, durability, diversification,
flexibility, and adaptability. Considerations of these conditions and constraints have to be given atall stages of
system design and development. Speedof evaluation is perhaps the most striking
requirement. For example, Tao et al. (8) estimated that an on-line apple grading
system may have to examine at least 3600 fruit/min. They describeda computer
vision system sorting 3.5 million fruit in an 8-hour day, which consisted of two
separate lighting unitswith eight camerasand one processing and control console
unit. This type of machine is being widely installed in the packing industry for
sorting applies, citrus, peaches, tomatoes,
and various other fruitsand vegetables.
IX. SUMMARYANDFUTURETRENDS
Computer vision techniquesplay a significant rolein fulfilling the needs of rapid
and nondestructive sensors in food quality evaluation. The increasing demand
for real-time or high-speed food quality evaluationwill be met by recent developments in high-speed DSP integrated cameras and frame grabbers. Commercially
available DSP chips (e.g., Texas Instruments C-40, C-44,
and C-80; Intels I860) offer flexibility, expandability, and upgradability suitable for parallel processing of images. Image processing analysis operation is a better candidate for
parallelism. Therefore, parallel processing can be integrated for high-speed and
real-time applications using the DSP chip.
At present, many commercial DSP
integrated frame grabbers are available that can be configured for parallel processing ( 1 13). Dedicated high-speed processors also offer alternate solutions for
high-speed and real-time imaging applications.
Recentdevelopments in busarchitecture,such as compact PC1 (CPCl),
high-speed networks for image transmission, and cost-effective storage devices
will add to the increasing integrationof real-time imaging systems for food quality evaluation ( 1 14).
The development of portable, cost-effective, miniature laser systems permits the implementation of structured lighting techniques for extracting 3-D informationforfoodproducts.Moreover,ongoingtechnologicaladvancements
have made 3-D cameras available from commercial sources. With the capabilities
of high-speed processors and cost-effective 3-D imaging software, food sectors
will encounter increased 3-D computer imaging for automated quality evaluation
and inspections.
Significantgrowth in thetechnologicalcapabilities of software has occurred along with their user-friendliness and adaptability. One does not need to
know high-level programming in order to developa specific application. Capabil-
92
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1.
INTRODUCTION
100
101
the reactions; (e) suppression by carbon-dioxide; and (f) the drop in intensity
produced by continuous illumination with time.
Contrary to the interpretation of Strehler and Arnold (2) that the luminescence phenomenon is a consequence of the reversibility of some of the early
enzymatic photosynthetic reactions, the early processes following light absorption were observed to be nonenzymatic in nature (8,9). Later investigations suggested an interpretation of the physical processes leading to DLE and photosynthesis in terms of semiconductor theory (10,l I). The emitted light was attributed
to an electron transition between the first excited singlet state of chlorophyll and
the ground state (9,12). However, the luminescenceof DLE is an extremely complex temperature-dependent process, suggesting that a multiprocess mechanism
may be involved. Based on their temperature-dependent DLE studies on Chlorella, Scenedesmus, and spinach chloroplasts, Tollin et al. (9) showed that the
early processes following light absorption are purely physical, whereas the later
stages of emission are enzymatic in nature. Some investigators (13-15) have
suggested that DLE is regulated by the rate of the electron transport reaction,
and others ( 16- 18) have indicated that it is controlled by the rate of photophosphorylation (light-induced esterification of compounds with phosphoric acid).
Typically, induction of delayed light would show a very fast increase to
the initial level, then a slow increase to the peak, and a slow decrease toa steadystate level (2,7,19).
111.
FACTORSAFFECTINGDLE
A.
Wavelength of Excitation
700
102
600
500
400
800
nm. However, the wavelength dependence may vary slightly based on the measurement system used.
B. ExcitationIntensity
In general, the intensityof DLE bears a direct relationship to the excitation intensity until saturation occurs. Itoh and Murata (7), however, observed an opposite
trend with spinach chloroplasts. Additional illumination has little effect in prolevel (22-24). For
ducing an increase in DLE intensity beyond the saturation
oranges, for example, DLE intensity levels off after
an excitation intensity of
2750 lx (Fig. 2 ) . This aspect is desirable for DLE measurements since small
changes in excitation will not interfere with quantitative measurements of DLE,
it
provided the illumination is sufficiently above the saturation point. However,
has been shown (7,24) that higher illumination intensity lowers the time required
for saturation. Total excitation energy required to obtain maximum DLE is differto obtain high DLE intensity
ent for each product. Optimal measuring criteria
for various fruits are listed in Table 1.
C. Excitation
103
2000
4000
6000
Fig. 2 Effect of exciting illumination intensity on DLE from green oranges after different decay periods: A, 0.7 s; B, 1.0 s; C, 1.5 s; D, 2.0 s. (Data from Ref. 22.)
phases of DLE decay (9,13,14). The most rapid known has a half-life
of about
one ms and is popularly termed millisecond delayed light emission (19). Certain other systems have much longer half-decay times. The decay curves
of Chlorella luminescence are shown in Fig. 3. The decay appears to be faster at lower
temperatures than at higher temperatures. For a given decay period, maximum
DLE intensity is reached within the first 2-4s. Longer excitation times gradually
degrade DLE intensity. Therefore, it is advisable to provide short (about 2-4 s)
but strong excitation (to assure saturation). Temperature has a profound effect
Table 1 OptimalMeasuringCriteria
for ObtainingHighDLEIntensitya
Excitation
(min)
Temperature
Illuminationperiod
Product
Dark
Tomato
Satsuma orange
Persimmon
Japanese apricot
Banana
Papaya
Measured after 75-s delay.
Source: Ref. 38.
10
>20
15
(1x1
Time (s)
(C)
13-17
550
2750
2800
1-32
3-6
4-7
1-3
23-28
>20
>SO0
>IO
18-25
2750
>5500
15-22
1-2
2-4
104
1
'm
0.8
5 0.6
-E
W
&
0.4
0.2
0
0
Fig. 3 DLE decay curves from Chlorella at 28C (A) and 6.5"C (B) (Data from Ref. 2 . )
on the shape of DLE decay. In general, the lower the temperature, the faster the
decay of DLE intensity. Jacob et al. (24), however, observed an opposite trend
of faster decayat higher temperature in immature oranges. The effect
of temperature on DLE is discussed further in a following section.
D. DarkPeriods
In order to eliminate the effect of previous illumination on DLE intensity, the
product is kept in a dark chamber fora certain length of time between successive
on the same product.
illuminations when more than one measurement is made
In some instances, the product may be subjected to dark periods after excitation
but prior to DLE measurement. Based on the studies of DLE in tomatoes (20),
Satsuma oranges (22), and tea leaves (21), definite relationships between the
preconditioning dark periods and DLE have been established. Usually, short dark
periods (<1 min) cause a reduction in DLE intensity while longer dark periods
(about 10-15 min) bring about some recovery of DLE intensity. Regarding chlorophyllderived from spinach chloroplasts, the illumination after varying dark
periods showed initial DLE peaks of varying levels depending on the duration
of dark periods (7). The effect of a dark period before excitation on DLE intensity
of yellow persimmons excited at 5500 Ix is shown in Fig. 4.
105
10
20
Fig. 4 Effect of dark period before excitationon DLE intensity from yellow persimmons
excited at 5500 lx as a function of decay periods: A, 0.9 s; B, 1 .0 s; C, 1 .5 s. (Data from
Ref. 24.)
for DLE detectionin tomatoes. Chuma and Nakaji (20) obtained highDLE intenrind, the interior being scooped
sity from tomato flesh 6 mm thick below the
out.
A direct relationship between the area of excitation and the intensity
of
emitted light has been observed (20,22,24). Figure
5 represents the linear relationship between the area of excitation and the DLE intensity of green tomatoes at
different decay periods. Also, a larger area of excitation resulted in a faster DLE
decay. Therefore,it is necessary to carefully define the areaof excitation for each
product and for each quality attribute to obtain reproducible results. This would
have a bearingon developing automatic grading systems for fruits and vegetables.
F. Temperature
TemperatureeffectsonDLE,asobservedearlier,areextremelycomplex
to
(9,22,25). Chlorella luminescence shows a temperature dependence similar
that of an enzyme-catalyzed reaction (Fig. 3). The amplitude and decay kinetics
of DLE are temperature-dependent in the time range of seconds (3,8,9,15). DLE
2
3
4
5
Illumination Area (cm)
Fig. 5 DLE intensity vs. area of excitation in green tomatoes excited at 5500 Ix. (Data
from Ref. 20.)
intensity is generally maximum in a narrow range of sample temperatures. However, the variation from maximum DLE intensity is dependent on the particular
of DLE from green
product. Figure 6 represents the temperature dependence
tomatoes and bananas, respectively. Peak intensities are at 16 and 25C for green
tomatoes and green bananas, respectively.Also, higher temperatures havea more
profound effect on DLE from tomatoes than from bananas. The temperature dependence of DLE, however, is not a major problem when agricultural products
encounter relatively small temperature changes (24). Abbott and Massie (26) related temperature and duration of exposure to chilling temperaturesof cucumber
fruit to DLE; Chuma et al. (22) observed an increase in DLE from orange peel
up to 30C and a sharp decline reaching almost zero level beyond 40C; and
Nakaji et al. (21) found a strong dependence of DLE on the temperature of tea
leaves. Terzaghiet al. (27) reported that the temperatureof maximum DLE upon
chilling was strongly correlated withlateral phase separation temperature but was
about4Cloweronaverage.Thephenomenonobserved
in chilling-sensitive
plants during chilling a minor component below freezingis lateral phase separation. These investigations indicate that large temperature changes have a definite
effect on DLE, which can be used to predict certain quality attributes.
107
.. .
3.5
n
10
15
25
20
30
35
Temperature ("C)
G. ChlorophyllContent
DLE is probably produced by all vegetables, fruits, and plant materials undergoto the chlorophyll concentration of the
ing photosynthesis and can be related
product (24). This is perhaps the most important aspect of DLE from a practical
viewpoint because the change in the level of chlorophyll content is a primary
20
40
60
80
100
Time (s)
Fig. 7 DLE intensity at different stages of maturity of lemons: A, Dark green: B, light
green: C, silver, D, yellow. (Data from Ref. 24.)
Table 2 Empirical Relationships Developed for DLE from Various Products as a Measure of Chlorophyll Content or Peel Color
Based on Chlorophyll Content
0
Q)
Correlation
Product (variety)
Empirical relationship
+ 0.298
(W
Measurement conditions
Ref.
content (pg/
0.92
20
Dependent variable
C-chlorophyll
I00 cm')
Tomato (Japanese)
DLE = 0. I63 C
Tomato (Duke)
DLE = 0.0121 C
0." 13
0.86
DLE = 0.0896 C +
0.318
0.86
Papaya (Hawaiian)
DLE
0.92
Orange (Satsuma)
Banana (monkey)
DLE
DLE intensity, V .
0018 P
1.054
C-chlorophyll content of
peel (pg/100 g fresh
weight)
GPC-grade
of peel color
21
29
22
P
v)
FP
23
a
a
a
'CI
1.
(CI
109
IV.
QUALITYEVALUATIONBASEDON DLE
1.
110
111
only a trace of DLE (23). The grade of peel color of bananas had been related
to DLE intensity (Fig. 8), which indicates the possibility
of using DLE measurements to determine the state of maturity of bananas. Other quality attributes of
bananas such as sugar content and firmness were also related to DLE. As the
sugar content of the flesh (S, Brix) of bananas increased from 1 to 20% during
ripening, the intensity of emitted light (Y, volt) decreased almost linearly as:
-0.773
(1) S
+ 3.26
Y(2)= 14 F
3.50
DLE intensityalso has a significant correlation with firmness for other fruits
such as apricots and papayas (38).
DLE characteristics of the chlorophyllof green tea leaves werealso investigated to serve as a means of nondestructive estimation of the quality of tea and
tobacco leaves (38). Each ingredient valueof leaf color (hue, value, and chrome)
wasfound to correlate highlywith DLE intensity of fresh leaves. The linear
relationship between the chlorophyll content of fresh leaves and DLE intensity
could be used to estimate the quality of tea leaves.
Abbott and Massie (26) and Abbott et al. (39) related the temperature and
duration of exposure to chilling temperatures of cucumber and bell pepper fruit
and coleus to DLE measured at nonchilling temperatures. They observed that the
DLE measurement would help determine the mechanisms of chilling injury and
the methods of ameliorating chilling injuryof cucumbers, bell peppers, and other
chlorophyll-containing tissues (40). Besides chilling injury, DLE measurements
can also be used to inspect fruits and vegetables for mechanical stress during
Color Grade
112
harvesting (41) and injury from sunburn or air pollution before visible symptoms
develop (42). Based on the effects
of quarantine heat treatments on DLE measurements on papayas (43), Forbus and Chan (44) investigated the potential for DLE
as a biochemical indicator of papaya heat treatment. The DLE system was not
as sensitive to deleterious effects
of heat on the ethylene-forming enzyme system.
However, they concluded that DLE measurements provided a quantifiable, nondestructive means for measuring the primary deleterious effects of heat damage.
Forbus and Senter(45) evaluated Saticoy cantaloupes at six stagesof maturity for differences in DLE, chlorophyll, and soluble solids. DLE varied significantly and predictably by stage of maturity, indicating its potential as a rapid,
nondestructive method of measuring cantaloupe quality.
Forbus et al.(29) developed an index for separating ripe and unripe papayas
(Fig. 9). Working with papaya maturity ranks, they found that ranks 9 through
28 (mature green through three-quarters ripe4 days after harvest) couldbe sorted
more accurately than ranks 1 through 9 (immature green 4 days after harvest).
Half-ripe or riper papayas (maturity ranks21 through 28) are the most susceptible
in
to fruit fly infestation(43).Therefore,DLEmeasurementscangreatlyaid
identifying and removing infestation-prone fruits and thus assure quality in large
for DLE
commercial shipments. Forbus and Chan (44) demonstrated the potential
0.9
I
.b
u)
c
0.8
Q)
c
m
0.7 Overhalf-ripe
K
UI
.-
Under half-ripe
w 0.6
21
n
25
0.5
27
0.4
5
10
15
20
25
30
Fig. 9 Regression of DLE intensity vs. papaya maturity (I' = 0.92). Nutnhers 9 through
27 on the tigure represent papaya maturity rank arranged in order of increasing tnaturity.
(Data from Ref. 24.)
113
V.
There have been varying designs of DLE measurement units to evaluate fruit
and vegetable quality (20,24,29,43). Jacob et al. (24) reported one of the earliest
instrumentation systems for continuous DLE measurement and sorting fruits. Forbus et al. (53) developed an experimental DLE meter for horticultural crops.
According to them, DLE measuring systems must incorporate the following components: (a) a light source for illuminating a sample
to activate DLE, (b) a totally
dark enclosure in which to isolate the sample for measuring DLE after illumination is removed, (c) a photomultiplier tube mounted inside the totally dark enclosure for detecting light emitted from the sample, (d) an amplifier for applying
of emitted
low-intensity signals, and (e) a recorder for recording the intensity
light as a function of time.
The basic components of instrumentation and measurement units for autoto most optical and/or
matic quality evaluation based on DLE are very similar
spectrophotometric units. A detailed description of various detector properties
and specifics of associated electronics to measure photoluminescence and other
optical radiations can be found in an excellent multivolume treatise on optical
radiation measurements (54-56). However, an attempt is made here to broadly
unit. The overall
outline various components of a typical optical measurement
measurement unit can be divided into four major systems
to facilitate: sample
illumination, viewing and sensing, signal processing, and sorting.
A.
IlluminationSystem
Incandescent lamps (tungsten filament and tungsten halogen), arc lamps, andfluorescent lamps have commonly been used as sources
of radiation in optical instruments. Occasionally, low-power lasers have also been used as
light sources in
optical systems (57,58). Laser light is highly intense, coherent and directional,
has a well-defined beam diameter, and
is readily adaptable for optical use (57).
However, its use may be limitedby its monochromatic nature. Primarily, a source
114
Panigrahi
and
Gunasekaran
B. SampleViewingandSensingSystem
The sequence of operations in this system
is singulation, conveying and orienting,
and scanning and sensing. The singulation stageis incorporated into systems that
of discrete objects is formed
evaluate individual products. At this stage, a bulk
in a noncontiguous single file. Even though there is no established theoretical
basis for the study of the process of singulation, Henderson and Shawver (61)
described singulation as a three-stage process. First, the objects received in bulk
are spread into a disordered monolayer. Second, the monolayer is organized into
rows. Third, the objects in the rows are separated to a specified spacing between
them.Acompendium
ofsingulationandassociateddevicespublishedby
Shawver and Henderson (62) is an exhaustive reference on this subject. Conveying the singulated materials at the proper orientationis critical for an accurate
evaluation of quality. Since most agricultural and biological products are irregularly shaped, orienting them properly is not easily achieved. An orienting and
conveying device for sorting dried prunes developed by Burkhardt and Mrozek
(63) is a good example. Gaffney (64) suggested incorporating techniques
that
measure the size and shape of objects into the design of a conveying and orienting
unit. Another important aspect is the speed of conveying, which could affect the
sorting efficiency.
115
C.SignalProcessing
System
Signal processing is an essential part of the measurement unit. Often the ability
of the signal processing componentwill dictate the designof the rest of the system
(39). In a broad sense, signal processing simply means transforming the photodetector response into a more useful form to aid in decision making. For this, it can
or
be as simple as direct reading of the electrical signal coming from the detector,
it can be as involvedas requiring sophisticated mathematical analyses ofmeasurement data performed with the aid
of large digital data processing facilities.
In
116
Panigrahi
and
Gunasekaran
processing photoluminance or fluorescence data, simple manipulation of measured values from some meter or analog-to-digital converter
is inadequate. In
this case, the domainof data analysis may extend from the direct detector output
to the digitization process and
finally to the conversion of these quantities to
information of analytical interest (65). For this reason, greater reliance on signal
processing is a necessity. Typically, a microcomputer will be dedicated for this
with specially developed software and hardware incorporated into the system. In
general, this system provides overall control, calibration, and operation
of the
unit and helpsto display, record, and/or store
the optical characteristics measured.
D. Sorting System
At this stage, this is the actual classification according to the information gained
from the signal processing system. This may be grouping fruits or vegetables
into different grades or rejecting defective samples. Prompted by the microcomputer signal,a solenoid could actuate a deflecting vane (24)or an air gun (62,66).
of
A pneumatically operated sliding gate could also be used to direct the flow
defective products (66,67). Sorting speed should match the conveying speed, and
to cause mechanical damage to the
theunitshouldbecarefullydesignednot
objects being sorted.
VI.
FLUORESCENCE
117
First Exc
SI -
v=4
v=3
v=2
so -
Fig. 10 Schematic energy level diagram for a diatomic molecule. The potential energy
level of a diatomic molecule are indicated with the various vibration levels represented
as v = 0. I , 2, 3, and 4 on each curve. (Reprinted from Ref. (6). p. 3.)
118
h
where E = energy (J), h = Plancksconstant (6.63X
J . s), c = velocity
of light = 3 X 10 (m/s), and h = wavelength (m) (6).
The energy of the photons in the visible spectrum is 146-607 KJ/mol and
they cause electronic transition (6).The resulted fluorescence lasts for typically
I O ns (6). The time for which the fluorescence phenomenon exists is called fluorescence lifetime (6,104).
Fluorescence has two characteristic spectra-an excitation spectrum and
an emission spectrum. The excitation spectrum refers to the relative efficiency
of different wavelengths of exciting radiation to cause fluorescence. The shape
of the excitation spectrum should be identical to the absorption spectrum and is
independent of wavelengthof fluorescence measurement. The emission spectrum
represents the fluorescence spectral data.
It is the relative intensity of emitted
radiation at various wavelengths (6).
A.
Types of Fluorescence
Fluorescence may be classified as either primary or secondary. Primary fluoresto the intrinsic or native fluorescence, also known as autofluorescence, refers
cence characteristics of an object/product that fluoresces under UV or other electromagnetic radiation. Many plant products, botanical tissue components, animal
tissues including meat and fish products, and other food products possess autoSecondary fluorescence, also known as influorescence characteristics (6,l 13).
duced fluorescence, refers to the fluorescence characteristic of an object obtained
byaddingspecificfluorescenceactivatorscalledfluorochromoorfluorescent
stains or fluorophores. Some nonfluorescent objects show fluorescence characteristics after undergoing a chemical reaction or after the addition of chemical reagents (104,113).
In some cases, fluorescence may be classified as (6):
1.
Stokes fluorescence-thereemission
of lessenergeticphotons
that
is normally
have a longer wavelength than absorbed photons, which
observed in solutions.
119
ql,, ( I
e)-"k
(4)
where q = quantum efficiency, I,, = radiant incident power, a = molar absorptivity, b = path length of the sample, and c = molar concentration (6).
The quantum efficiency or yieldq is furtherdefinedasthe
ratio of the
emitted to the absorbed number of quanta (6). The higher the values of q, the
I,, could cause
greater the fluorescenceof a compound. However, higher intensity
photodecomposition of the sample(fluorophores),thusreducingfluorescence.
Therefore, a source of moderate intensity is used (e.g., mercury or xenon lamp)
(6). For very dilute solutions, Eq. (4) reduces to Beer's law:
KqIoabc
(5)
B. AdvantagesandLimitations
Fluorescence and phosphorescence are well suited as analytical tools for food
quality evaluation becauseof their extreme sensitivity and specificity. Concentration of substances as low as I in 10"' can be detected. The main reason for this
extreme sensitivity is that the emitted light is measured directly and can be increased or decreased by changing the intensity of the exciting radiation. The
120
specificity of fluorescence is due to the fact that there are few components that
fluoresce compared to those that absorb light energy, and two wavelengths are
used in fluorometry compared to only one in spectrophotometry. Many biological
and food products tend to have unique fluorescent characteristics. Two compounds that absorb radiation at the same wavelength will probably not emit at
the same wavelength. The difference between excitation and emission peak could
range from I O to 280 nm (6).
There are, however, the following limitations
of fluorescence as an analytical tool (6):
1.
C.Detection
of Fluorescence
1.
121
radiation to interact with the sample. (In the cases where laser is used
as the light source, a filter may not be necessary.)
3. A detector and other associated electronics including A/D converter.
The detector will detect the emitted spectrum/fluorescence. The A/D
converter and associated electronics will convert the emitted signal into
equivalent digital form.
4. A host computer, which could host the detector assembly and store the
digitizedfluorescenceinformationforstorageandadditionalprocessing.
5 . Absorbance filter to allowtheemittedsignal
in theselectivewavelength range before the signal is digitized.
1. Illumination Sources
Manybroadbandlightsources
(e.g., xenon lamps, UVlight,mercurylamps,
xenon-mercury lamps, and lasers) are used as illumination sources (6,104). High
intensity discharge lamps such as mercury and xenon lamps are mostly used as
illumination sources (6,104,105). Mercury lamps provide sharp spectral output
and many times are used for calibration purposes along their expected lifetime
in high- and low-pressure format. Xenon
in average (105). They are also available
lamps provide continuous spectral output, generally from 270-400
nm (UV zone)
(104). Xenon gas is contained under high pressure in xenon lamps (104,105) and
therefore should be handled carefully. Xenon and mercury combination-based
(104). It is
xenon-mercury arc lamps provide higher output than a xenon lamp
always recommended to obtain detailed spectral output from the manufacturer
before selecting a particular light source.
Promising research is being carried out by various entities, and it is anticipated that UV light emitting diodes (LEDs) will be commercially available soon.
of the unique propertiesof
Another typeof illumination source is laser based. One
lasers is their ability to emit high-energy coherent lightof a specific wavelength.
of laser
Depending on the required wavelength and cost, the appropriate type
can be selected.
2. Detectors
122
rescent spectra will depend on the type of detecting material used in the detector
(which is dependent on emission wavelength, active area of the detector, noise
equivalent power, and detectivity) (106). For fluorescence (emission) measureGe (germaments in the visible and near-infrared range, generally Si (silicon),
nium), and InGaAs(indium-gallium-aresenide) type detectors are used (107). For
of light is low, new types of
many fluorescence measurements where the level
photo-receivers such as femtowatt receivers (New Focus, Inc., CA) can be very
of food products, nanosecond phouseful. For fluorescence lifetime measurement
todetectors (New Focus, Inc., CA) with typical rise time of 1 ns may be appropriate (108).
To obtain fluorescent images, different types
of cameras/computer imaging
systems are used. A computer-based fluorescence imaging system differs from
a standard computer imaging system mostly by the image acquisition system or
camera. The camera oftenis incorporated with a microscope. The most commonly
used camera for a fluorescence imaging system is an intensified charge coupled
device (ICCD) video camera. The detectors on these cameras are cooled to provide high sensitivity and spatial resolution with video rate (30
frameds). Other
cameras that are used for fluorescence imaging use cooled and UV-enhanced or
back-illuminated CCD detectors (109). UV-enhanced or back-illuminated CCD
provides enhanced sensitivity in the UV or visible band to acquire low-light fluorescence images ( I 09). Fluorescence images can also be obtained with digital
signal processing (DSP) integrated CCD camera system. DSP circuitry allows
the integration of information over a user-defined amountof time (generally from
0.01 s to several minutes) to obtain high-quality images. This type
of system
works well with applications where the amount of light (fluorescence emission)
a
is relatively high. Fluorescence imaging can provide more information than
spectrofluorometer such as spatial distribution of intended fluorescence information. Thus, fluorescence imaging can be very useful for food quality measurements.
VII.APPLICATIONS
Many food, agricultural, and biological materials possess autofluorescent characto fluoresce (i.e., secondary fluorescence) by
teristics, and many can be made
in a number
adding appropriate fluorophores. These properties have been used
of quality evaluation applications.
A.
AutofluorescenceTechniques
Separation of bran (pericap, aleurone, and fatty germ) from wheat flour (endosperm) is performed in many wheat-processing operations. Chemical analysis of
123
ash along with the color check are conducted to measure the effectiveness
of
separation (1 IO). However,thismethod is verytediousandtime-consuming.
Munck (1 IO) proposed a rapid methodin which the autofluorescence characteristics of different components of wheat seeds were used. He showed that the aleu350 nm wavelength light,
rone autofluoresces at 420 nm (blue) when excited with
and the pericarp fluoresces at 540 nm (yellow) when excited at 450 nm ( 1 IO).
A similar method was usedto study the components of sorghum via fluorescence
microscopy (68).
Mycotoxins (toxic fungal metabolites) in food products have been linked
with different forms of fatal and chronic illnesses for humans and animals (70).
Theyhavebeenreportedtobefound
in manyfoodproducts
at any stage of
production (70). Wood and Mann (70) have described the use
of fluorescence
Le., aflatoxins, citrinin,
techniques to detect and quantify selected mycotoxins,
ochratoxin A, and zearaleurone. Traditional detection methods for these mycotoxins have been either thin layer chromatography (TLC) or high-performance liquid
chromatography (HPLC) (70). The fluorescence characteristics
of aflatoxins (BI ,
B2,G1,andG2).citrinin,ochratoxin,
andzearaleuroneare listed in Table 3
(70). Because of the tediousness of the process involved
in TLC and HPLC,
investigation is being carried out by one of the coauthors (Panigrahi) to develop
a fast, nondestructive method for detection and/or quantification of mycotoxins.
He is using a personal computer-based
fiber optic spectroscope to investigate
fluorescence characteristics of wheat samples infected with mycotoxins and to
develop techniques to quantify the amount of mycotoxins present. Similarly, Lil1 ) studied the relationship between aflatoxin concentration and moislehoj et al. (7
ture at harvest and bright greenish-yellow fluorescenceof different corn hybrids.
Panigrahi et al. (72) investigated the autofluorescence characteristics
of edible beans for rapid, nondestructive evaluation. There are many types
of edible
beans: navy, pinto, red, black, etc. As with all other food and agricultural products, cracks or splitsin edible beans are undesirable andneed to be detected and/
or quantified. The conventional approach of crack detection in navy beans in-
Mycotoxins
Toxins
Aflatoxins
Citrinin and ochratoxin A
Zearalenone
Source: Adapted from Ref. 70
Excitation
wavelength
Emission
wavelength
340-380
254
440
400-700
308
400-700
124
volves dying them with a red dye and then counting the
of visible
numberdefects/
cracks. Use of red dye is justified because this enhances cracks and defects for
easy identification. The problem with this approach is that dyed beans are not
of
generally used for human consumption. It was found that the endosperms
the bean autofluoresce under UV light
(72). The fluorescence imaging system
(72) included a long-UV lamp for illumination.
developed for crack identification
CCD
The autofluorescenceof the beans was imaged using a low light-sensitive
camera. The acquired images were processed to quantify and assess the extent
of defect. Figure 11 is a typical image of a bean showing the bright defective/
cracked region that autofluoresces.
Autofluorescence has been used for quality characterization of fish meat,
to separate fishbone from meat, to quantify lean in animal carcasses, to detect
parasites in meat products, etc. (73-80). When excited at 340 nm, the fishbone
of a number of fishes (sod, whiting, haddock, plaice, flounder, and sole, etc.)
360 nm. The
fluoresces at 390 nm, but fish meat has a fluorescence peak at
intensity of fluorescence from the bones is also much higher than that from the
fish meat (Fig. 12) (81). These differences have been used to evaluate the quality
of fish fillets. Commercial fishbone detectorsare available that employ autofluorescence. The fluorescence intensity from fishbone decreases with the thickness
of the fish meat covering the bones. For example, when the flesh thickness of
0 to 2 mm, the relative fluorescence intensity from
cod and plaice increased from
the bones decreased almost linearly from 100% to about 10% (Fig. 13). Therefore, present technology is suitable for surface or near-surface detection, which
125
100
75
50
25
0
350
450
400
500
Wavelength (nm)
Fig. 12 Fluorescence emission spectra of fishbone and fish meat when excited at
340
nm. (Ref. 81.) (Reprinted with permission from Jourrzul of Food Protecfion. 0 Interna-
0.5
1.5
Thickness (mm)
Fig. 13 Decrease in relative fluorescence intensity from fishbones with flesh thickness.
(Ref. 8 I .) (Reprinted with permission from Journal of Food P rorecrion. 0 International
Association for Food Protection.)
126
ide value (PV). TBS and PV values do not reliably relate to lipid oxidation in
meats (86), particularly in freeze-dried meat (86). Nakhost and Karel (88) used
fluorescence techniques to assess the effectiveness of antioxidantTBHQ (monot-butylhydroquinone), reduction of headspace oxygen, and compression on the
of oxidized beef
storage stability of freeze-dried meats. Fluorescence spectra
showed maximum excitation and emission at 350 and 440 nm, respectively. The
nonoxidized beef showed three peaks in the excitation spectrum at 308, 3 18, and
350 nm and a broad peak in the emission spectrum at about 475 nm. The use of
in beef samples,
antioxidant (100 ppm TBHQ) greatly decreased the fluorescence
and the fluorescence spectra of oxidized beef showed maximum excitation of
350 nm and emission excitation at 440 nm (89).
In the case of fruits and vegetables, as with
DLE, trends in chlorophyll
contentwerefollowedusingwhat
is knownaschlorophyllfluorescence.For
apples (90) and broccoli (91), fluorescence parameters were found suitable for
evaluating quality and maturity. Thus, use of chlorophyll fluorescence may be
suitable for maturity sorting of chlorophyll-containing fruits and vegetables on
commercial packaging lines (90). Woolf and Lasing (92) studied
avocado fruit
skin fluorescence following hot water treatments and pretreatments used as possible postharvest disinfection techniques. They reported that chlorophyll
fluorescence clearly reflected the effects of heat on the photosynthetic systems in
avocado fruit but did not detect the alleviation of heat damage by pretreatments.
Fluorescence spectra of fruit juices have also been proven useful quality
evaluation criteria (93,94). Seiden et al. (93) correlated the fluorescence spectra
of correctly
with soluble solids in apple juice and two apple cultivars as a means
to clasclassifying them. Similar fluorescence chemometrics were also suitable
sify and predict quality and process parametersof white sugar solutions and thick
juice samples of sugar beets.
B. InducedFluorescenceTechniques
In induced (secondary) fluorescence methods, food products are made
to fluoresce
by adding fluorochromes or by producing specific fluorescent reaction products
in the tissue or by applying fluorescent-labeled biological molecules (lectins antibodies) with specific binding affinity for the given food constituent. Table 4 lists
commonly used fluorochromes for food applications. The useof secondary fluorescence characteristics has been reported for many applications including the
use of calcoflour to detect glucans in malting barley, seed fixation system, use
of fluoroscamine to detect sprouting, and morphological and microchemical analysis of cereals (95,110). Fulcher et al. (96) provided a comprehensive overview
of fluorescence microscopy and its application to characterize nonstarchy carbohydrates and for routine analysis of products and processing conditions in industrial laboratories. Barnes and Fulcher (97) further reported about measuring fat
pplications
127
Table 4 CommonlyUsedFluorochromesfor
Food Applications
Fluorochrome
Safranin
Aniline blue
Congo red
Diphenylborinic acid
Acid fuchsin
Acridine orange
Acriflavine
Ruse Bengale
Calcoflour white
Auramine
Starch
Cellwalls
P-Glucans
Flavonoids
Storage protein
Microbiology, bacteria
E. coli
usinginducedfluorescence.According
to theirstudy(97),whenNileRed
(a
fluorescence dye) was added, aqueous suspension of wheat germ showed strong
fluorescence (Fig. 14). The absence of Nile Red, however showed almost no
fluorescence (Fig. 14) (97). The defatted powdered wheat germ (with Nile Red)
showed significant less fluorescence than normal wheat germ (Fig. 14) (97). This
implied the effect of fat content in emitting fluorescence. This postulation was
verified from the findings of Barnes and Fulcher (97) (Fig. 15). Figure 15 shows
strong dependency of fluorescence intensity on fat content of wheat flour mill
(measured by soxhlet extraction). Useof Nile Red for fluorescence measurement
of lipid and oils can be atool for quality control vegetable oil (97). The different
oils/lipids with dissolved Nile Red showed different emission wavelengths and
fluorescence intensities (Table5 ) (97). Tryglycerides (with Nile Red) were found
to have higher emission wavelengths than those
of polar monoglycerides and
unesterified fatty acids (with Nile Red) (97) (Table 5 ) . Fulcher et al. (69) examined the aleurone layer in barley seeds using fluorescence microscopy.
Fluorescence has also been used with cytometry for rapid analysis of food
microorganisms. The fundamentalprinciple of flowcytometry is asfollows:
a stationeryoptical
The sample to be examined is allowedtoflowthrough
sensing system in the form of continuous stream (using a flow cell). The optical
sensing system consists of a light source (excitation source) and a detector. The
of the
detector detects the optical signal representing the specific characterization
microorganism present in the sample (98). Maximum
flow rates up to a 0.5
mL/min and cell countratesupto
1O,OOO/s usingflowcytometryhavebeen
reported by Pinder and Godfrey (98). Fluorescent labeling technique, along with
flow cytometry, has shown potential for many structural and functional parame-
128
550
450
.-E
ln
c
3 .
10
Fig. 15 Fluorescence intensity (arbitrary units) vs. fat content of mill streams from a
commercial wheat flour mill. (Ref. 97.) (Reprinted with permission.)
129
Fluorescence Emission
ensity'
(nm) max.Lipidhegetable
(nm) max. oil
Excitation
Mono-olein
Oleic acid
Triolein
Monolinolein
Linoleic acid
Trilinolein
Corn oil
Olive oil
Palm oil
508
522
SO4
526
520
507
505
SO6
515
600
596
567
600
600
589
519
58 1
59 1
48
65
27 1
46
63
121
200
20 1
160
ters for food products. The structural and functional parameters are cell size, cell
shape, DNA and RNA content, surface sugars, total proteins, enzymes activity,
intracellular pH, DNA synthesis, etc. (98). Fluorescent flow cytometry has been
used to identify bacteriaby using light scattering and nucleic acid staining. Identification of food pathogens including Salmonella and Campylobacter bacteria has
been reported by immunofluorescent labeling (98). Other applications that have
usedflowcytometryinclude
analysis of fruit preparation, milk products, and
brewing yeast (98,99). Shelf life predictionof salads and fruit juice hasalso been
performed using flow cytometry (98).
Fluorescence cytometry, fluorescence microscopy, and other fluorescence
spectroscopic techniques have been presented
in more detail in other standard
sources (100,101).
VIII. SUMMARY
DLE and fluorescence are valuable techniques for evaluatinga variety of quality
factors.Allfruits,vegetables,andplantmaterialsundergoingphotosynthesis
probably produce DLE. However, the intensity and duration of the emitted light
vary widely, depending upon many factors. Because
of the strong dependence
of DLE on chlorophyll content, variationin DLE can be expected among different
varieties of the same product. Similarly, not all materials exhibit fluorescence,
and inducing fluorescence should be done carefully so as not to add agents that
would render foods unsafe. Fluorescence is also strongly dependent on environ-
130
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juice and beet sugar by fluorescence spectroscopy and chemometrics. Zucker Ind
120(1):970-981,1995.
9s. RG Fulcher. Fluorescence microscopy of cereals. Food Microstructure 75(
I): 167175.1982.
96. RG Fulcher, PJ Wood, SH Yiu. Insights into food carbohydrates through fluorescence microscopy. Food Technol (Jan): 101- 106, 1984.
97. PJ Barnes, RG Fulcher. Fluorometric measurement of fats. In: L Munck, ed. Fluorescence Analysis in Foods. New York: John Wiley and Sons, Inc., 1989.
of food micro98. AC Pinder, S Godfrey. Fluorescence cytometry for the rapid analysis
organisms. In: Food Process Monitoring Systems. AC Pinder, G Godfrey, eds.
New
York: Blackie Academic and Professional, 1993.
99. L Jespersen. Use of fluorescence staining and flow cytometry for analysis of brewingyeasts (Snccharornyces cerevisiae). DissertationAbstractsInt57(
I ) : 1 IO,
1996.
100. OS Wolfbeis, ed. Fluorescence SpectroscopyNew Methods and Applications. New
York:Springer-Verlag. 1993.
101. J Slavik.FluorescenceMicroscopyandFluorescentProbes.NewYork:Plenum
Press,1996.
102. LO Bjorn, AS Forsberg. Imaging by delayed light emission (photoluminography)
as a method for detecting damage to the photosynthetic system tested on leaves of
Hibiscus,Oxalistetraphylla,tobacco,Fagussilvatica,Polypodiumvulgareand
beans. Physiol Plants 47(4):215-222, 1979.
103. ESundborn,LOBjorn.Phytoluminography:imagingplants
by delayedlight
emission (for the study of pathological disturbances). Physiol Plants 40( 1):39-41,
1977.
136
106.
107.
108.
109.
1 10.
111.
112.
113.
1.
INTRODUCTION
I 38
,"\
Pterpaf,
Economic
Inputs
,Qua ~ t y
I
/I
\\
I
I
Alternatwe
Store fol
ater supp y
4
p$
ar et
X-Ray Imaging
139
whether the product should be marketed immediately, stored for supply later on
to maximize profit, used for making alternative products, or discarded.
Automation of the quality evaluation process could generate significant
economic benefits to growers, packers, and processors. Our overarching view of
the decision support system for food systems is shown in Fig. 1. The component
enclosed by the dotted line in Fig. I , concerned with internal quality, is the primary subject of this chapter.
Various energy-based imaging techniques have been
tried in the past for
the detection of internal defects with varying degrees of success. The most appropriate image analysis technique that can relate image features to product quality
is, in many cases, yet to be identified. An emphasis should be placed on the
development of better image analysis and decision-making techniques for the
widest range of inspection applications. The goal of this chapter is to emphasize
x-ray techniques in the food industry and present some fairly promising case
study results and review relevant applications.
Image Acquisition
where
I
I,,
=
=
45 4 r e e s
180 degrees
315 degrees
90 degrees
es
360 degrees
Fig. 2 Three-dimensional schematic (a) of the x-ray imaging system showing two modes of operation: line scan (LS) and CT scan. In the LS
mode, the object moves across stationary source-detector assembly. The LS voxel is indicated along with a corresponding LS pixel in (a). In the
C T mode the source-detector assembly rotates around the stationary object as indicated in (b). Fig. (b) is an end view of Fig. (a), rotated the
indicated degrees. The CT voxel is indicated, as is the corresponding pixel in (a).
X-Ray Imaging
141
The measurement of the x-ray absorption coefficientp (or some parameter related
to p) is of paramount interest in x-ray applications. The image is in effect a map
of p. X-ray absorption in food products is partitioned into solids pg and water
p wcomponents, as shown below:
where
vu, = x-ray absorption coefficient of water at the specified energy level (m")
p, = x-ray absorption coefficient of the solids at the specified energy level
pw=
ps =
pSgw=
p,$\ =
(m")
mass of water per unit volume of sample (kg/m3)
dry bulk density of the sample (kg/m')
density of water (assumed constant, kg/m3)
particle density of dry solids (assumed constant, kg/m')
Shahin
142
and
Tollner
B. ProductOrientation
Product orientation has a direct effect on the feature characteristics. Orientation
relative to the scanned plane affects both shape and brightness of features. These
X-Ray
143
C. Image Noise
The image analysis processis essentially one of extracting desired image features
(if present) from the vast arrayof possible undesirable image features. The undesirable features arise from a variety of sources: the instrument, image artifacts,
image features not related to the quality attribute at hand, and lack of fit of the
decision engine algorithm and the distribution of the desired feature. Each proto decrease the
cessing step in an image processing protocol must be designed
into nondesirnondesirable features without transforming some desirable features
able features. At this point we consider instrument-induced noise and image artifacts. Other noise sources will be considered in the discussion of feature extraction and decision engine selection.
During the process of image acquisition, noise is introduced inadvertently
to theactualsignal,whichmustberemoved
or at least minimized to extract
features related to the quality of the product. Instrument-induced noise is in addition to noise associated to unrelated features of interest in an ideal x-ray image.
An x-ray image may be subject to instrument noise and interference from several
sources, including electrical sensor noise, channel errors, spatial sampling and
Shahin
144
and
Tollner
X-Ray imaging
145
111.
X-rays have been usedin the food processing industry for several years. Schatzki
et al. (1 3) demonstrated a technique for determining the density of lettuce heads
al. (14) demonstratedthatx-raytransmission
priortoharvesting.Dieneret
through bruised apple tissue was less than the transmission through nonbruised
tissue, although the typeof bruise was not clearly specified. X-ray machines have
also been used for detecting hollow heart in potatoes (1 5) and split-pit defect in
peaches ( 16). In food processing plants, x-ray-based technologies have success(17). Direct transmission
fully detected stones, bones, metal, and other objects
x-radiology can be useful for evaluating overall internal quality of several food
products. However, the resulting information from these techniquesis unsatisfactory for analyzing specific point locations of a fruit because of inherent volume
averaging effects. The U.S. Department of Agriculture (USDA) now uses linear
array x-ray scanners for detecting food in baggage at some airports.
X-ray technology hasalso been applied for detectionof watercore in apples
(18). Whiledetectionwaspossible,thevariation
of pathlengthorradiation
a very short length at the edge of the fruit to a
through the whole fruit, from
maximum just outside the center, produced an irregularly exposed radiograph.
of
Theapplication of x-radiographyforquantifyingphysicalproperties
fleshy fruits requires appropriate correlations between the physical property
and
x-ray absorption. Tollner et al. (19) present results for apples. Such correlations
enable the useof x-ray CT and lineararray scanners to nondestructively quantify
physical properties of food products. X-ray CT can effectively simulate linear
array scanner action; thus, most discussion is focused on tomography.
a
X-ray CT imaging is a proven method for nondestructively evaluating
cross section of an object. CT greatly reduces volume averaging compared
to
linear array scanner. Each point on a CT image represents a small volume in the
Shahin
146
and
Tollner
scanned plane by the x-ray, while a point on a transmission x-ray image (film)
represents a volume average of many volume elements between the x-ray source
and the film or detector array. X-ray CT enables detailed evaluation of relative
x-ray absorption coefficients over a defined plane within a fruit. One can also
simulate direct transmission (line scan) x-radiography with
a CT scannerby summing results in selected directions. Thus CT scanners are ideal for studying xray absorption properties of fruits.
X-ray CT was used to image the interior region of Red Delicious apples
(19). X-ray absorption properties of
under varying moisture and density states
apples with watercore disorder were different than apples without watercore disto the
order. The increased absorption of x-rays in watercore apples was due
increased water content of the tissue. CT image of watercore apple cross section
clearly revealed the disorder and severity results from the image.
The above studies were
mainly oriented toward manual detection of various
defects from x-ray images. Studies of this type are necessary to determine feasibility of defect detection and to learn the defect signature. X-ray-based defect
detection can at the least provide a machine assist for graders and other quality
control personnel. Once manual defect detection is possible, one may then cona decision
sider automating the feature extraction process and subsequently using
engine to classify the images based on the degree of defect.
IV. FEATUREEXTRACTION
Feature extraction is the applicationof various image processing operations such
as morphological operators, edge detecting with various edge detectors, thresholding, pattern matches, pixel counts, and pixel moments, etc. Operations are not
necessarily in this order, nor do specific protocols include all operations. The
development of the protocol for specific applications is as much an art as it is
science.
One in effect decides at the feature extraction juncture what is and is not
feature noise. The automation process should
begin with considerable input from
those experienced in the physiology of the disorder or defect in question. This
is especially true with issues related to moisture distribution and factors related
to handling and processing, which may affect the distribution of moisture. One
should be familiar with appropriate grade standards and extensionsto these standards, which are typically imposed by individual processors and packinghouse
operators who happen to be the client or customer. One should have experience
in manually extracting the feature of interest before attempting to do same with
computerized feature extraction. Judicious selection
of x-ray energy level and
product orientation relative to the x-ray source is always appropriate. These decisions should be determined in the manual extraction phase.
X-Ray Imaging
147
A. Watercore in Apples
Shahinetal.(20)summarizedacasestudyinvolvingwatercore
in apples.
Watercore is a cosmopolitan disorder that occurs sporadically, prevalent in some
years and absent in others. It occurs only later in the season in mature apples.
In addition, watercore occurs onlyin susceptible cultivars, and generally the characteristic symptoms appear only when the fruit is attached to the tree. According
is described as
to the U.S. grade standard for apples, damage due to watercore
invisible watercore existing around the core and extending to watercore in the
vascular bundles; or surrounding the vascular bundles when the affected areas
surrounding three or more vascular bundles meetor coalesce; or existingin more
than slight degree outside the circular area formed by the vascular bundles; or
any externally visible watercore. A normal fruit has 20-35% of the total tissue
volume occupied by the intercellular air space. In apples with watercore this large
air space is filled with a liquid. The liquid in the air spaces also increases the
density of the fruit. Tollner et al. (19) reported that density of the whole apples
increased with increasing severity of watercore disorder, however, the difference
was not always statistically significant. These changes
in fruit density and absorption characteristics with watercore have been employed in nondestructive detection of disorder. They (19) used x-ray CT to image the interior region of Red
Deliciousapplesundervaryingmoistureanddensitystates.X-rayabsorption
properties of apples with watercore were different from apples without watercore
disorder. The increased absorption of x-rays in watercored apples was due to
increased water contentof the tissue. CT images of watercore apple cross sections
clearly revealed the disorder severity. Results from the image analysis correlated
well with visual observations on the cut-open apples. Throop et al. (21) reported
a classification standard based on natural features for apples to define watercore.
Watercore can be thought of as groups of watery tissues, starting at the
center of the fruit and growing outward. In an x-ray image, the regions corresponding to watercorewill appear as bright regions consisting of adjacent pixels.
This a priori knowledge canbe of great importance to identify features indicative
of watercore. The likelihood principle along
with the laws of perceptual organization can be very useful in determining fruit quality from the image features. This
implies that the area of these brighter regions or blobs might be a good measure of watercore severity. Based on this assumption, fruit area in the processed
image was used as an indicator of watercorein apples (22,23). Since morphological operations are especially good for blob extraction, morphological opening
was used to extract the area features from line scan images
of red delicious apples.
Morphological opening is a combination of erosion and dilation operations. Erosion operation eliminates the false features, whereas dilation operation makes
sure that the true features remain intact. A structuring element approximately the
size of the prefiltering mask is appropriate.
Shahin
148
and
Tollner
Morphological image processing is a powerful tool for examining bloblike shapes and areasin images. McDonald and Chen(24) described some applications where morphological image processing was used for size distribution,
shape discrimination, and texture analysis of agricultural commodities. Throop
et al. (21) presented an algorithm to separate old and new bruised tissues from
stored apples using NIR reflectance. Apple images were first low-pass filtered to
removesmallsurfacecharacteristicsandthennormalized.Theshapes
of the
bruised areas were evaluated based on a shape factor. Morphological closing was
used to eliminate unwanted isolated details. Recognition of agricultural objects
by shape using morphological erosionof the absolute gradient of the x-ray image
was described by Schatzki et al. (25).
The ratio of the fruit areas measured before and after processing may be
an important feature in order to eliminate the effect of fruit size. Variation in the
fruit image intensities may be important due to the fact that watercored fruits are
expected to show more variation in the fruit image as compared to good fruits.
Therefore, standard deviationof the gray values withina fruit image may provide
someinformationaboutwatercoreseverity.Otherapplicationsnodoubtmay
cause other areal features to come to the fore. Typical results before and after
prefiltering plus application of the morphological operator to apple line scan images are shown in Fig. 3.
Image transforms are mainly usedto represent images in compressed form.
This makes the transform coefficients potentially good indicators
of fruit quality.
The Karhunen-Loeve transform (KLT) is the optimum transform that minimizes
the mean square error in the reconstructed image (26). The KLT yields a set of
uncorrelated coefficients, but it is slow to compute. However, the discrete cosine
transform (DCT), which is muchfaster to compute, closely approximates the
information packing ability of the optimal KLT (27). The discrete wavelet transform (DWT) is another commonly used transform in image processing applications. The DWT generates coefficientsthat are local both in frequency and spatial
(28).
domains; a characteristic that the discrete Fourier transform (DFT) lacks
Both DCT and DWT have been widely used in the general areas of image compression and feature extraction applications.
11 was hypothesized that textural features extractable by transforms such
as those above would provide additional information to a decision engine. The
choice of a particular transform i n a given application, however, depends on the
computational resources available and the a1nount of error that can be tolerated.
Information packing ability of DCT is superior to that of DFT and WHT. Although this is usually true for most natural images, the KLT, not the DCT, is the
preferred transform in the information packing sense (29). However, because the
KLT is data dependent, obtaining the KLTbasis images is a nontrivial computational task. For this reason, the KLT is seldom used in practice. Instead, DCT,
whose basis images are fixed, is normally preferred because it is fast and closely
149
X-Ray Imaging
I
Fig. 3 Results of applyingthresholding and morphologicaloperatorto raw line scan
of apples: (a) original line scan image of apples; (b) image following thresholding and
morphological processing.
approximates theKLT. Ahmed et al.(30) first noticed that the KLT basis images
closely resemble the DCT basis images. As the correlation between adjacent pixels approaches unity, the basis images become the same for both the KLT and
DCT (31). The DCT has found widespread applications in speech and image
processing for the purpose of data compression and feature extraction (32) and
Shahin
150
and
Tollner
noise filtering (33). However, no application of DCT for area defect detection
was found in cases of food products.
Wavelets are a set of mathematical functions, called basis functions, that
form a compact description of a signal. These basis functions can be used to
of resolution. As such, wavelets compress
represent signals at multiple levels
images and recover information from even the murkiest images. Wavelets also
isolate the location as well as the scale of features in a signal/image; thus, they
can encode rapidly changing signals in compact form. Details on wavelet transforms are givenby Daubechies (34). Wavelet transform has been used widely for
feature extraction purposes in a variety of applications. Agricultural applications
include grading beef carcasses (35) and picking green peppers in a field of green
leaves (36).
B. Bruises in Apples
Quality defects such as surface bruising decrease market value
of fresh fruits.
of the fresh market fruit producTender fruit varietiesthat make up a large portion
tion are especially susceptible to bruise damage during mechanical harvest and
postharvest handling. According to Studman et al.(37), on the average more than
15% of apples do not qualify as US extra fancy due to bruise damage during
orchard transport. As much as 29% of apples have been reported to have some
bruising after harvest. In order to maintain fruit quality, bruised apples must be
separated during the grading operation. At present, apple sortingis done through
human visual inspection that is inconsistent, inefficient, and subjective. Additionally, bruises in dark-colored fruits such as Red Delicious apples are verydifficult
to see, especially when the fruits are moving on the conveyors, thus further hinderingvisualinspection.Applesarethethirdlargestfruit
crop i n theUnited
States (38). Hence, substantial economic benefits could be achieved by timely
redirection of bruised fruits for alternative processing, proper utilization of storagespaceforstoringonlygoodfruits,andreducinglaborcostsformanual
sorting.
Impact stress occurs when the productis dropped from a sufficient distance
to cause injury. This type of injury is generally seen as bruising. With bruising
the injury is restricted to the interior flesh of the tissue and in many products
may be only initially detected as a water-soaked area after peeling.
With time,
in
the exposure of the damaged cells to air in the intercellular spaces results
thetypicalbrowning
symptoms. The damaged tissue mayeventuallybecome
desiccated, thus opening the possibility
of x-ray imaging. X-ray imaging
is a
leading contender becauseof its abilityto detect both external and internal defects
such as surface bruises and watercore (39,40). Apple defect classification from
x-ray images warrants major emphasis. Shahin et ai. (41) found that edge detection in the zone just under the fruit skin correlated with bruise presence and
X-Ray imaging
151
severity. Figure 4 shows line scan images of apples prior to prefiltering (Fig. 4a)
and after prefiltering and Roberts Edge operators were applied (Fig. 4b). Figure
4c shows the effect of substituting the median filter for the Gaussian filter. The
Gaussian filter was ultimately chosen based on subsequent testing. The edge corresponding to the skin edge was removedby further image subtraction operations
(results not shown).
C.Diseases
in Onions
Sweet onions are an important specialty crop grown in the Vidalia region of
of onions,sweetonionslackpungencycomGeorgia.Unlikeothervarieties
pounds (42). As a result, they are readily susceptible to disease inoculation and
tend to have a shorter shelf life. Several decay-causing organisms can possibly
(43,44). Mechanical injuries
invade the onion bulbs at any time via soil or air
occurring during spraying and harvest operations may damage the bulbs leading
to infection. Quality defects such as internal decay, ring separation, and internal
of onions. These physiological changes cause
sprouting decrease the market value
voids and/or other tissue change, which alters moisture distribution. X-ray imaging has shown promising results for detecting internal defects
in fruit and vege(45), pests (39), watercore
tables such as interior voids and foreign inclusions
(20), and freeze damage (46).
X-ray imaging and image processing techniques were employed (47) to
identify poor quality onions. They emphasized theneed for better image analysis
techniques for improved accuracy of classification. In light of the physiology of
the onion bulb and physics of the line scan operation, edge detection techniques
are expected to perform well for enhancing internal defect features in x-ray imin eggs (48) and bruise
ages. Success of edge detectors for enhancing cracks
damage in apples (49) provided a reasonable likelihood of success with disease
detection in onions (50) sincethe feature of interest in each case appearsin images
as a line or edge. Figure 5 shows the results of line scanning onions before (Fig.
Sa)andafter(Fig.5b)prefilteringandfeatureextraction.Arealfeaturesplus
various transform coefficients provide a suite
of potential inputs to a decision
engine for classification purposes.
D.MiscellaneousStudies
Long-established feature extraction approaches for optical images (e.g., see Ref.
51) may be applied to x-ray images. X-ray image feature extraction research
is
of baby
ongoing at several laboratories. Problems including the sex separation
chicks (52), insects, bruises, rots and browningof apples (39), and other general
agricultural products ( 5 3 ) are being researched. Techniques ranging from simple
segmentation to sophisticated watershed delineation algorithms (54) are under
152
X-Ray Imaging
153
Fig. 5 Line scan image of onions before and after Gaussian filtering and Roberts edge
detection. (a) Original line scan image of onions: dark features indicate defects. (b) Processed image showing features of good and defective onions:bright arcdlines insidebulb
boundary indicate defects.
154
evaluation. The numberof laboratories for x-ray imaging research for food products continues to expand.
V.
DECISIONENGINES FORFEATURECLASSIFICATION
A.
Bayesian Classifier
X-Ray Imaging
155
density function. The closer this assumptionis to reality, the closer the Bayesian
classifier approaches the minimum average loss in classification. The Bayesian
classifier implements a decision function of the form:
dj(x) = p(x/oj)P(oj)
1, 2, . . . , M
where a pattern x is assigned to class mi if di(x) > dj(x) for all j f i. P(wj) and
p(x/oj) are the probability of occurrence and the probability density function of
class w,, respectively. M is the number of classes the pattern can be assigned to.
Assuming a Gaussian probability density function, the Bayes decision function
is given by:
d,(x) = In P(wj) - 0.5 lnlCjl - 0.5[(x - rn,)TC;(x
mj)]
(4)
where mj and C, are the mean vector and covariance matrix, respectively.
In
addition to the normality assumption, the Bayes discriminant function assumes
that the group covariance matrices are equal and the output changes linearly with
the input variables.
Outputs from typical discriminant analyses enable one to analyze the relative contribution of the various inputs. Inputs not contributing meaningful information need not be further considered, thus simplifying the model. Datasets for
discriminant analyses are comprisedof the image features plusan expert classification using destructive techniques. Model development generally is performed
using a subset of the data.The remainder of the data are used for model validation.
The primary outputof interest is the classification of each sample product. Percent
accuracy of the classification along with the percent of false positives are generally reported. Classification accuracy may range anywhere from random (e.g.,
number of categories divided by 100%;very poor) to 90% plus, considered to be
very good. In cases where the expert standardis hand inspection, 90% accuracy is
to assure
considered excellent. Percent accuracy represents the systems ability
a good product, and false positives (100% accuracy) represent a tangible loss to
the system in that a good product is rejected.
The significant image features found to be significant contributors
in an
apple watercore study were blob area, mean grey value, and the tenth discrete
depending
cosine coefficient (20). Prefiltering may or may not be appropriate,
on the feature of interest. Selection of the prefiltering mask can be completed
once one has determined the significant image features contributing
to meaningful
classification. In the case of apples with watercore, Shahin et al. (20) justified
the size of the structuring element used in the morphological processing based
on the image noise statistics (20). Results from the watercore study are summarized in Table 1 . For the most part the classification was generally acceptable by
modem standards. Results for the moderate watercore category were worse
than either the none or the severe category. Some of the moderate cate-
156
Table 1 Classification Results for Bayes Classifier Applied to Red Delicious Apples
with Watercore
No/mild
Moderate
Severe
Total predicted
False
positives
Predicted class
actual
Watercore
No/mildclass
Moderate
Severe
10
4
0
52
m;
2
19
19
actual
(%)
(%I
58
14
63
1618
90
83
50
89
21
6. Fuzzy Logic
Fuzzy logic is based on fuzzy set theory introduced by Zadeh
(56). Fuzzy logic
is a nonparametric decision engine that can encompass nonlinear relationships.
Biological systems are complex in nature, and when idealized to develop representation by traditional models, they can distort to the extent where the models
are not representative and have little
use. Fuzzy logic methods present alternatives
for modeling complex biological systems
with a remarkably simpleway of drawing precise conclusions from vague, ambiguous, or imprecise information.
The initial stage of fuzzy model development depends to some extent on
intuition and the designers feel for the nature of the problem. Design
of a
fuzzy model does not require a well-defined mathematical model of the system
to be modeled. It can start with a general idea of how the model should work.
The idea may consistof defining input variables and their membership functions
(fuzzification), a set of rules to define the model behavior under different conditions (fuzzy inferencing unit,FIU), and an output variable to translate fuzzy output of the FIU into a crisp value (defuzzification).
Several fuzzy models have been developed to predict the response of biological systems. Among several applications, fuzzy logic has been used for predicting yield for prescription farming (57), estimating forest parameters from
image data (58), analyzing plant structure(59), diagnosing tomato diseases(61),
predicting peanut maturity (60), and sorting tomatoes(62) and apples (23) based
on quality.
The dataset for fuzzy classifier development consists of image features plus
expert data. Fuzzy approaches do not enable a ready evaluation of significance
X-Ray
157
C.NeuralNetworks
Artificial neural networks (ANNs) provide an alternative way of modeling sysor ill-definedinput-outputrelationships.ANNclassifiers
temswithunknown
in a manner similar to humans. The
have the capability of classifying patterns
power of a neural network lies in its ability to model nonlinear relationships and
in realits robustness to deal with noisy and incomplete data commonly found
life situations. ANNs are capable
of forming highly nonlinear decision boundaries
and have an intrinsic power to generalize. Neural networks look for patterns in
develop the ability to correctly
training sets of data,learnthesepatterns,and
classify new patterns.
The multiple-layer feedforward network (MLFN) was originally described
byRumelhart et al.(63). It is themostwidelyusednetworkarchitecturefor
problems that can make use of supervised training (learning through examples).
The MLFN can require longer training periods, but execution of the trained network takes relatively little time. Hence, real-time applications are best served by
the MLFN model (64). Details of neural networks can be found in a number of
publications (65-67).
Neural networks have been successfully used for food quality evaluation
and related applications. Boudolf (68) used the neural network approach to predict peanut maturity from nuclear magnetic resonance (NMR) data.
Pate1 et al.
(48) successfully developed a neural network for crack detection in eggs using
computer vision. Elizondo et al. (69) developed
neural
a network model to predict
flowering and physiological maturity dates of soybean. Das and Evans (70) used
computer vision and neural networks to separate fertile and infertile eggs. Deck
et al. (71) compared neural networks and statistical methods for sorting potatoes
into various classes using machine vision.
The dataset for theneural net classifier development is identical to that for
the Bayesian analyses except that insteadof a model building set and a validation
data set, the data are divided into model building, model testing, and validation
datasets. The MLFN requires that a test set be accessed for limited testing in the
model building phase. Using the same three image features used with the Bayesian and fuzzy classifier development discussed above, Shahin et al. (20) found
158
Table 2 Classification Results for Neural Classifier with Four Hidden Nodes
Predicted class
Watercore observed
No/mild
class
Nolmild
Moderate
Severe
Total predicted
False
Total
Accuracy positives
observed
(%)
(%I
Moderate
-3
3
0
58
Severe
- 3
14
2
13
58
95
57
5
38
18
19
90
Observed watercore seventy was assigned based on visual judgement of the cut fruit.
D. OtherClassifiers
Bayesian, fuzzy, and neural net classifiers tend to represent classifier types
in and
themselves do not comprise the entire universe
of classifiers. A feature extraction
methodcalledthemaximumrepresentationanddiscriminatingfeature
(MRDF)was reported(72) to classify good and bad pistachio with
nuts accuracies
be an advanced nonlinear implementaat or above90%. Their method appears to
tion of the Bayesian method. They found that adding the quadratic component
to the linear model increased accuracy, which may explain why a number
of
are
studies (40,41,50) reported increased accuracy with neural nets. Neural nets
known to represent nonlinear data fairly well.
X-Ray Imaging
159
VII.
FUTURETRENDS
X-ray technology has been usedin the past in the food industry for foreign matter
detection,such as screeningmeat/poultryproductsforextraneousmaterials.
However, single energy x-ray technologyis incapable of identifying small defects
and contaminants. Small defects and contaminants are of considerable interest
(73) because of lack of availability of sufficient number of qualified inspectors.
This is often because the variance in mass density between the defect/contaminant and the productand the product noise confound positive identification. Adding an additional scanning energy is being proposed to address these concerns.
Dual energy scanning allows imaging based on molecular weight and mass
density. One could use images at each energy level to further enhance features
of interest. Simultaneous measurementof moisture and soil density has been done
using dual energy level monochromatic sealed gamma sources for many years.
Aylmore (74) developed a dual energy gamma CT system, which successfully
detected plant moisture uptake and root growth. They developed an image or
160
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5
Nuclear Magnetic Resonance
Techniques and Their Application
in Food Quality Analysis
R. Roger Ruan and Paul
L. Chen
1.
INTRODUCTION
Laboratoryanalysis
of foodqualityofteninvolvesextensivepreparation
procedures,duringwhichfoodsareseverelydisturbed
by size-reducing,deforming, or diluting steps. This
is very unlike the way
in which food consumersevaluatethequality
of foodproducts (Le., foodsareconsumedwhile
theirintegrity is generallyintact.).Onemaythereforeask:Arethecurrent
chemical and physical methods for food quality measurement reliable? Naturin a completely
ally it would be desirable to be able to analyze food quality
nondestructiveandnoninvasiveway.Nuclearmagneticresonance(NMR)
a task.NMR
techniquesareamongthosemostcapableofperformingsuch
techniques,includingNMRspectroscopyandNMRimaging,ormorefreto nondequentlytermedmagneticresonanceimaging(MRI),canbeused
structivelyandnoninvasivelystudythechemicalandphysicalpropertiesand
phenomena, anatomical structure, and dynamic processes of food materials and
products.
In this chapter we will introduce the basic principles and methodologies
of proton NMR spectroscopy and imaging techniques and present some examples
of practical applications of the techniques.
165
166
II.
A. NuclearMagneticResonance
o = ?Bo
(1)
Fig. 1 Magnetic moment (p)precesses about the bulk magnetic field Bo with an angle 8.
167
themselves in either the same direction or the opposite direction as the field. In
the classic view described above, the alignment of the dipoles is determined by
whether the nuclei precession is in a clockwise or counter-clockwise direction.
A nucleus that has its spin aligned with the
field will have a lower energy and
more stable than when it has its spin aligned in the direction opposite to the field
(Fig. 2 ) .
The energy difference BE is proportional to the strength of the static field
and the population difference:
and
<
-112
........................
Aligned anti-parallel
Zen, field
2:w
ti
........................
Fig. 2 NuclearZeemansplitting
quantum number I/:.
168
energy by the nuclei. This resonance effect is therefore termed nuclear magnetic
resonance.
B. VectorDiagrams of Magnetization
The magnitude of the magnetization caused by the RF pulse can be visualized
through the vector model (Fig. 3). For protons with I = %I/?, the magnetic quantum number m I takes two values, + I / ? and --I/?, that is, the spins align in either
the same or opposite direction with Bo. Thenumber of spins pointing in the same
direction as Bo is greater than that pointingin the opposite directionas B,)because
the former alignment represents a low-energy or stable state. The individual spins
precess about the Bo at the Larmor frequency. The net magnetization of the spin
system is M. The length of the vector M corresponds to the difference in population between the two energy states.
We can put this vector model in a laboratory coordinate with a set of axes
as shown in Fig. 4. The applied magnetic field Bo is traditionally parallel to the
z-axis, and the net magnetization, M,, also points in the z-axis direction. If the
spin system remains in the Bo field long enough, the number of spins oriented
with the field reaches an equilibrium value. This value is termed Mo and is the
largest possible value of magnetization.
Because the net magnetization is precessing so fast, visualizing its motion
is difficult. One solution to this problem is to use a rotating frame to replace the
stationary frame. The condition is that the rotating frame rotates about the z-axis
at exactly the Larmor frequency. Therefore, within the rotating frame, the net
Fig. 3 Vector modelof magnetization of protons in the presenceof a permanent field Bo.
169
magnetization does not appear to be precessing and is thus easier to follow. The
rotating frame uses x, y, and z to label the three axes as shown in Fig. 5.
Visualization of interactions between applied RF field B , (in a direction
perpendicular to Bll)and spin magnetization becomes much easier with the rotating frame. Figure 6 shows a series of situations when different RF pulses are
applied to the spin system placed
in Bo field. In the equilibrium state (a), the
net magnetization along the z-axis has its maximum value, M, = MI].The net
magnetization vector can be flipped by an angle a from the z-axis if individual
x-y
nuclei absorbed RF energy and shifted to the higher energy state (b). An
170
y = o
z'
%= "0
plane component is introduced by the RF pulse with a value of M,, = MI, cos
a, while z'-axis net magnetization is M, = M,, sina. It can be seen that flipping
M, and increases Mxy.The net
the net magnetization from the z'-axis reduces
magnetization can be flipped to any angle by application of a suitable RF pulse.
The 90", 180", and 270" RF pulses in the x'-direction, for example, will rotate
the net magnetization to the ??'-axis, z'-axis, and $-axis, respectively. After the
application of an RF pulse, the net magnetization will return to its equilibrium
state. During the return, the net magnetization along the $-axis and on the x'-y'
plane changes with time, in the form of recovery or decay. It is a process where
the excited spins release their energy and go back to the stable state or a process
in which the excited spins interact with each other and lose their phase coherence.
These processes are termed relaxation processes, which will be discussed next.
The ability to flip the net magnetization through RF pulses allows us to
manipulate magnetization so that the characteristicsof the nuclei can be observed
and detected. Sometimes, a sequence of RF pulses is used to generate the magnein this chapter.
tization signal. We will discuss the topic later
C.
171
RelaxationProcesses
After excitation, the spins return to their equilibrium states or equilibrium population distribution during the periodof free procession. The majority upward transition population, that is, the originally lower level population, returnsits to
equilibriumbylosingenergy
in theform ofan RF waveviavariousradiationless
transition processes called "relaxation processes."The RF wave signal
is characterized by the Larmor frequency of the nucleus and can be received and recorded
by the NMR instrument (RF-receiving antenna).
of a
The magnitude of M, and M,, versus the time after the application
90" pulse is plotted in Fig. 7. The magnetization along the z-axis, M,, indicates
a recovery or growth of the signal, which was first flipped to the y-axis direction
with M, = 0 by the 90" pulse. The recovery of M, is in exponential form and
eventually equilibrates to Mo. The x-y plane magnetization, M,,, is at its maximum value (M,,) immediately after the 90" pulse, after which it decays to 0.
The recorded signal of recovery or decay is characteristic of the relaxation
processes. There are two kinds of relaxation processes: spin-lattice (or longitudinal) relaxation and spin-spin (or transverse) relaxation.
The time constants describe these exponential relaxation processes and are known as relaxation times.
"_
Mo
<
nz
. .
172
Rl
T,
where I takes the value of 1 and 2 for spin-lattice relaxation and spin-spin relaxation, respectively.
Figure 7 also shows that M, reaches its equilibrium after approximately
five Tls, suggesting that the time to reach complete relaxation should be greater
than five times the spin-lattice relaxation time constant. This
is important in NMR
experiments, for which complete relaxation is often required.
1. Spin-LatticeRelaxation, T,
The lattice here means the environment surrounding the nucleus, including the
remainder of that molecule and other solute and solvent molecules. The energy
of the resonant nucleus is transferred to the various components of the lattice as
additional rotational, transnational, or vibrational energy until thermal equilibrium is reached between the nuclear spin and the lattice. The more the lattice
components rotate at the resonance frequency, the more
efficient or faster the
spin-lattice relaxation. In other words, there is a distribution of rotation frequenTI. Because
cies in a sample of molecules. Only the Larmor frequency affects
of
the Larmor frequency is proportional to B,,, T I will also vary as a function
magnetic field strength. In general, T I is inversely proportional to the number of
molecular motions at the Larmor frequency.
2. Spin-SpinRelaxation, T2
Chemical exchange or mutual exchange of spin states by two neighboring spins
is the typical form of spin-spin relaxation, although the sources contributing to
spin-lattice relaxation also lead to spin-spin relaxation. Unlike spin-lattice relaxation, spin-spin relaxationis an adiabatic process, and the redistribution
of energy
among thc spins doesnot change the numberof nuclei i n the higher energy level.
173
3. RelaxationMechanisms
Excited spins may reach equilibrium state with their environment (lattice)
in varito equilibrium:
ous ways. The following sources can cause a spin system to come
Dipole-dipole coupling caused by magnetic moments of other nuclei
Paratnagnetic relaxation caused by magnetic moments of unpaired elcctrons
Anistropic electronic shielding due to angular variations i n the electronic
shielding of the stationary B,, magnetic tield
Electric quadrupole relaxation causcdby electric quadruple moment of the
nucleus being irradiated
Spin-rotation relaxation as a result of molecular magnetic monuments
for many
Dipole-dipolecoupling is thedominantrelaxationmechanism
spin systems and involves neighboring spins (magnetic dipoles). This interaction
is referred to as dipole-dipole coupling because the motionof one spin is coupled
into the motion of the other. Each of these spins is affected not only by the B,,
tield but also by the magnetic moment of the other; the latter is dependent on
the magnetic moments of the two spins and the distance and relative orientation
between the two spins. For more details about rclaxation mechanisms, the reader
is referred to the texts of James and McDonald (2), Field (3). and Farrar (4).
D. RelaxationTimesandMolecularMobility
Both T , and Tz are related to molecular mobility in a system. T2 is proportional
to the rate of rotational motion of thc molecules. As a general rule,Tz is inversely
proportional to the molecular weight. A larger T2 generally means greater mobility. As we mentioned earlier, spin-spin relaxation is affected by a chemical exchange process. which is especially true in biological systems like foods. Chemicalexchangetakesplacebetweendifferentsites
of differentmobilities.For
example, protonso f water exchangewith protons ofmacromolecules. and protons
of less mobile water
of more mobile water molecules exchange with protons
molecules. etc. The rate of exchange has great influence on the T? value. If the
exchange is very fast, the differencein T? of the two exchanging sitesis averaged.
On the other hand, if the exchange rate is low, a broad distribution of T2 and
even two separate Tz sites can be expected. The spin-lattice relaxation process
is slightly different, although in normal situations a larger T i also means greater
mobility. For very fast motion, T i approximates T?. If the motion is extremely
slow (e&, at very low temperature or whenthe system is very rigid), T , increases
with decreasing mobility whereas T2 continues to decrease. Therefore, rigid or
solid-like materials could havea very short T? but a very long T i . We will discuss
174
1. Measurement of T,
Because magnetization along the z-axis is difficult to detect, T, measurement is
impossible by using a single 90" RF pulse. Instead, the common approach is to
use an inversion-recovery pulse sequence
to generate the detectable signal.
In
RF pulse is followed by a 90"
the inversion-recovery pulse sequence, a 180"
RF pulse. The 180" pulse inverts the magnetization
to the -z-direction while
maintaining a magnitude -Mo. If the 90" pulse is applied immediately after the
180" pulse, the detected magnetization, M,,, will be equal to Mo. If we apply
the 90" pulse at some time T after the 180" pulse, the net magnetization has a
chance to decay somewhat due to the T , relaxation. Thus, the detected signal
along the y-axis will be slightly smaller than M,,. If this process is repeated with
longer T values, the delay times between the 180" and 90" pulses, the relationship
between the TI relaxation and time can be established through the following equation:
M,(z) = M(,(1
2e-T"'l)
(5)
Tz describes the decay of M,, and is readily detectable. A 90" RF pulse flips the
magnetizationalongthey-axiswithamagnitude
of M,, = M,). M,, starts to
175
....................................................................................
Time
"0
r...................................................................................
shrink following the application of the 90' pulse in exponential form as mentioned earlier (Fig. 7). The decay curve is normally termed free induction decay
or FID and can be described by the following equation:
M,,
M,,e -'IT?
176
RcgonII
RcgonI
Time0 +y-
Time A +y+.y+y+-y
... ....
TlmcB
....
Regon111
TotalNct
$-.$X
... ....
. . . ..
(A)
... .....
y T : y
RcgonI
y"
.....
RegonIII
$-.
.+y+.Y+>........
+y
.....
Region11
+y
TotalNeI
........
....
...
(B)
Fig. 9 Decay of x-y plane magnetization M,, in perfect ( A ) and imperfect (B)magnetic
field.
is perfectly homogeneous, all spins are rotating at the same frequency and pointof therelaxationprocess,although
ing in thesamedirectionatanytime
the net magnetization vectors shrink at later times because of T? relaxation. In
suchconditions i t is said that all the spinsare in phase orhavemaintained
phase coherence. In this condition, the net magnetization of the entire spin system at anygiventime
is equal t o the net magnetization of anyspins in the
system and can be described by the true
Tz. On the other hand. when the magin the
netic field is imperfect(Fig. 9B), thex-yplanemagnetizationvectors
to M,, afterthe 90" pulse,shrink by thesame
threeparts,initiallyequal
in slightlydifferentdirections
at
amountdue to the Tz relaxation.butpoint
times greater than 0 because the spins i n the three parts of the sample are rotating at different frequencies. This condition is termed dephase. and the spins
in thethreeparts
aresaid to losetheirphasecoherence.Becausethevectors
do not point in the same direction, the total net magnetization vector is shorter
thanthe total fortheperfectmagnetsystemthesametimeafterthe
initial
pulse.
The characteristics of such relaxation in an inhomogeneous magnetic field
is determined by TT, which includes contribution from natural molecular interactions (T?) and magnetic field inhomogeneity-related component (T2,,J:
177
However,because it isimpossibletodetermine
T?,,,withasingle 90" pulse,
90" pulse have been
many methods involving additional pulses after the initial
is to rephrase
developed for measurement of true T?. The common approach
or refocus the spin magnetization some time after the
initial pulse. Spin-echo
pulsesequence ( 5 ) is acommonmethodforT2measurement.Thespin-echo
sequence consists of two pulses, a 90" and a 180" pulse, in the form of 90"7-180" (Fig. 10). Figure 1 I illustrates the principle of spin-echo method. When
the spins are placed in the stationary magnetic field Bo, all the spins align with
the B,,-direction or z-direction (Fig. 1 IA). After the application of a 90" pulse,
to M,, (Fig.
all the spins are aligned along the v-axis, giving a nonzero value
1 le). M,, commences to decay because of T? relaxation and inhomogeneity
in themagnetic field. The field inhomogeneitycausestheindividualspins
to
in alossofphasecoherence,as
precess in differentfrequencies,resulting
in the x'-y' plane, as some spins move
discussed earlier, and they will fan out
faster (f) and some slower (s) (Fig. 1 IC). If after a time T a 180" pulse is applied,
the spins will be flipped 180" about the x'-axis, as shown in Fig. 11D. Because
theindividualspinsare
still precessingatthesameindividualfrequenciesas
before, the spins will
all be in phase again at time 2~ with the orientation in
the -v'-direction (Fig. 1 1 E). This is called refocusing or rephrasing. The continuing processing of the spins causes the spins again to lose their phase coherence
(Fig. 1 IF).
The rephrasing of the spins causes a free induction signal to build
to a
maximum (i.e., refocus) at time 2 T. This signal is termed spin-echo or
SE ampli-
90"RF
180"RF
Pulse
Pulse
"-c
178
90" pulse
Y'
X'
03)
180" pulse
Dephase
X'
(C>
Rephase
X'
(E>
Fig. 11
X'
(F>
tude. However, the dephasing process occurs not only because of the field inhomogeneity but also involves decay due to the natural transverse relaxation with
a time constant T2.If the 180" pulses are applied at different times, decay in SE
amplitude will be observed, characterizedby T2. Therefore,T2can be determined
by carrying out a separate pulse sequence for each value of t and detect the SE
amplitude at time 2 2 . However, the spin-echo method is limited in its range of
applicability because of the effect of molecular diffusion. The precise refocusing
of all the spin magnetic moments is dependent upon each nucleus remaining in a
constant magnetic field during the timeof refocusing experiment ( 2 ~ )If. diffusion
causes nuclei to move from one part of an inhomogeneous field to another, the
echo amplitude is reduced.
179
Carr and Purcell (6) proposed a variation of the simple Hahln's spin-echo
method, which reduces drastically the effect of diffusion. Their method involves
a 90" pulse at time t = 0 followed by a series of 180" pulses at times t, 3'1, 5t,
. . . , which refocus the individual spins to form echoes at times 2t, 4.t. 6t. . . .
However, the inaccuracy in the 180" pulse width can cause some errors in the
as the
spin-echo magnitude, which can be cumulative, becoming more serious
number of 180" pulses in the Can-Purcell sequence increases. The Carr-Purcell
sequence was modified by Meiboom and Gill to eliminate the errors. The CarrPurcell-Meiboorn-Gill or CPMG pulse sequence is the most common method for
Tz measurement. It uses the same pulse sequence as the Cam-Purcell technique,
but the 180" pulses are applied along the positive y'-axis. Thus, all of the subsequent refocusing is along the y'-axis, and a l l of the echoes are positive (Fig. 12).
The drawback of the CPMG experiment is that it takes a long time and is
unable to detect spins that relax so fast that their signal ceases to exist before
CPMG begins data acquisition. Therefore,the one-pulse (90") experiment is still
useful in obtaining information about the fast decay typically solid component.
to magnetic
For solid samples, T2 is so short that the apparent relaxation due
field inhomogeneityI/T?,,, is unimportant,and 1/TT = 1 /Tz + I/T,,,, 1/T2, or
Tz J: T?:.
F. MRI Methodology
MRI is an extension of NMR. It provides additional spatial information regarding
spins.The first publishedMRimage is credited to PaulLauterbur (7) in the
Chen
180
and
Ruan
r(Bo
+ xG,)
= Wo
+~xG,
(8)
or
where x is the position in x-axis direction and G, is the field gradient applied in
the x-axis direction. Here we say the frequency is spatially encoded. Field gradients can be appliedin all three directions. The symbols for a magnetic
field gradient i n the y- and z-directions are G, and G,. Similarly we have
181
S
I
Fig. 13 Application of a field gradient can spatially encode the signals from different
spatial positions. The three black spots represent three tubes of water in the sample.
Equation (8) forms the basisfor all magnetic resonance imaging methods.
The backprojection reconstruction imaging methodwill be presented in thenext
section to illustrate how the MR image is produced.
2. ProjectionReconstructionImaging
This method is of great historical importance since the first MR image was pro(7). Backprojection reconstruction is an extension
duced by Lauterbur in this way
of the frequency encoding discussed above. A one-dimensional linear
field gradient is applied at many different angles covering an arc of at least 180" and up
to 359". An NMR spectrum is recorded at each angle (Fig. 14A). Once a set of
data are recorded, the data are backprojected through space using a computer
program, and an image is reconstructed (Fig. 14B).
Chen
182
and
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3. FourierTransformImaging
Fourier transform (FT) imaging is currently the most widely usedMRI technique.
in 1974 by
The first two-dimensionalFouriertransformimagewasproposed
Kumer et al. (8). In FT imaging, both amplitude information and phase information are acquired to spatially encode the signal. The amplitude information (frequency encoding) is obtained by using the originalfield gradient, G,, from which
x can be calculated. Phase encoding is achieved by applying a phase encoding
gradient just before the originalfield gradient is turned on, and at the right angle
to the original gradient, normally in y-direction, denoted by G , or Go. The phase
encoding gradient is raised from zero in a series of small successive steps (time
duration t). Phase angle Cp is given by:
Cp
= 'Iy
1:
G,(t)dt
(12)
183
will get a data set containing tn rows and n columns. By using Fourier transform,
we can produce an t n X tz image.
4. SliceSelection
Data for production of MR images are collected from spins of a plane through
an object. This plane will have certain thickness and is therefore termed a slice.
An MRI instrument is capable of selecting a slice of certain spatial location and
thickness. Slice selectionis of great importance to signal-to-noise ratio and image
resolution. Slice selectionis achieved by applying a one-dimensional, linear magnetic field gradient during the period that the RF pulse is applied. A 90" pulse
applied in conjunction with a magnetic field gradient will rotate spins, which are
look
located in a slice or plane through the object. To picture what this would
to examine
like if we had a cube of small net magnetization vectors, we need
of frequencies.
the frequency content of a 90" pulse. A 90" pulse contains a band
This can be seen by employing the convolution theorem. The frequency content
of a square 90" pulseis shaped as a sinc pulse. The amplitude of thesinc function
is largest at the frequencyof the RF, which was turnedon and off. This frequency
will be rotated by 90", while other smaller and greater frequencies will be rotated
by lesser angles.
The application of this 90" pulse with a magnetic field gradient in the xdirection will rotate some of the spins in a plane perpendicular to the x-axis by
90". The word "some" was used because
some of the frequencies have a B ,
less than that required for a 90" rotation. As a consequence, the selected spins
do not actually constitute a slice.
5. Pulse Sequences
The basic procedure for productionof a two-dimensional MR image can be summarized into five stages:
I.
2.
3.
4.
Excitethespins of selectedslice
Apply a phase encoding gradient for
a fixed time
Apply a frequency encoding or read gradient and collect 12 data points
Increment the value of phase encoding gradient and repeat steps
I to
3 t n times
5. Perform two-dimensional Fourier transform on the data to produce an
n z X n image
184
RF
Frequency Encoding
or Read Gradient
Signal Detection
angle imaging, echo planar and hybrid imaging, etc. Due to limited space, these
techniques will not be discussed in this chapter.
NMR Analysis
in Food Quality
185
111.
The intensity and motional properties of protons are two of the most important
NMR properties from which a number of analytical methods have been developed. The abundant presence of protons in foods makes them excellent image
of
contrast agents, enabling nondestructive observation of the internal structure
food products. The initial signal intensity (before decay or relaxation) is directly
proportional to the number of nuclei present. Therefore, it is possible to measure
the moisture and fat contents of a food product by measuring its proton signal
intensity. Through MRI, mapping of moisture and fat in food products can be
achieved. Similarly, the relaxation time constants,
T I and Tz, can be related to
molecular mobility of food products, as discussed earlier, and canalso be mapped
out using MRI. Previous research has demonstrated that variations in relaxation
is interaction between liquid and
times arise from many reasons. Among those
solids in the system. The interaction between liquid and solids is governed primarilybymoisture content, composition, particle size, structure, temperature,
and physical state of the system. Therefore, relaxation times can
be used to probe
many aspects of food systems.
A.
MRI has sinceits invention mainly been used to noninvasively visualize the internal structure of the human body for diagnosis purposes. In recent years MRI has
186
Raw
Cooked
Fig. 17 MR images of eggs. The brighter the image, the stronger the signal.
187
intensity from egg, which may be attributed to the tighter structure and stronger
18 demonwater binding due to heat denaturalization of egg proteins. Figure
strates the possibility of using MRI to acquire three-dimensional images, although
the procedure for getting a three-dimensional image is very complicated.
Chen
188
and
Hard
Ruan
Bruised
Soft
of an affected fruit, and the area most affected was between 5 and 10 mm from
19 also shows that the bruised tissue had
the center toward the stem end. Figure
stronger signal intensity, probably due to increased water mobility in the bruised
tissue.
Figure 20 shows two MR images of kiwifruits, one unripe (left) and the
other ripe (right). These images are T2-weighted. Inwords,
other they have partial
information on the mobility of water because of the way the imaging was conducted. In this type of MR image, the high intensity is attributed to both the
amount and mobilityof protons in the sample. In the unripe,firm fruit (left), the
Raw
Fig. 20 M R images of unripe and ripe kiwifruit.
Mature
189
core has a very low intensity, suggesting that this part of tissue is very firm and
low in moisture content. In contrast, the tissue surrounding the core appears
very
bright, indicating a soft texture with alot of moisture. The signal intensity of the
As the fruit ripens, the
outer flesh layer is between the core and middle layer.
signal intensity in the fleshy portion increased dramatically while
the intensity
in the core remained low, forming a sharp contrast between the fleshy and core
tissues. The three images shown in Figure 19 are cross sections of strawberries.
The effect of ripening on the NMR signal intensity of strawberry is similar to
that in the kiwifruit.
C.
Water and fat have a profound impact on the sensory quality of food products.
Equally important in this matter is the distribution of water and fat throughout
the food product. Distribution of water and fat in foods may be uneven due to
improper processing, handling, and redistribution
of water and fat during proin variations in texture and chemicessing, storage, and transport. This may result
cal reactions, resultingin inhomogeneous product quality.In addition, since water
is essential to the microbiological activitiesin food products, uneven distribution
of water may cause deterioration in regions having higher moisture contents.
MRI is anidealtoolforinvestigating
the distribution of water and fat.
However, it is a challenging task to separate signals of water protons from those
of fat protons since protons in both the water and fat molecules contribute to the
NMR signal in a conventional proton imaging sequence. Differentiation between
moisture and fat is essential to the acquisition
of MR image of protons from
watermoleculesonlyandfromfatonly.(Thetermswater-onlyandfatonly will be used here to refer to MR images of protons from water molecules
only and from fat molecules only, respectively.) In medical applications, numerous techniques have been proposed to generate moisture-only and fat-only imof proton signal while supages. Basically, these techniques promote one type
pressing the other type of proton signal. The principle of these techniques lies
in the difference in relaxation times and/or resonance frequencies between water
and fat protons.
The protons in water and fat molecules are found in different physical and
chemical environments and, correspondingly, have slightly different resonance
frequencies. The difference in resonance frequency between water and fat, also
termed chemical shift, is about 3.5 ppm or about 700 Hz in a 4.7 T magnetic
field. Their relaxation times usually are not the same. Most current moisture and
fat separation techniques in MRI take advantage of the differences in resonance
frequency or relaxation times and can be classified into chemical shift select-
190
(4
(b)
191
-Water Saturated
-Fat Saturated
-No
20 22 24
Saturation
18 10 1612
14
ppm
Fig. 22 NMR spectra of cheese. Bottom: No suppression was applied. Middle: Fat was
suppressed. Top: Water was suppressed.
ration leaves the spin system in a state where no net magnetization of unwanted
component is left while the wanted component remains entirely unaffectedin the
form of longitudinal magnetization. A conventional spin-echo imaging sequence
is then applied to obtain the desired proton image.
192
cules are high compared to the time scale of the thermodynamic measurement.
Unfortunately, most food systems usually are nonequilibrium systems and, during
processing and storage, are maintained or brought intoa state of thermodynamic
instability, a state perhaps better designated as pseudo-stable since the unstable state may last longer than the normal lifetime of the product. Therefore, a
concept rootedin equilibrium thermodynamics, such
as a,, cannot fully represent
in
most food systems and should only be
used to describe systems that exist
equilibrium with their aqueous environments (e.g., water vapor) (28). Some researchers have begun to use relative vapor pressure (RVP) instead
of a, because
a, is in fact indicative of the rate at which water migrates out of the system.
Another defect of the aH.concept is that when different solutes are used to lower
the a, of a system, the response of the system in terms of the reaction rates or
stability is different. This suggests that measurement of a, is insensitive to the
solute-solute and solute-water interactions, whichin fact have a profound impact
on the reaction kinetics of the system.
The availability of water isa manifestation of how freely water molecules can participate in reactions or how easily water molecules diffuse to the
reaction sites to participate
in the reactions. The availability of water can be
reduced by adding certain chemical compounds, such as sugars (30,31). The reduced availability of water has been attributed to the increased viscosity of the
systems (3 1-34) or increased hydrogen bonding between water
and sugar molecules (35). Many researchers have found that the mobilityof water, as measured
by N M R and other techniques, is related to the availability of water in complex
systems (31,351-41). The higher the mobilityof water, the higher the availability
of water. Very mobile water molecules take
a long time to reach their equilibrium
state, or relax very slowly, thus having a small relaxation rate( R , or R?) or long
relaxation time (T, or T2, TI= 1/R, and TI = 1/R2). Figure 23 shows that added
sucrose suppresses the spin-spin relaxation time constant
of sucrose solutions.
N M R has been proven to be one of the most successful techniques to measure
the mobility of water, hence the availability of water (31,35-41).
193
1700
1300
900
500
100
0
3010
20
40
50
60
Fig. 23 Spin-spin relaxation time constant (TI) decreases as sugar content increases in
a sugar (sucrose) solution.
stood. In a heterogeneous system, spins exist in a wide variety of different environments, giving rise to a spectrum of relaxation times. In addition, chemical
and diffusive exchange, an important factor affecting spin-spin relaxation proa variety of T2 values, assuming there is a wide
cess, would also give rise to
range of exchange rates within the heterogeneous system (43-45). Therefore, the
measured relaxation decay is a sum of contributions from all spins, which have
sampled many different environments, or exchange with other spins at different
rates during the course of NMR experiment (46). It is thus reasonable to assume
that a continuum of relaxation times would arise from a continuum of different
exchange rates and different environments
in which spins exist. Some researchers
in various
have used the continuum models to follow the relaxation behavior
systems, although they focused on the state of water instead of that of biopolyal. (44) claimed that the
mers that interacted with water (44,46-51). Lillford et
continuous distribution of relaxation times is a better representation of the information content of the relaxation experiments.
The continuum approach seeks
a continuous distribution of relaxation times
and effectively adjusts a continuous variable number of degrees of freedom to
the minimum value necessary for a given data set. The CONTIN computer program of Provencher (52,53) has been used by researchers to process noisy data
Chen194
and
Ruan
where A is the amplitude at delay time t and AI, is the amplitude at equilibrium.
Usually, for heterogeneous systems like wheat flour dough. a multicomponent model (56) is used:
Equations ( 13) and (14) work well with simple systems. Both models have
been developed based on several assumptions, such as some predetermined number of discrete components in the decay curve (41).
WhittallandMackay (47) proposedamethodcallednonnegativeleastsquares (NNLS), which uses a continuum approach to analyze this type of data.
The general integral equation describing multiexponential relaxation is
J=
is:
,=I
where A,, is a matrix with elements exp(t,/TJ), and M is set large enough SO as
not to bias the solution into small number of relaxation times. Because of the
noise contamination, Eq. 18 cannot be solved exactly.
A non-negative least-squares algorithm has been developed for
this kind
of relaxation time analysis. in which extra constrains are incorporated into the
matrix A of Eq. 18 to alter the discrete character of the basic least-squares solution. A general form is to minimize
2. Mono-Exponential Decay
Proton relaxation is normally in exponential form, and the relaxation time con1I.E that
stants can be determined from the decay curves. We know from Sec.
T I can be determined by running an inversion recovery or saturation recovery
experiment, and T2 by a 90" pulse or a CPMG pulse experiment. Take the 90"
196
t
Fig. 24 Freeinductiondecay (FID) curve.
pulse experiment, for example; the resultant FID curve is normally represented
by:
In A(t) = InA.
1
-
T2
where A(t) is the amplitude at timet and A. is the amplitude at equilibrium. The
plots of A versus t and In A versus t are shown in Figs. 24 and 25.
3. Multiexponential Decay
Figure 25 is a straight line as expected from Eq. (20). However, in many cases,
the plot of In A(t) versus t is not a straight line, as shownin Figure 26, suggesting
t
Fig. 25 Semi-log plot of mono-exponentialFIDcurve.
197
that there are two or more groups of water molecules that relaxat different rates.
The relaxation curve cannot be described by using the mono-exponential model
[Eq. 141. Instead, the multicomponent model described by Eq. (15) must be folin isolated amplitudesand relaxation time constants.
lowed. This model can result
Figure 27 shows a three-component model.
198
45
4. Continuum Model
The example given here is from
our study of state of water
in wheat flour dough.
90"
Dough samples of different moisture were prepared and measured using the
and CPMG pulse sequences. Analysis of the data obtained from the 90" pulse and
CPMG experiments using the CONTIN package resulted in spectra (continuous
distribution) of T2 (Figs. 28,29). The x-, y-, and z-axes are T2value, amplitude,
T2 spectra computed
and moisture content, respectively. Figure 28 shows the
from data obtained from the90" pulse experiment.At moisture contentsof 12%
to 28%, two peaks (PI and P2) appear on each spectrum. Water molecules covered by these two peaks can be regarded as two groups having distinctly different
1 to 66 ps, proton
mobility. Because theT2values of these two groups range from
signals falling into these two groups can be regarded as from the solids (proteins
and carbohydrates) and/or water molecules
very close to the solids. Below moisture content of 23%, the increase in the area of the second peak and decreasein
average T2of individual peaks could be attributed to the increased available binding sites of the swollen flour substrates as a result
of addition of water (58). The
disappearance of the first peak at moisture above 23% may be due to the same
199
45%
O.OEMO\
loo
403
,~,
6579
-c
..--,
reasons explained earlier. The increasein both peak area and T2 above moisture
content of 23% suggests that at 23% moisture level, all the water-binding sites
two
on the flour solids have been hydrated, and the additional water would be
or three layers away from these binding sites and therefore exchange and relax
more slowly.
The CPMG experiment was intendedto detect proton signals having relatively longer spin-spin relaxation time than the one-pulse experiment. The analysis of the data obtained from the CPMG experiments indicated that at moisture
content below 18%, no signal was detected, suggesting the dry flour had little
mobile water. At and above the 18% level, there were one to three peaks (P3,
P4, and P5) on the spectra (Fig. 29). T2values shown in Fig. 29 range from lo2
to IO5 ps, suggesting that the detected signals were from water molecules with
relatively high mobility. The number and size of peaks increased and the mean
T2values shifted to the right (increasing
T2value), with moisture content increasing up to 28%. The appearance
of new peaks suggests that new physical and
of addition of
chemical environments were formed within the system as a result
Chen
200
Ruan and
SD
c.v.% = y x 100
X
+Pl
+P2
"
P
3
+P4
+PS
10
20
30
40
50
Fig. 30 Coefficient of variation of spin-spin relaxation times determined using the continuum model (see Figs. 28 and 29 for labels of PI, P2, P3, P4, and P5).
201
like and tightly bound water protons were associated did not change very
much while the moisture content increased substantially.
The coefficients of variation of those longer T?s (the three peaks shown in
of 40%, which
Fig. 29) showa gradual and slow increase below moisture content
of
may be a result of gradual formation of the bicontinuous network structure
dough and uneven distribution
of water among the flour constituents, as discussed
earlier. Upon reaching a moisture contentof 40%, which is more than the normal
dough moisture level, the coefficients of variation rose sharply, suggesting that
theexcesswater mayhavecreatedveryinhomogeneousdoughmorphology,
which should be further investigated.
202
chain of the polymer molecule are first mobilized before the whole molecule
starts moving. Further heating causes the entire molecule to move and become
liquid. From this discussion we can differentiate two types of motions: internal
or segmental motion and external or molecular motion. Both motions can be
regarded as Brownian. At a certain temperature,
both motions are frozen; at
higher temperatures both motions can be activated. These two situations correspond to the solid state and liquid states, respectively. What happens
if the polymer is placed at a temperature where only the segmental motion
is activated while
the molecular motion is frozen? The activated segments correspond to the liquid
state, while the molecule as a whole, with the mobility frozen, corresponds to
the solid state. This state, which is indeed a mixture of liquid and solid, is called
the rubbery state. On further heating the polymer in the rubbery state becomes
a highly viscous liquid and starts flowing; this state is called the viscofluid state,
the transition taking place at the flow temperature Tf (Fig. 31).
Polymers can have two types of motions: segmental and molecular. How
can we relate these motions to the NMR properties? Can we determine the Tg
by detecting the motional characteristicsof the polymer? Does waterplay a role
here?
NMR measuresprotonrelaxationcharacteristics,
Asdiscussedearlier,
which can be related to the mobility
of the proton-containing molecules. In a
liquid state, a long relaxation time constant indicates high mobility and increases
with increasing temperature. When the system is moving toward a solid state,
for instance, due to suppressing of temperature, the relaxation processes behave
somewhat peculiarly. We have mentioned that the spin-lattice relaxation is a process of energy release by the excited spins to the lattice (the entire molecular
0Motion
not activated
Motion
activated
203
system). To facilitate an efficient energy release by the excited spins, the lattice
In the solid state, the number of
has to oscillate at the resonant frequency a,,.
molecules oscillating or rotating at the resonant frequency olIis very small, suggesting a very inefficient spin-lattice relaxation. Therefore, the dynamic contribution to the T I decay is very small, causing TI to become very long again. On the
is very short due to the decrease
in static contribuother hand,in the solid state, Tz
tion to transverse-component dephasing.
The dynamic motion of spins (or molecules) can also be characterized by
a correlation time z,, which is, roughly speaking, the minimum time required for
the nuclear magnetic moment to rotate one radian (I/? 7t of a complete circle). In
general, T, for a nonviscous liquid is very short. With water, for instance, T, is
about 10"' s. On theotherhand, T, forsolids is verylong,about
s. Within
Z~ and the more quickly a perlimits, the slower the motion, the longer will be
Z, and relaxation time
turbed spin system will relax. The relationships between
constants can be described as follows:
(25)
Fig. 33 NMR relaxation time constants (TI and Tz) as a function of reciprocal absolute
temperature ( 1 IT).
205
10000
+T1
(20M H z )
+T1(200
MHz)
0.01
'
'
0.0001
l.E-10
'
"""'
'
1 .E-09
'
"""'
1 .E-08
"""'
'
1 .E-07
'
'"'.rl
1 .E-06
Tc (s)
where fll is the frequency of the NMR equipment related to the static magnetic
field Bo, i.e.,
BecauseformostcommercialNMRanalyzers,
fo is in therange of 10-600
MHz, o,]
is in the range of 60-3800 MHz. Therefore, for T , minimum to occur,
z, (= 0.616/o11)has to be in the range of 10""-10~* s.
Another model for calculation of z,, the Debye-Stockes theory, relates T,
to molecular size (with radius of a), medium viscosity 11,and absolute temperature T:
zc = 4na3q/3kT
(28)
where k is theBoltzmannconstant.Thismodelpredictsthatcorrelationtime
increases with larger molecules, viscous solutions, and low temperature. In other
words, within certain limits, the relaxation time constants for large molecules,
viscous solutions, and low temperature systems are small. On a qualitative basis,
Eq. (28) predicts a linear dependence of T, on T / q for some liquids (75).
However,thesemodelsare
not alwaysapplicable to complexsystems.
Many synthetic polymers, which have more than one group on the main chain
andprobablymorethanonecorrelationtime,undergomultiplerelaxation
Chen
206
and
Ruan
1000
T,minimum
100 :
I
~
10
100
200
I
300
400
Temperature (K)
Fig. 35 Spin-latticc relaxation time constant (T,)as a function of absolute temperaturc
for maltodextrin samples (DE = 5; moisture content = 25%).
-GE
207
n
W
1 0 0 ;:
100
10
150
200
250
300
350
400
Temperature (K)
Fig. 36 Spin-lattice relaxation time constants as a function of absolute temperature for
maltodextrin samples (DE = 25; moisture content = 29.2%).
100
n
rA
3.
Transition
point
10
100
200
300
400
Temperature (K)
Fig. 37 Spin-spinrelaxationtimeconstantsasafunctionofabsolutetemperature
maltodextrin samples (DE = 5 ; moisture content = 25%).
for
and
208
Ruan
Chen
1000 c
t
.
8.9%
+10.4%
+12.8%
10
'
200
250
300
350
400
Temperature (K)
Fig. 38 Effect of moisture content on spin-lattice relaxation time constantTI for maltodextrin (DE = 5).
209
1.35
A 14.70%
1.25
-3
1.15
1.os
x18.30&
0.95 I
250 200 150
300
350
400
Temperature (K)
Fig. 39 Effect of moisture contenton spin-spin relaxation time constantTzfor maltodextrin (DE = 5 ) .
a shift of the transition point to the lower temperature as the moisture content
of the samples increased.
210
ing spatial information about the thermal dynamic or dielectric properties of the
materials. On the other hand, MRI can generate spatial information about the
nuclear relaxation characteristicsof the material, which can be related to the glass
transition process as discussed earlier in this chapter. Therefore,
if we can estabL = 1,2) encoded with
lish a relationship between relaxation time constants (TL,
the spatial information and temperature (T), i.e.,
TL(X, Y) = f(T)
(29)
M C = 25%
M C = 25%
MC 1Wo
M C = 18%
47C30C-5C
23C13C 6C
211
IV.
FUTURE TRENDS
NMR and MRI have found use in other areas not mentioned specifically above.
of
NMR is widely used for rapid determination
of moisture and fat contents
oilseeds. Use of MRI to measure rheology of food products has also been
reported. Attempts were also made to combine MRI and mathematical modeling
techniques to study simultaneous heat and mass transfer.
Efforts are needed to address the relationships between NMR properties
and sensory quality attributesin food systems so that NMR can be used for rapid,
nondestructive, and noninvasive evaluation of food products. There are also
a
need to develop affordable NMR hardware and more sophisticated analysis software.
GLOSSARY
180"pulse: RF pulse designed to rotate the net magnetization vector 180" from
the static magnetic field.
90" pulse: RF pulse designed to rotate the net magnetization vector 90" from
the static magnetic field.
B,,: Conventional symbol for the main magnetic
field in MRI system.
Carr-Purcell (CP) sequence: Sequence of a 90" RF pulse followed by a repeated 180" RF pulse to produce a train of spin-echos.
Carr-Purcell-Meiboom-Gill (CPMG) sequence: Modified CP sequence with
90" phase shift in the rotating frame of reference between the 90" pulse
and the subsequent 180" pulse to reduce accumulating effects of imperfections in the 180" pulses.
Correlation time: The minimum time required for the molecule
to rotate one
radian.
Echo: A form of magnetic resonance signal from the refocusing of transverse
magnetization.
Equilibrium: Themagneticstate of anobjectthat is fullymagnetized by a
static magnetic field.
Fourier transform imaging: A mathematical procedure used in MR that converts a time-domain signal into a frequency- or spatial-domain signal or
image. It is analogous to the way that our ear distinguishes or separates
out separate sounds or frequencies from noise we hear. Our eyes do
not
212
work this way. If we see a mixture of blue and yellow we see the color
green, not the original blue and yellow.
Free induction decay (FID): A form of magnetic resonance signal from the
decay of transverse magnetization.
Frequency encoding: Use of a magnetic field gradient to produce a range of
frequencies along the MR signalto provide information on spatial position.
Gradient: Amount and direction of the rate of change in space of some quantity
such as magnetic field strength.
Larmor frequency ( 0 ) : The frequency at whichmagneticresonancecanbe
excited.
Lattice: Environmentssurroundinganucleus
or spin.
Magnetic moment: Measure of the magnetic properties of an object or particle
that causes it to align with the static magnetic field.
Magnetization: A vector quantity measuring the net magnetic moment
of nuclear spins per volume of a sample.
NMR signal: Electromagnetic signal i n RF range produced by the precession
of the transverse magnetization of nuclear spins.
Precession: A rotational motion of a spinning body about an axis
of a vector
whose origin is fixed at the origin.
Pulse sequence: A series of RF pulses and/or magnetic field gradients applied
to a spin system to produce a signal representative of some property of the
spin system.
Radiofrequency (RF): Electromagnetic radiation lower in energy than infrared. RF is in the range of 10 to 100 MHz.
Relaxation rates and time constants: After excitation, the spins tend to return
to their equilibrium state at certain rate. This rate is called relaxation rate.
The reciprocal of relaxation rate
is relaxation time. There are two relaxation
processes: spin-lattice or longitudinalrelaxation. I t is the return of longitudinal magnetization to its equilibrium value after excitation through exchange of energy between the spins and the lattice with a characteristic
time constant termed spin-lattice relaxation time T I .The second is called
the spin-spin or transverse relaxation process. in which the transverse component of magnetization vector, which is at right angles to the static magloss of transnetic field. decays towards zero. The characteristic time for
verse magnetization to zero is termed spin-spin relaxation time
T:.
Spin or nuclearspin: The intrinsic magnetic momentum of an elementary particle such as a nucleus responsible for
the magnetic moment. A fundamental
property of matter responsible for MRl and NMR.
Spin-echo: Reappearance of an MRIsignalaftertheFIDhasdisappeared.
It is the result of the effective reversal of the dephasing of the nuclear
spins.
213
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Nondestructive detectionofwatercoreinapplewith
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nuclear magnetic resonance imaging. Sci Hort 35(3-4):227-234, 1988.
1s. B Zion, P Chen, MJ McCarthy. Nondestructive quality evaluation of fresh prunes
by NMR spectroscopy. J Sci Food Agric 67(4):423-429, 1995.
16. JL Maas. MM Millard, MJ Line, GJ Galletta, JL Maas, GJ Galletta. Nuclear magnetic resonance microimaging of strawberry fruit. Acta Hort 348:375-377, 1993.
17. BR Rosen. VJ Wedeen, TJ Brady. Selective saturation NMR imaging.
J Computer
Assisted Tomography 8:813. 1984.
18. A Haase. J Frahm. WH Nicke,DMatthaei.IH
NMR chemicalshiftselective
(CHESS) imaging. Phys MedBiol30:341.1985.
19. PJ Keller, WWJ Hunter, P Schmalbrock. Multisection fat-water imaging with chemical shift selective presaturation. Radiology 164539. 1987.
20. RC Semelka, W Chew, H Hricak, E Tomei. CB Higgins. Fat-saturation
MR imaging
of the upper abdomen. AJR 155:I I 1 I . 1990.
21. J Mao, J Gao, H Yan, JR Ballinger. Susceptibility artifact reduction in fat suppression. MRM 33582-587. 1995.
22. K Chang, RR Ruan, PL Chen,RG Fulcher, E Bastian. Moisture, fat and temperature
mapping of cheese block during cooling using MRI. Paper N0.956535 presented at
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83-127,1957.
24. JA Troller. Influence of water activity on microorganisms in foods. Food Technol
(5):76-83,1980.
25. S Schwimmer. Influence of water activity on enzyme reactivity and stability. Food
Technol (5):64-74, 1980.
26. JA Troller. Water activity and food quality. In: TM Hardman, ed. Water and Food
Quality. London: Elsevier Applied Science, 1989: 1-3 I .
27. F Frank. Hydration phenomena: an update and implications for food processing industry. In: H Levine, L Slade, eds. Water Relationships in Foods-Advances in the
1980s and Trends in the 1990s. New York: Plenum Press, I99 1 : 1- 19.
of food safety and quality? Trends Food
28. F Frank. Water activity: a credible measure
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29. L Slade, H Levine. Beyond water activity: recent advances based on an alternative
approach to assessment of food quality and safety. Crit Rev Food Sci Nutr 30: 115360,1991.
ORFennema, ed.FoodChemistry. New York:
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31. H-M Lai, SJ Schmidt. Mobility of water in various sugar-water systems as studied
by oxygen-I7 NMR. Food Chem 4655-60, 1993.
32. MJ Tai. A Suggett, F Frank, S Ablett, PA Quickenden. Hydrogen of monosaccharides: a study by dielectric and nuclear magnetic relaxation. J Solut Chem 1 :131151,1972.
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of sucrose-water interaction. J Chem SOC Faraday Trans 1(82):451-456, 1972.
34. GW Padua. Water States Associated with Skim Milk Components as Determined
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determined by deuterium and oxygen-17 nuclear magnetic resonance.
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to "0 and '>CNMR data for carbohydrate solutions. J Agric Food Chem 37: 14591465,1989.
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79. YH Roes, M Karel, JL Kokin. Glass transitions in low moisture and frozen foods:
effect on shelf life and quality. Food Technol SO( I I ):95- 108. 1996.
Ultrasonics
John Coupland
The Pennsylvania State University, University Park, Pennsylvania
David Julian McClements
University of Massachusetts, Amherst, Massachusetts
1.
INTRODUCTION
218
However it is established, the relationship between how a consumer and a spectrometer see a material is likely to be weak. An analytical basis for any technique is therefore preferable because it clearly identifies the relationship between
material properties and instrumental readings. Second, different regions
of the
spectrum are sensitive to different physical and chemical structures present.
In
general, a higher frequency sees faster events, which most typically occur at
smaller scales.
Mechanical waves are a series of mechanical disturbances that propagate
as stresses and strains in the physical bonds of the material. The speed and efficiency of the transmission is sensitive to the nature of the bonds and masses of
the molecules present and therefore to composition. There are two distinct types
of ultrasonic waves; the most commonly used in food nondestructive evaluation
(NDE) arelongitudinalwaves(Fig.
la). In this case,thedeformations of the
material occur in the direction of transmission of the wave. In the second case,
a shearing action, causing
shear waves, the wave passes through the material with
deformations normal to the movementof the wave front (Fig. lb). Combinations
of shearing and longitudinal propagation are also possible. Shear waves are very
strongly attenuated in fluids, and because they cannot propagate far, they are very
rarely used to characterize food materials (typically largely liquid).
It is important to distinguish between the low-powered ultrasound used for
NDE of materials and the high-powered ultrasound used for homogenization,
welding, cell disruption, etc. In sensing applications, the deformations caused by
the passing wave are small-ideally within the elastic limit
of the material and
Direction of Propagation
Ultrasonics
219
* The attenuatlon coefficient of a material can be expressed as Nepers or decibels per meter, Np.m
220 McClements
and
Coupland
where a,is the classical attenuation coefficient, y = C,/C,, 6 is the thermal conductivity, and q is the viscosity. In systems whereit is applicable, classical theory
can be usedto measure any of these useful physicochemical properties. However,
often the measured attenuation is higher thanthat predicted classically due to the
scattering of sound by small particles (see Sec. 1II.D) or the presence of certain
chemical equilibria.
Additional energy can be lostif there are chemical equilibria present whose
position is affected by the ultrasonic wave. These additional (nonclassical) losses
can be measured and have been applied to the measurement of fast chemical
a material at equilibkinetics (2). When the compression wave passes through
rium, the change in temperature and pressure displaces the balance of reactants
and products. The equilibrium seeks to reestablish itself in the new conditions,
and its capacity to do so depends on both the speed of the reaction and the frequency of the sound. At low frequencies the temperature-pressure gradients are
so slight that the reaction remains in equilibrium and at high frequencies the
reaction cannot proceed fast enough to respond to the rapid fluctuations. Under
these conditions there is little excess sound absorption, but at intermediate frequencies thereactionposition
is constantlyshiftingandthere
is alargeabsorbance peak and a corresponding relaxation in velocity. By measuring the energy loss (attenuation) as a function of frequency over the relaxation process, the
rate constant, k,, of the reaction is given by:
1
2nfC= - " 2 k , d s
z
where Kc is the equilibrium constant,f, is the center frequency of the relaxation,
T is the relaxation time, and C is the concentration. The rate constant can then be
calculated froma plot of relaxation time against the square root of concentration
if
the equilibrium constant can be calculatedby macroscopic methods. This method
is appropriate for reaction times of the order 10-5-10"" s. Slower reactions can
also be followed if the reactants have a different velocity than the products, although other methods such as optical spectroscopy are generally preferred.
Ultrasonics
221
II. METHODS
A varietyof experimental designs have been developed to measure the ultrasonic
properties of food materials (2,3).
All share the common elements
of an electrical
signal generator, which is used to generate vibrations in an ultrasonic transducer,
and another transducer (or the same one after a time interval) to reconvert the
acoustic energy back to an electrical signal, which is then digitized for analysis.
A. Pitch-and-CatchMethod
Perhaps the simplest and most widely used implementation of these elements is
the pitch-and-catch or sing-around pulsed sound method (3-5). The two
major variations of this device are (a) pulse-echo-the sound is reflected from
a metal plate and detected at the original transducer (Fig. 2b)and (b) through
transmission-the sound from one transducer is detected by a second (Fig. 2a).
If the ultrasonic properties of the material are reasonably frequency independent
(nondispersive), velocity can be calculated from the time taken for the pulse to
travel the known pathlength (from a water calibration) and attenuation from the
(1). If required, the frequency depenlogarithmic decrease in energy with distance
Precise
Pathlength
Imprecise
Pathlength
2:
Fig. 2 Diagram of some typical methods of making ultrasonic measurements: (a) and
(c) are pulse echo methods using a single transducer to produce and detect the acoustic
signal; (b) and (d)are through transmission methods usingone transducer to produce and
another to detect the signal. (a) and (b) are methods using a fluid cell which can be precisely calibrated; (c) and (d)are measurements on irregularly shaped objects whereit may
be impossible to precisely know the pathlength.
222
and
Coupland
McClements
Ultrasonics
223
attenuate the signal. If transmission measurements are impossible, another approach is to measure the reflectance coefficient (proportion of normally incident
energy reflected) at an interface and use it to calculate the ultrasonic properties
of the materials from the following equation:
1 into
where R,? is thereflectioncoefficient of a wave passing from material
material 2 and z is the acoustic impedance of the material (=cp) ( I ) . Reflectance
measurements have also been used to measure the surface smoothness of food
materials (8).
B. Imaging
Most people are familiar with the use of ultrasonic imaging techniques from their
medical applications, for example, in prenatal care. These same principles have
been applied extensively to foods to provide information on the spatial heteroa transducer
geneity of food components. Acoustic images can be generated from
fixed to a robot arm that is placed in a tank filled with a suitable couplant (e.g.,
water) with the material under investigation. Operating
in either pulse-echo mode
or through transmission mode (Fig. 2 ) , the transducer records echoes from the
front surface, back surface, and internal structures in the material from a series
of X-Y positions. Each of the recorded waveforms is known as an A-scan.
A set of A-scans can be used to generate an image by assigning a color to either
the magnitude of the signal at either a fixed time (B-scan) or a selected feature
of the signal (e.g., second echo magnitude, time between successive echoes) (Cscan). The B-scan represents a slice through the material, whilea C-scan is more
useful for identifying the changing properties of a component. Both imaging approaches discard a large quantity of the information in each A-scan and should
be used critically. A diagrammatic illustration of image acquisition is shown in
Fig. 3.
In many cases, most especially large (e.g., whole carcasses and animals)
and water-sensitive materials, it is not appropriate to use the scanning tank approach described above. Medical imaging techniques developed forin vivo measurements on human patients (9) have proved useful in these cases-particularly
for muscle foods.
By using very high-frequency ultrasoundit is possible to achieve resolution
approaching optical microscopy. Acoustic microscopy has been used on occasion
with foods and other soft materials, for example, the detection
of sealworms
and bones in cod fillets (10).
224
A-scans
(time of second
marked echo). Good
data recovery
B-scan
(amplitude in
window)
Some data loss
111.
APPLICATIONS
A.
Simple Solutions
1. Binary Mixtures
One of the most successful groupof applications of ultrasound in food characterization is the determination of the composition of binary mixtures. The ultrasonic
velocity in ideal mixtures can be calculated as a volume-weighted sum of the
in Eq. (2). Nonideal behavior is an
density and adiabatic compressibility used
indication of association or segregation of components of the mixture and is difficult to predict a priori. So, a more practical approach to concentration determination is to prepare a standard calibration curve and use this for similar unknown
samples. Some typical velocity-concentration curves for common food materials
are shown in Fig. 4. Ultrasonic velocity measurements have been usedto measure
the solids concentration in fruit juices (1 1)and can easily be used to measure the
concentration of most two-component mixtures [e.g.. salt in brine (12), alcoholin
spirits (13), solids in skimmed milk (14)].
225
Ultrasonics
1650
"
1"
5
10
15
20
Concentration (weight%)
25
2. Ternary Mixtures
For many simple solutes (e.g., salts and sugars) thereis little temperature dependence in the concentration incrementof velocity, butin other cases (especially fats
and alcohols) the temperature increment is negative while that of water is positive (T < 76C). In the latter cases, at a critical temperature the speed of sound
in the solute is identical to that of the solvent, and velocity is independent of the
solutioncomposition.Thisproperty
is veryuseful in concentrationmeasurements. Consider solutes 1 and 2 , the former sharing a critical point T, with the
solvent; then cTC= f($z) and cT+TC= ($,, @).By developing two calibration curves
at the two measurement temperatures, it is possible to measure the composition
to measure alcohol
of a three-component system. This approach has been used
and solids in wine (13), fat and solidsin milk (14), and fat and proteinin fish (16).
In the absenceof a critical point, orif temperature is not variable, multicomponent
mixtures require additional nonultrasonic measurements for complete characterization; for example, Anton-Paar (Graz, Austria) have developed a nondestructive
method based on simultaneous velocity and density measurements.
B. Lipids
1. Liquid Oils
Food lipids are a mixture of various types of triacyl glycerols along with minor
components including cholesterol, mono- and diacyl glycerols, and vitamins. The
226
2. MeltingBehavior
The pure chemical components of food oils have a wide range of melting points
in the range commonly encountered during food processing, use, and storage. As
they are a mixture, the combinationof colligative properties and mutual solubility
means the observed melting behavior of food oil occurs over a wide temperature
range (20). The solids content of fatty foods is related to their perceived quality
(e.g., gloss in chocolate, stabilityof emulsions, textureof butter). Ultrasound can
be used to measure the volume fraction of solid fat mixed with liquid oil as the
velocity of sound is much less in liquids than in solids. A typical melting profile
for a sample of chocolate is shown in Fig. 5. The solid fat (SF) content can be
calculated as:
"_
cf ct
1200 LO
10 20 30 40 50 60
Temperature (OC)
Ultrasonics
227
where c is the measured ultrasonic velocity and c, (c,) the velocity in pure solid
fat (liquid oil) extrapolated to the measurement temperature. This equation was
developed from Eq. (2)
by assuming solid fats and liquid oils have similar density
and behave ideally as a mixture (21). Ultrasonic measurements give very similar
data to conventional methods such as NMR and DSC (22). This technique has
been applied to the measurement of solid fat in adipose tissue (21) and oil-inwater emulsions (22).
C. Polymers
Polymers and their aggregates play an important role in determining the stability
and textural characteristics of many foods, and there have been several attempts
of polymer
to use ultrasonic measurements to characterize the bulk properties
networks and the structure of isolated molecules in solution. The attenuation of
sound by a hydrocolbid solution is due to classical, scattering, and relaxational
(fast physicochemical reaction) losses. By measuring the attenuation overa wide
frequency range, it is possible to some extentto separate these effects and ascribe
changes in attenuation to molecular and scattering events. Unfortunately, the relaxations occur over a very wide frequency range and several instruments are
required to capture an entire spectrum. In oneof the most complete studies, Choi
and coworkers (23) measured the spectrum of bovine serum albumin from 0.11600 MHz at pH 1.5-13.2 using five techniques to cover the entire range. They
noted excess absorption at acid and alkali pH due to carboxyl and amino group
proton exchange reactions and structural fluctuationsin the molecule. Other studies using single or narrow frequency ranges for attenuation measurements are
less able to define which molecular events are causing the measured changes but
have had some empirical success.
Audebrand and coworkers (24) studied the gelation of alginate and amylose
by ultrasonic spectroscopy. While velocity was unchanged during gelation, attenuation increased in a manner similar to the real part of complex viscosity (G')
and, in the caseof amylose, turbidity. The time axis of the functions was different
to different molecular profor the three assays consistent with their sensitivity
cesses. Attenuation was shown to become more dependant on gelation at higher
frequencies (100 > 80 > 50 MHz). Small changesin 5 MHz velocity (-2 m.s")
were observed at temperatures close, but not identical, to the gel point of gellan
measured by a mechanical test (25). Measurements of velocity and attenuation
of a-amylase
at lower wavelengths (2 MHz) were used (26) to measure the action
on starch. The attenuation of the material decreased linearly with the number of
bonds broken, and this was ascribed to the (unmeasured) change
in viscosity;
measured velocity was unaffected by enzymatic action.
Coagulation of casein micelles to form a self-supporting network is a crucial stage in cheese manufacture. After a period of reaction, the cut-time, the
coagulum is cut and excess water allowed
to drain out. The cut-point is often
228
defined by the expertise of the cheese maker but can be defined as the time at
which the viscosity of the material sharply increases. Ay and Gunasekaran (27)
showed that the ultrasonic attenuation coefficient
a , at a frequency of 1 MHz,
of milk decreases at a decreasing rate with coagulation and the turning point of
a polynomial equation fitted to the measured data (daldt = 0) provided a similar
time to the accepted rheological method. Velocity showed no significant change
during coagulation, although there wasa very large variation in reported data (of
the orderof ? I O m.s) (28). In another studyof protein aggregation, the attenuation coefficient of solutions of broad bean legumin proteins reached a maximum
at pH values near the isoelectric point
( - 9 , probably because the proteins formed
aggregates that scattered the sound (29). This approach was also used to study
the effects of dextran on limiting the isoelectric precipitation of the same protein
(30).
In summary, it seems that high-frequency attenuation is most sensitive to
the state of food polymers and hencemany of their bulk properties. The velocity
of sound in polymer solutions is largely frequency independent and relatively
insensitive to aggregation.Velocitychangeshavebeenexploited
to a much
greater degree in measurements of individual molecular compressibility via Eq.
(2). The compressibility of a molecule in solution is measured as the change in
solution compressibility on adding one molecule of solute to pure solvent; in
practice this is achievedby extrapolating measurements made in a series of dilute
solutions. [It may also be possible to make measurements at higher concentrations, therefore requiring lower precision, if the scattering of sound is accounted
for (31).] The compressibility of a molecule in aqueous solution depends on (a)
its intrinsic compressibility and (b) the compressibility
of the associated water
molecules and is therefore very sensitiveto the hydration of polymers in solution.
The intrinsic compressibility of the protein (believed to be due to a cavity in
the molecular structure) is less than the surrounding water, while the bound surface water is less compressible than the bulk (32). This model gained support
from a molecular dynamics simulation (33), which further suggested that the
intrinsiccompressibility of the polymer (i.e., the cavity) was identical for
the two globular proteins studied (superoxide dismutase and lysozyme).
If this
observation is generally true for globular proteins, then ultrasonic measurements
can be used to directly measure their hydration state.
The surface hydrationof a protein is largely a measure of surface hydrophobicity; an important parameter governing the functionality of food proteins (34).
It is therefore unsurprising that compressibility correlates with protease susceptiof unfolding (35) and that there is a
bility, foaming capacity, and free energy
measurable change in compressibility on thermal or guanidine hydrochlorideinduced denaturation (32). However, empirical attempts to predict the compressof its constituent amino acids have met with
ibility of a protein from the properties
only limited success (36), suggesting that we are a long way from understanding
mechanistically which properties of a protein are measured by compressibility.
Ultrasonics
229
D. Dispersed Systems
Many foods are dispersed systems, importantly emulsions (e.g., mayonnaise, soft
of colloidal
drinks),foams (e.g., beer,carbonateddrinks),andcombinations
structures (e.g., bread dough, ice cream). When waves pass through the material
inhomogeneities, there is an interaction known as scattering, which is dependant
on the physicochemical properties of the two phases as well as their size, shape,
concentration,spatialdistribution,andthefrequencyoftheultrasoundused.
Some of the ultrasound is directed out of its path and so is not detected, and
some is lost as heat as the scattering is not completely efficient. Consequently,
the measured ultrasonic frequency spectra contain a relaxation, which can be
related back to the physical properties of the material under evaluation.
In certain
cases it is possible to understand the acoustic lossesin terms of various scattering
theories, but if this is not possible, empirical relationships may frequently be
developed.
1. Emulsions
Scattering of sound by emulsion droplets is relatively easy to solve analytically
as the particles are spherical, and their typical size (-pm) is much less than the
wavelength of the ultrasound (-mm), so the long-wavelength approximations to
scattering theory are applicable (37). Under these conditions, sound is scattered
by emulsion droplets by two important mechanisms:
I.
Thermalscattering: The particleandsurroundingmediumarecompressed to different extents by the wave, and the resulting temperature
difference causes a heat flux. Thermally scattered energy radiates in
all directions around the particle (Fig. 6).
2. Viscous scattering: The particle oscillates in the pressure gradient because it has a different density than the continuous phase (Fig.6). This
oscillation leads to the generation of a secondary wave by the particle
that has a cosine dependence on angle.In addition, the particle oscillation is damped by the viscosity of the surrounding liquid and some of
the ultrasound is lost as heat.
Neither mechanism is completely efficient, and there
is significant energy loss.
Using relatively few assumptions, it is possible to develop an analytical expression for scattering from a single particle.
The effect of finite concentrations of
particles can then be accounted for using scattering theory
(38) or a core-shell
a function of particle
model (39) to calculate the bulk ultrasonic properties as
of the component
size distribution and concentration and the physical properties*
phases.
* Viscoslty. density, thermal conductivity, thermal expansion coefficient, specific heat, and ultrasonic
velocity and attenuation.
230
Monopolar scattering
_".
"....___
Dipolar
scattering
Droplet
Fig. 6 Diagram illustrating the scattering of ultrasonic waves by emulsion droplets. The
main mechanisms are droplet pulsation due to differences in thermal properties with the
continuous phase and oscillation due to density differences. These mechanisms scatter
monopolar and dipolar waves respectively.
The bulk properties of common food are well documented in the literature
(40), so, for given ingredients, it is possible to predict the ultrasonic properties
of any size/concentration emulsion. Typical results for a model food emulsion
are shown in Fig. 7.
At all frequencies the velocity and attenuation are dependent
on concentrathe spectra upor down),but over a critical
tion (changing the concentration moves
range of frequencies there is also dependence on particle size. Therefore, using
n
-".
1460
0.03 a,
0
w.
0
,E 1450
0.02
C
0.01
.-0
5
C
0.00
B
a
-7 -6 -5 -4 -3 -2 -1 0 1 2 3
log df
Fig. 7 Velocity and attenuation ofa 10%corn oil in water emulsion ( his the wavelength
of the sound, other symbols are defined in the text). (Calculated as described in Ref. 38
using data from Ref. 40.)
Ultrasonics
231
high- or low-frequency measurementsit is possible to measure droplet concentration and use this value to measure size from measurements in the central region.
It would of course be preferable to record the entire spectrum and solve for size
and concentration simultaneously. Particle size measurements using either velocity or attenuation agree with laser diffraction scattering measurements
in pmin concentratedemulsions(volumefraction,
Q < 0.5)
sizedfoodemulsions
(41,42). This is a particularly important application of ultrasonic NDE, as the
information obtained cannot be readily measured by other methods. Commercial
instruments based on these principles are available from various suppliers.
An interesting extension of this method arose from one of its limitations.
When the scattered waves from one particle interact with those from another
(multiple scattering) less energy is lost. This occurs when the average particle
separation decreases to a critical level, either at high concentrations when the
measured attenuation increases less rapidly than the simple theory would predict
or in flocculated emulsions. Detection of flocculation in emulsions is particularly
of physical deteriorationof a product.
important, as it is frequently the initial stage
McClements (43) was able to detect flocculated emulsions with this method before they began to visibly cream.
When the particles are charged, there is additional attenuation due to ionic
friction between the moving particle and its counterions, which generates an
A full soluA/C voltage that can be measured alongside the acoustic attenuation.
tion of the viscous, thermal and electroacoustic scattering losses allows calculation of particle size and surface charge (c-potential) (44). This method showed
good agreement with previously published values for casein micelles
in skim
milk (45).
Ultrasonic imaging has been widely used to study the creaming of food
emulsions under gravity. In its simplest form this method merely relates the vea known pathlength as a function of time
locity for the sound to pass through
and position to the volume fraction (46), but by measuring the scattering effects
it should also be possible to determine size separation under gravity (47).
2. Foams
The concentration, size, and growth of air cells
in bread, fruit, dairy products,
beers, and wines are vital to the perceived quality of these products. Ultrasound
is very sensitive to dispersed gases and would seem an ideal investigative tool,
but in practice the attenuation due to the resonant scattering of the bubbles is so
strong that transmission measurements are not possible at high frequencies
(>O. 1
MHz). Measurement of the surface reflection coefficient is a more practical apa
proach to capturing the important frequency dependence of scattering from
bubbly liquid, and the potential of this method has been demonstrated for some
whipped food materials (48).
232
E. Muscle Foods
Ultrasound has been used for a number of years to measure the fat content of
various live animals and carcasses. Such whole animal studies are beyond the
scope of this work, but the principle
of the measurement is similar to the unknown
pathlength pulse-echo method described above (Fig. 2d). A transducer is
held
against the back of the animal and a pulse of sound fired through the surface
layer and an echo recorded from the fat/muscle interface. The fat thickness corre(49-51).
lates with overall fat content and other carcass and meat properties
Alternatively, pattern recognition techniques developed for medical imaging devices may be used (52,53).
Another imaging method that has seen some success
in imaging muscle
foods is elastography(54). In this method, an A-scan is recorded before and after
the material is slightly compressed
by the transducer. Pressing the transducer into
the material causes the material to be deformed, the softer materials more than
the harder, and the relative movements of the peaks can be tracked by crosscorrelation techniques. In this method isitpossible to get imaging across a plane
in the material based on Youngs modulus. Ophir and coworkers(54) have used
this approach to distinguish between fibrous muscle and perimysial tissue and to
visualize a healed traumatic injury in beef samples. By calibrating the analysis
with material of known properties, it is possible to make absolute measurements
of Youngs modulus. A typical elastography image of a meat sample is shown
in Fig. 8, in which the light bands represent bands of collagen and the dark areas
fibrous muscle.
Fig. 8 Elastographic image of beef muscle; differentiation is based on the elastic modulus of the material. The white areas represent connective tissue and the dark myofibrillar
muscle. (Image kindly donated by Rhonda Miller, Texas A&M University.)
Ultrasonics
233
(58).
F. Plant Foods
The ripening and deterioration of vegetable products is associated with changes
in chemical composition and mechanical properties that might reasonably be expected to change the ultrasonic properties of the material. However, the correlations between ultrasonic and quality parameters are often weak for fruits and
vegetables because the theoretical link between acoustic propagation and strucof
ture is poorly understood. The acoustic properties are an unknown function
vegetable material properties including size concentration and distribution of air
cells, the cytoplasmic composition, the mechanical properties
of the cell walls,
and the intercellular bonds, while the perceived quality (e.g.,flavor, crunchiness)
is probably a very different function of physicochemical structure. Methods that
more closely mimic the consumers use of a food (e.g., compressional tests, GC
analysis of volatiles) are likely to correlate better with perceived quality, but
because these are inevitably destructive, acoustic methods have been frequently
considered. A good example of the limitation of acoustic measurements was seen
in recent (59) measurementsof the velocity, attenuation(37 kHz), and other properties of carrots cooked for different times (0-15 rnin). Measured velocity decreased linearly with strain at failure, Youngs modulus, solids content, and density, butthecorrelationswereweak
(r = -0.69,-0.62,-0.46,and-0.29
234
respectively), while attenuation showed still weaker positive and negative correlations with the same parameters.
Practically, vegetables are difficult to measure because not only are they
irregular in shape and variable in composition, but they also frequently contain
intercellular air cells, which scatter ultrasound and cause unmeasurably high attenuation at high (->20 kHz) frequencies (60). Adequate transmission measurements are possibleat lower frequencies, butthis is not ideal as there is less spatial
resolution and beam spreading and wave-guide effects can reduce the precision.
in the
Despite these limitations, many researchers have reported some success
ultrasonic NDE of plant foods. Cheng and Haugh (61) identified whole potatoes
with hollow heartas they absorbed more low frequency (0-75 kHz) sound energy
than the healthy samples. The very-low frequency sonic resonance (<1 kHz) of
whole apples decreased monotonically during the accelerated ripening process
in a similar manner to texture measured bya puncture test (62). Self and coworkers used low-frequency ultrasonicsto follow ripening in banana (60) and avocado
(63) and to determine the elastic modulus of apple tissue (64).
G. Miscellaneous
One of the very few concerted uses
of shear wave ultrasound in food systems
was made by Lee and coworkers (65), who made measurements on cheese and
dough (0.5-1 MHz) and comparedthe results with conventional oscillatory rheological measurements. Both devices showed similar trends in complex rheological properties with composition, but because the devices operated over different
frequency ranges, quantitative comparisons could not be made. The success
of
thisstudy is surprising, because ultrasonic waves
of this frequency would be
expected to be very highly attenuated in a gas-containing mixture such as dough
and shear waves would not be expected to propagate in fluid media.
The time of flight for a 1.25 MHz ultrasonic pulse across a pipe was used
to detect fouling at the pipe surface (66).
The sensor could measurefilms between
0.5 and 6 mm thick, the lower limit being set by the wavelength
of the ultrasound
and the upper limit by the acoustic impedance
of the materials. When implemented in a pilot-scale UHT milk plant, results were unaffected by flow rate (0I O L/min).
The crispness of several types of cookies and crackers as measured by a
sensory panel and fracture testing correlated with ultrasonic velocity (67). Ultrasonic absorption has been used to measure the texture of wafer sheets (68).
There have been some attempts at using ultrasonic imaging to detect the
growth of spoilage organisms in packaged milk (69) and other liquid foods (70).
Good images could be recorded through plastic and metal packaging materials
(paper contains air cells that strongly attenuate the sound) (70), and the speckle
density could be correlated
to conventional plate counts. However, only very
Ultrasonics
235
IV. FUTURETRENDS
A survey of the literature in this field reveals more than 40 years of ultrasonic
(71,72). This
methods of foodcharacterizationbasedonultrasonicprinciples
concerted effort has produced remarkably few practical applications, most importantly on-line concentration meters, carcass evaluation tools, and, most recently,
emulsion particle-sizing instruments. This developmental effort must be better
focused to meet actual rather than perceived needsin the food industry. In developing a method it is important to remember the strengths and weaknesses of
a given technique as summarized for ultrasonics in this work. It is also worth
remembering that a new method will not be adopted based on its novelty alone;
it must provide information that is not readily attainable by existing technology
or be in some way more practical (e.g., cheaper, easier
to use). Additionally, food
a measurement device must maintain
is an intrinsically variable material, and
sensitivity to the important variable (e.g., crunchiness in apples) while ignoring
changes in other variables (e.g., variety, size, insect damage). Food-processing
plants are rarely static, single-product operations, and the sensor must be flexible
enough to account for this. For example, a juice-bottling plant may switch bea day, and
tween several product types over different lines over the course of
any analytical controls must be equally easy to recalibrate or their potential for
improved quality and production times will be
lost in the inconvenience of operation.Bearingthesepoints
in mind,wewouldliketosuggestsomeareaswe
believeultrasound-basedinstrumentationcouldbeused
in thenondestructive
evaluation of foods:
236
Ultrasonics
237
GLOSSARY
Attenuation coefficient (a):The loss of acoustic energy per unit distance.
Couplant: Ultrasound is strongly attenuated in air and a liquid couplant (often
water of a proprietary gel) is needed to facilitate the transmission between
the transducer and sample.
Delay line: Apiece of material(frequentlyPlexiglasorquartz)designed
to
separate the large signal from the transducers
initial ring from later measurable echoes.
Dispersed system: A material that is inhomogeneous supramolecular scale but
homogeneous in bulk. Includes emulsions and foams.
Elastography: An imaging system based on the change in detected signal on
bulk deformation.
Emulsion: Aliquid-in-liquiddispersedsystem.
Foam: Agas-in-liquiddispersedsystem.
Frequency: The reciprocal of thetimerequiredforawave
to complete on
cycle. The frequencyof ultrasonic waves usedin food analysisis commonly
in the 10hs" region.
Imaging: Use of the spatial dependence of an ultrasonic signal to develop an
image of internal structure.
Impedance: A measure of the capacity of a material to transmit sound. Commonly measured as the product of density and ultrasonic velocity.
Resonator: An ultrasonic measurement system where the properties of interest
are measured from the constructive and destructive interference
of a continuous wave with itself.
Scattering: The process by which the direction
of transmission of ultrasonic
energy is changed by interaction with microscopic inhomogeneities.
Transducer: Mechanical device to convert electrical to mechanical (ultrasonic)
energy or vice versa.
Ultrasound: Mechanical vibrations similar to sound but at frequencies far beyond the range o f human hearing (>20 kHz)
Velocity (c): The speed of sound in the direction of transmission or detection.
Wavelength: The distance between points of equal amplitude on a wave. Ultrasonic waves used in food characterization typically have a wavelength of
the order of a millimeter.
238
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7
Firmness-Measurement Methods
Yen-Con Hung, Stan Prussia, and Gabriel 0.1. Ezeike
The University of Georgia, Griffin, Georgia
1.
INTRODUCTION
A.
DefiningTexture
B.
Importance of TexturelFirmnessEvaluation
Food texture is understood to be the sensory manifestation of kinesthetic properties (6) and depends on particle size, shape, structure, and rheological parameters
( 5 ) . The quality and acceptability of any food are influenced considerably by
textural and structural properties normally associated with it. In relation to food
quality, the importance of texture can be best demonstrated by food product advertisements. Some words that have been used to describe desirable textural quality include crisp, crunchy, tender, juicy, creamy, firm, spongy, and smooth. Conversely, some descriptors that have been usedto characterize undesirable textural
quality include soggy, sticky, gummy, soft, fibrous, tough. and dry.
243
Hung et al.
244
Texture is also one of the key factors in determining harvest date and can
of foods (7). For example, Washalso be used for determining the market grades
(8) requirethatRedDeliciousapplesatthetime
of
ingtonStateStandards
shipping have firmness values
of at least 53 N (12 Ibf) as determined by the
Magness-Taylor (MT) puncture test. Texture has also been used as an indicator
to crackers) also
for freshness of seafood (9). Processed foods (from hotdogs
need to maintain certain well-defined textural quality to meet consumer expectations. In manufactured foods, texture has also been used as an indicator for the
consistency of products.
as measuring
Bourne (10) summarized texture-measurement instruments
either force, distance, time, energy, multiple units, or chemical composition. An
overview was also provided by Giese (1 1) on different food texture-measuring
methods. However, there are more textural parameters than can possibly be covered in detail in one chapter. Firmness is a popular term for hardness ( 5 ) and has
been described as the force necessary to attain a given deformation. Firmness
has also been used as an indication of quality as well as a parameter for sorting
operations. For the remainder of this chapter, the discussion is focused on firmness.
C.Overview
of Firmness-EvaluationMethods
245
Firmness-Measurement Methods
Sensory
(Subjective, methodolog ,
psychology, physlologyr
Instrumental
(Objective)
Destructive
(Llmited sample size)
Nondestructive
(On-line, more samples)
Acoustic
Human
I Judgment I
netic resonance (NMR), etc.Dull ( I 5) defined the term nondestructive to indicate that the sample (of any physical form) can be analyzed to obtain meaningful
of
data by some means in such a way that the physical and chemical attributes
the sample are not altered. This means that the sample, after testing, can be subjected to subsequent uses or activities such as food manufacture, handling, storage, or direct consumption without loss.
After considering the various approaches used for categorizing firmness,
we have decided on two broad categories: methods having mechanical contact
and those having no mechanical contact with the food.
II. MECHANICALCONTACTWITHPRODUCT
A.
DestructiveFirmnessTests
The standard measure of food firmness is conducted with a penetrometer developed by Magness and Taylor (16). It is a mechanically driven probe used to
penetrate the food products with the maximum required penetration force giving
an indication of firmness. In addition to the Magness-Taylor penetrometer, Studman and Yuwana ( 1 7) presented a new firmness-measurement method based on
the moment necessary to rotate a blade attached to a spindle after it has pushed
246
Hung et al.
into the fruit. After the blade has been pushed into the fruit to the desired depth,
the fruit is slowly rotated manually about the axis of the spindle. When the fruit
tissue fails, the maximum angle of rotation is read and the value is used to determine firmness. The resistance to the moment is assumed to be due entirely to
the crushing of the cells by compression and shear. This device requires less than
IS seconds to complete and record each measurement, is able to take measurements at specified depths, and is able to sense a wide range of fruit firmness by
using different sizes of blades for firm (smaller blade) and soft (larger blade)
fruits. There is no need to clean the device between tests, and no need to remove
the fruits skin.
Another modified force-deformation method was investigated by Murillo
et al. ( I 8) using peaches. A cylindrical plug measuring 1.5-2.0 cm long was cut
from each peach 2-3 mm below the skin surface on the blush side using a 2-3
cm (internal diameter) cork borer. Using the Instron Universal Testing machine
with a load cell of 50 kg at cross head speed of 20 mm/min. the specimens were
cut axially through the plug with a 1.36 mm diameter wire probe, and a steadystate force was noted to represent firmness.
The Magness-Taylor methodhas a key advantage of many years of history
that have led to its use as a standard against which other methods are compared
in order to determinetheiracceptability.However,the
main disadvantage of
methods based on this principle is that the probe test is destructive to the product
being measured and only provides test results from a sample that
is used to represent the lot. Therefore, this method cannot beused to measure firmness of every
item going through a packing or processing line.
B. NondestructiveForceMethods
Destructive tests for firmness continue mostly because suitable sensors are not
available for nondestructively measuring firmness
of all items in a lot. Consequently. considerable effort has been made for finding a nondestructive method.
The specific principles employed in many of the instrumental methods are nearly
as varied as the products.
1. Based on Human Touch
As indicated before. manual separation/sortingis the only practical method available now for firmness sorting of foods. Hung et al. (19) used 100 untrained consumers to rank five peaches with different firmnesson a hedonic scale from most
firm, very firm, firm, less firm, to least firm by touching the peaches. A total of
160 freshly harvested peaches werefirst ranked by an expert judge into five firmby 100 untrained
nessgroups with 32 peachespergroupandthenevaluated
Firmness-Measurement Methods
247
2. Based onForce-Deformation
Mechanical force-deformation is the conventional means of evaluating the firmness of fruits and vegetables.The instruments measure either force or deformation
or both (dynamic force-deformation). For nondestructive firmness evaluation, the
force applied needs to be at a level that results in no damage to the product.
Mehlschau et al. (20) measured the maturity of pears nondestructively using two
19 mm diameter steel balls to apply a constant force against the opposite sides
of the fruit.
A portable device developed by Timm et al. (21) employed two parallel
surfaces to slightly compress the fruit to measure the firmness of sweet and tart
cherries, blueberries, and strawberries. The device was mounted on an aluminum
frame on which a stepping motor (incremented 0.1 mm per pulse at a constant
rate of 8.85 m/s) was used to control speed. A computer was used to control the
stepping motor and to acquire data from a load cell.
it
Mizrach et al. (22) developed a mechanical thumb device and used
to measure force-deformation of tomatoes and oranges. The method is based on
creating a slight elastic deformation of the fruit peel using a spring-loaded pin
3 mm in diameter. Two models of this device were tested, one operated
in a
go-no-go mode in which a fruit peel deformed as the pin penetrated into the
fruit and caused the movement of a pivot arm against a spring adjustedby a load
presetscrew. An adjustable small movement (e.g.. 0.2
mm) of the pivot arm
is adactivates a microswitch, thus preventing damage to the fruit. The spring
justed to a certain cut-off stiffness value that is equivalent to the stiffness of a
fruit of desiredfirmness.Fruit is consideredfirmwhentherecordedforce
is
greater than the predetermined spring load. The other mode of the device allows
continuous deformation of the peel with a pin connected to a flat spring to which
a strain gage is bonded. The deflection of the flat spring under load indicated
fruitfirmness,withfirmerfruitsshowinghigherdeflection
of thespringand
higher output voltage from the strain gage.
The two sensing devices described
above were placed on a sorting line and tested with oranges and tomato fruits
248
Hung et al.
with good results. The on-line system was implemented onan endless conveyor,
which moved the fruits towards the sensing
device attached to a pivot arm that
was free to move up or down depending on the fruit size.
Takao and Ohmori (23) implemented a similar concept an
foroff-line computer-automated system using either deformation for a preset force or maximum
on kiwifruit and melon.
force for a preset deformation. Tests were conducted
They used different compressing plunger shapes and diameters to accommodate
different fruit shapes and diameters, respectively. The plunger speed was set
at
50 mm/min and the plunger diameter ranged from 8 to 12 mm.
to nondestructively
Botta (24) used a unique force-deformation concept
measure the firmness (and resilience) of Atlantic cod fillets, which could also be
adapted for fruits and vegetables. A portable instrument was developed to apply
a force of 100 mN at 20 mm/s to just touch the surface of the product placed
on a rigid flat plane, using a 2.5 cm diameter probe.The force was then increased
Di) the prodto 4.9 N, and the probe was made to depress (deformation distance,
uct for one second. The force was then removed and the product was allowed
D,, was measured. A texture
to rebound for one second, and the rebound distance,
(firmness and resilience) index (D,/D,) was then calculated.
Davie et al. (25) used a digital micrometer for nondestructive measurement
of kiwifruit firmness. A 5 mm diameter hemisphere probe was attached near the
end of a horizontal lever such that it could depress a fruit placed under it. The
mass of the lever and its assembly on the product side are counterbalanced using
two weights (0.55 kg for coarse adjustment and 0.031 kg for
fine adjustment)
attached to metal rectangular channel made to slide along the part of the lever
on the opposite side of the cross bar. A digital micrometer was mounted between
the probe and the central pivot (micrometer spindle was 217 mm from the central
pivot and 63 mm from the probe), with its tip touching the top surface
of the
lever. The resting forceof the probe on the fruit surfacewas adjusted to a slightly
positive value using the coarse andfine weights. After zeroingof the micrometer,
a mass of 0.1 kg was applied on the probe manually and the deformation was
(SC) was calcurecorded every 5 seconds for 30 seconds. The softness coefficient
lated as the slope of deformation versus natural logarithm of time. Tests with 28
fruits were correlated with Effegi penetrometer firmness (f) using the following
equation:
where k, and k2 were coefficients.In a second prototype, they (25) used a digital
micrometer fitted with a motorized system to raise or lower the probe ( 5 mm
diameter hemisphere). After contacting the fruit surface, the micrometer was preset to zero and a mass of 0.1 kg was applied. A computer and data logger were
Methods
Firmness-Measurement
249
used to acquire a burst of three micrometer deformation readings per second for
5 seconds.
Dawson (26) described a nondestructive firmness tester for kiwifruit based
on the displacementof a small mass pushed against the fruit by
a spring. A device
for the measurement of berry firmness was reported by Armstrong et al. (27). A
230 mm diameter turntable that had 25 spherical indentations was driven
by a
stepper motor with precise 0.13 mm/step. A fruit wasplaced into each indenture
that had a small hole in the center by which a vacuum was applied to ensure the
to the stepper motor had a plate by
berry did not move. A load cell connected
which compression was applied to the fruit and the load cell signal after amplification was digitally convertedby a data acquisition system. All 25 measurements
could be completed in one minute.
All of the above-discussed methods directly measure the force and the corresponding deformation. However, Abbott and Massie (28) developed a system
to apply compressive force dynamically to apple and used the dynamic forcedeformation data in the range of 40-440 Hz to obtain a predictor of firmness.
A 1.25 cm diameter force transducer was statically loaded against the apple with
8.8 N. A two-channel dynamic signal analyzer generateda sine wave sweep(40440 Hz), which drove an electrodynamic vibrator through an amplifier. A signal
analyzerthencapturedthetimedomainsignalsfrombothforcetransducers
attached to an accelerometer, and the frequency response (ratio of output (force)
to input (accelerometer)) was used to calculate dynamic force/deformation from
each test.
The main advantage of force-deformation methods is that results give a
direct measure of product firmness. However, most direct contact methods have
limited speed and require mechanical contact with the product, thus increasing
the chances of cross-contamination of the bulk.
3. Based on Impact
Two general concepts havebeen adopted by different researchers, who have measured the firmness of fruits and vegetables
by the impact phenomenon. In the
first mode, the fruit is allowed to fall through space and impact a rigid surface,
often fitted with a force transducer. In the second mode, an impacter is made to
strike the product. Variations of these two concepts have also been explored by
also found that the impact of a fruit
some researchers. Many researchers have
on a rigid surface can be closely modeled by the impact of an elastic sphere and
that the firmness of a fruit could be obtained based on the impact force response.
Delwiche et al. (29) sorted peaches and pears into hard, firm, and soft categories by analyzing the force from the fruit impacting a plate supported by force
a rigid steel
transducers. The impact force was measured when fruit impacted
plate measuring 50 mm X 50 mm X 9 m m to which a piezoelectric force trans-
250
Hung et al.
ducer was attached. The transducer output was sampled at 1 1.62 kHz by a spectrum analyzer.
Technological advances have enabled greater ease in acquiring, analyzing,
and modeling of impact data required for improved sorting systems. Lichtensteiger et al.(30)investigatedimpactresponseforsortingtomatoesandother
to controlthe
sphericalviscoelasticobjects by incorporatingavacuumpump
release of a sample onto an aluminum plate (3.75 cm
in diameter and 0.32 cm
thick) glued to the top of the force transducer to increase the impact area and to
lower the natural frequency from 70 to 10.5 kHz.
Rigney et al. (31) developed an experimental machine to orient peaches
for on-line quality measurement as
an aid to more consistent results where a
precise presentation of the fruit is needed. Also, to overcome the variability aris(32) developed
ing from the influenceof angle and location of impact, Ozer et al.
a method based on multiple impacts. In their extensive study of nondestructive
firmness measurement of cantaloupes, Ozer et al. (32) used a drop test from a
fixed height of 2 cm to an impact surface. The impact response data of the fruit
was captured by a force transducer at a sampling rate of 10 kHz. The same fruits
were subjected to four multiple impact tests starting at the groundspot and moving
along the equator to locate two other points, and the fourth was at the blossom
end.Foreachimpact,theimpactparameter,
FT, wascalculated to represent
firmness:
where P, is the amplitude of the impact response (V), T,, is the starting time, and
T,, is the end time of the impact, s.
Theconcept of multipleimpacts on thesamefruitwasautomated
by
on afruitfirmnessgraderforkiwifruit(Softsort
SchaareandMcGlone(33)
grader) based on four impact stages on specially designed concave steps as the
into the receiving
fruit tumbled down an inclined chute. Unsorted fruit was fed
conveyor, which moved the fruits through a single lane over inspection rollers
where an operator removed fruit with external defects. The whole (unblemished)
fruit thentumbleddownaninclinedchutecomprisingfourseparateconcave
steps, each vibration-isolated (with sheets of rubber foam) from the grader frame.
Each concave step served as an impact sensor formed by folding an aluminum
sheet over a heavy steel base.A piezoelectric film sensor was glued to the underside of the aluminum step to sense the flexure of the aluminum during fruit impacts. The fruits were released at intervals to ensure that only one fruit was on
the sensor at one time. The voltage producedby the piezoelectric film as the fruit
impacts on a sensor is amplified and processed by a microcontroller attached to
each of the four sensors. The four microcontrollers transmit firmness measurements to a dedicated microcontroller, which runs the grader.
Methods
Firmness-Measurement
251
Bajema and Hyde (34) provided a guide for determining the suitability of
an impact-based nondestructive method for fruits and vegetables. They developed
an instrumented pendulum (3 m high) to study impact phenomena. The device
was equipped to record force and contact area profiles during impact at the rate
of I O kHz onto a flat rigid anvil. The anvil was fitted with a force transducer
and with a contact areasensor. Two precisely spaced infrared beams (beam interrupted by the sample) allowed approach and rebound velocities to be measured.
An accelerometer attached to the trailing end of the product measured deceleration during impact and free vibrations during rebound. The impact profile data
were sampled at I O kHz per channel. Using the setup, they developed schemes
for impact sensitivity for fruits and vegetables. Operating procedures for single
impacts, constant height multiple impacts (for determining bruise resistance), and
(to determine bruise threshold height) were
increasing height multiple impact
discussed.
Chen et al. (35) used an impact-testing system fitted with an accelerometer
and electromagnet and a computer-based data acquisition system to measure the
firmness of pears. Based on the theoryof elasticity, they determined that the time
(t) to reach peak force was given by
DIV
t =
1.47
(3)
of approach
A = Flm,
where F is the peak impact force and m, is the mass of the impactor. The impact
force F depends on impact velocity V, mass of the impactor, mass of the fruit,
fruit modulus of elasticity, Poisons ratio of fruit (0.49), radii of curvature of
fruit (35 mm), and impactor (9.5 mm). The firmness of the tested product was
defined as the ratio A h .
The impact method can be high speed. and some studies have demonstrated
good firmness sorting results; e.g., Chen et al. (35) achieved good sorting ability
on a prototype packing line
by measuring the deceleration of a low mass impactor.
However, a major problem associated with the measurement of firmness by the
impact principle is the uncertainty of the angle and location of the impact on the
fruit surface, especially when such surfaces are irregular.
4. Based on Impact-Rebound
In the preceding section, it was shown how some researchers have used the
reit, to directly measure firmness
sponse of a product to an impact either on, or from
of the product. A unique variantof this principle utilizes the characteristic trajec-
252
Hung et al.
where 0, is the number of onions in product exit, 0, the number of onions rejected, C, the number of clods in reject exit, andC , the number of clods remaining
with product. They also used a similar system to separate clods from potatoes
(38).
Although the impact-rebound method has the advantage of high capacity,
it is unsuitable for products having a wide variation in shape. In addition, it is
unsuitable for delicate products.
5. BasedonVibrationalCharacteristics
The vibrational characteristics of fruits and vegetables depend on their elastic
modulus (firmness), mass, and geometry.A form of impact-rebound is generated
when fruits are placed on a vibrating surface, and this principle has been studied
as a means of sensing firmness. In a review of nondestructive firmness measurement, Chen and Sun (12) found that prototype equipment had been built by Bower
and Rohrbach (39) for sorting blueberries and grapes by low-frequency vibration
(200 Hz). The fruit was placed in an L-shaped trough, one side of which was
vibrated at a fixed frequency and amplitude of 200 Hz and 0.3 mm, respectively.
Firm fruit bounced outof the vibrating trough and intoa collecting trough, while
softer fruit was conveyed along the trough by the vibration.
The problem of mechanical contact between the fruit and sensor appears
to have beenaddressed in a recentreport (40) on a uniquecommercialfruit
firmness sorter for apples, nectarines, and kiwifruit. The machine is comprised
of the fruit
of a conveying system, which allows nondestructive physical contact
ethods
Firmness-Measurement
253
and a sensor finger while the fruit was on-the-go at speeds of 2.5-7.5 fruits/
s per lane. An electrodynamic shaker wasused to vibrationally excite the bottom
part of the fruit. The root mean square (RMS) of the input signal was measured
in the shaker head while the RMS
of the output signal (fruit response) was picked
up with a miniature accelerometer attached to the sensor finger contacting the
top of the fruit. A firmness index (PFT) was defined as
PFT = X,/(X,,
X,)
where X,, and X, were the input and output RMS signals. Larger PFT indicated
firmer fruits, because softer fruits tended to attenuate the input vibrational energy,
while firm fruits pass a larger portion of the input vibrational energy.
Vibrational methods have the advantage of firmness evaluations based on
a response from the whole item. However, they can be limited by the influence
of geometrical differences, such as size. There is also therisk of stimulating
whole-fruit softening damage, depending on the regime of vibrational frequency
and amplitude employed. Also, a technical problem that needs to be solved is
theabilitytomaintaingoodmechanicalcontactbetweentheproductandthe
sensor, since this is necessary to faithfully transfer the product response signal
to the sensor.
254
Hung et ai.
ated by light-emitting diodes placed about 6.35 mm above the sensor. Firmness
was determined by the change in electrical resistance.
The main advantage of the soft touch methodis that it is intrinsically nondestructive. However, it is slow and difficult to integrate on-line, and in addition,
different product size may affect accuracy.
C. NondestructiveMethodsBasedonSecondary
Properties
Themeasurement of secondaryproperties (not force-deformation)greatlyincreases the number of possible approaches. Most of the research has been on
resonant frequencies, acoustic response, and ultrasonic methods. A major advantage of firmness methods based on secondary properties is that they evaluate the
whole product. Some applications can also utilize remote sensing with no physical contact with the product. However, general limitations include the fact that
some of the properties may not phenomenally relate to firmness, and i n some
methods product size may affect the readings. Also, in some applications the cost
of equipment may be high.
The challenge with the firmness-sensing methods using secondary properties is either to develop devices that are independently accurate or to develop
methods having very high correlations with the results obtained with force-deformation relationships of the product that are important to buyers and end users.
Also, the speed of methods that require mechanical contact or signal processing
A technical complication
should be addressed for on-line sorting applications.
of NMR is the need to avoid metallic objects near the vicinity of the sensor.
Methods
Firmness-Measurement
255
where id is the number of data points, t, ( = 14.2 ps) is the sampling period (70
kHz sampling/channel), and 0.5 was added t o account for the difference in sampling time between the two channels caused by the multiplexer of the A/D converter. Sound transmission velocity (V,l) was expressed as;
Vd = d/t,
where d is the distance between the microphones. The average of five measurements of Vd from different locations on each fruit was determined and used as
the nondestructive firmness.
Hung et al.
256
where f, and H, are the local values of frequency and amplitude of the signal,
respectively, and n is the total number of data points. The meritof this parameter
is that it does not depend on the specific resonant frequency, but uses the entire
spectrum of frequency response.
Mizrach et al. (50) measured nondestructive firmness of mango fruit by
A high-power, lowmeans of ultrasonic probes in contact with the fruit peel.
frequency ultrasonic pulser-receiver, a pairof 50 kHz ultrasonic transducers, and
a microcomputer and data acquisition system were used to generate the signal and
to collect data. The ultrasonic head with a transmitter-receiver system allowed
transmission and reception of ultrasonic signals, which passed through the peel
and the fruit tissue next to the peel. Exponential-type plexiglass beam-focusing
elements were used to reduce the beam diameter of each transducer on the fruit.
(A, dB/
They found that firmness (FR, N) can be calculated from attenuation
mm) as
FR = 48.21A2 - 408.71A
+ 878.16
(10)
3. Other Methods
Recently, nondestructive texture measurement of kiwifruit, pear, and peaches by
(LDV) hasbeenreported
(53). Individualfruitwas
laserdopplervibrometer
placed on a vibration generator, and a small amount of clay was applied directly
between the fruit and vibrator stageto ensure integrity of vibration transmission.
The vibrator generated inputs to the fruit in the frequency range of 5-2000 Hz
at 0.5 Hz increments. A laser beam reflected from the fruit surface to the sensor
head was used to measure the vibration at the upper surface of the fruit. A fast
to identify the phase shift between
Fourier transform (FFT) analyzer was used
ethods
Firmness-Measurement
257
the input and output of the vibrational signal. A phase shift at either 1200
or
1600 Hz was used to indicate firmness. Also, Muramatsu et al.
(54)used LDV
to measure the firmness
of kiwifruit and apples. They found that using the accelerFFT
ation output as reference signal gave more accurate results than using a
output signal as in their earlier study.
Some research has been done relating firmness to NMR data
of fruit. NMR
methods are based on a processof magnetic relaxation following external excitation of a fruit. The signal induced in the magnetic coil decays exponentially with
time. The relaxation data could be expressed in two forms, butmore
the important
mechanism is known as transverse relaxation (55).
In a recent review article, Clark et al. (56) gave an overview of magnetic
resonance imaging (MRI), including NMR applications in postharvest handling
and quality evaluation of fruits and vegetables. Kim et al. (55) investigated the
feasibility of using magnetic resonance imaging
to determine the firmness of
tomato. The principle of these techniques is based on many proton-containing
species, in particular hydrogen atoms in fruits and vegetables, which are capable
of absorbing radio frequency energy.In general, fruits were subjectedto magnetic
to decay exporesonance, and the signal induced in the receiver coil was allowed
nentially with respect to time. The relaxation allowed the spins to return to their
equilibrium levels, releasing their surplus energy into the surroundings.
The spectral data can be analyzed by two relaxation mechanisms. Longitudinal relaxation
(spin lattice relaxation) was expressed by (55) as
where, MY is the initial amount of longitudinal magnetization, M(t), is the magnetic moment at a certain time t, and T , is the spin-lattice time constant. Transverse relaxation (spin-spin relaxation) is given by
Hung et al.
258
D. FactorsAffectingFirmnessMeasurement
1.
Temperature
where, f , and f2 are firmness at T, and T2, the lowest and highest temperatures,
respectively. Jeffery and Banks (60) adopted this concept and obtained equations
to evaluate the effect of temperature on the firmness of kiwifruit measured destructively at 0 and 20C. They also found that fruit origins had no effect on the
relations between KITand f , or f2, suggesting
that within thescope of experimental
to be used for fruits from a variety of
error, the models may be robust enough
sources. In addition,temperaturesbelowzeromayyieldfirmnessvalues
that
differ significantly from those measured
at higher temperatures. Jackman and
rigidity of refrigerated
Stanley (61) found that the increased shear strength and
tomatoes may be dueto a chilling injury mechanism in which the cell wall structure binds together firmly.
Firmness-Measurement Methods
259
The shell effect was also found by Ezeike and Otten (62) in cotyledonous
seeds where an air layer separates the outer cotyledon from the seed. Abbott and
Lu (63) found that the stress, strain, energy at failure, and Youngs modulus of
apples varied with orientation at point
of measurement and location within the
fruit. Youngs modulus was higher in radial samples than in tangential and vertical samples. They also found that fruit variety was a contributing factor. For
example,DeliciousapplesshowedgreatervariabilitythanGoldenandRome
apples. Similar findings were reported by Maness and Brusewitz (64), who mea8
sured peach firmness using an Effegi penetrometer equipped with a standard
mm diameter probe, on samples taken from inner, middle, and outer regions of
the mesocarp at four angular positions around each peach half. Inner mesocarp
was firmer than outer mesocarp. Variations were also found between middle and
outer regions of the mesocarp and were cultivar dependent. Firmness was found
to decrease longitudinally from the stem endto the blossom end and latitudinally
from the suture to the cheeks.
Weexpect that firmnessmethodsbased on sensingtheresponse of the
outermost layers of fruit may be subject to the shell effect if the fruit has a structure with distinct outer and inner tissues. On the other hand, firmness methods
that excite the whole fruit (e.g., acoustic methods) are less subject
to the shell
effect.
3. OtherFactors
In their study of effects of mass and drop heighton kiwifruit firmness by impact,
(a measure of impact
McGlone et al. (65) developed equations for dwell time
duration) and peak force and found that the firmness parameter Cz (= f&, f,, is
the peak force and t, the dwell time) was independent of mass of the fruit. They
also found that fruit was not damaged even with repeated impacts
at 50 mm. Chen
and De Baerdemaeker (66) conducted studies with apples and Litchtensteiger et
al. (30) with spherical viscoelastic objects and tomatoes to optimize the parameters involved in impact firmness testing. These parameters also affect the performance of different firmness methods, which include impactor mass, fruit mass,
internaUexternal properties, maturity/damping, calculation method,
initial contact velocity, flat contact surface, elasticity of material, and stiffness factor.
111.
NO MECHANICALCONTACTWITHPRODUCT
A.
Concept
260
Hung et al.
B. Original Design
Extensive tests with the first prototype verified the validity of the laser air-puff
in its accuracy and repeatability for
a wide
concept and provided confidence
range of fruits, vegetables,and other products (68). Compressed air was supplied
through a pressure regulator to a tank located directly over pilot
a
actuated valve
with an attached 9.5 mm diameter nozzle (Fig. 2). A laser displacement sensor
was mounted at a 30" angle to the nozzle and aimed at the top of the product
that was supported under the nozzle in a specially designed holder with vertical
adjustment. A timer circuit controlled the duration of the puff (valve opening)
after a switch activated the circuit. The amount
of deformation caused by the
compressed air was measured using a laser displacement sensor and recorded
with a digital oscilloscope.
A second pressure regulator on the main air supply assured that consistent
pressure was delivered to the pilot side of the pilot actuated valve (ensuring repeatable speed of opening). A large tank (8 L) was used to minimize the drop
in pressure while the valve was open for 200 ms to produce the puff of air. The
laser displacement sensor (Keyence model LB 041) was mounted 40 mm from
the product surface (stand off distance). The laser beam was aimed at the center
of the deformation when the product was 2 cm from the nozzle. Vertical adjustment was provided for the product holder to assure that the top surface
was at
the set point regardless of variations in product size. Spherical objects were supported by three cups about1 cm in diameter and with a radiusof curvature slightly
larger than the object. The cups were supported on a ball-and-socket joint that
allows movement of the cups for accommodating products of irregular shape.
Several stepswere required to obtaina firmness measurement. Product was
placed in the holder, positioned under the nozzle, and the height was adjusted
until a voltmeter connected to the output of the laser displacement sensor was
zero (indicating the nozzle-product distance was2 cm). The pressure in the tank
was then set by adjusting the pressure regulator until the desired pressure was
reached as indicated by a precise digital pressure sensor (1.7 kPa resolution). The
switch for the solenoid valve was then pressed when the digital pressure sensor
indicated the desired value.
Firmness-Measurement Methods
261
Hung et al.
262
3000
A
E
3 2500
*
C
y = 6.51ffiX - 295.78
2000
0
5m 1500
6
5 1000
-E
i!
500
o f
0
100
200
300
400
500
Plots obtained for fruits and vegetables were also linear. Repeatable results over
a wide range of products and conditions indicated that the concept and design
was successful.
to holdthemainstructure,
A 1 m X 2 mtableorbenchwasrequired
oscilloscope, laser control box, electronics box, and volt meter. Although it was
it was somewhat difficult at 90 kg and with
possible to move the equipment,
several electrical connections. Due to the large size and complicated operating
procedures of thefirst prototype, theunit was redesigned to facilitate easier operation.
C. Improved Design
Figure 4 shows the improved prototype design of the laser air-puff unit, which
is about the size of a tower computer. The componentsof the system are similar
to the original prototype except that they are smaller in size and with electronic
interfaces. A notebook computer is used for controlling a pressure regulator and
a solenoid valve, for acquiring data from the laser displacement sensor, for anain a spreadsheet format. The software is
lyzing the data, and for storing data
a visual programming package called LabView from National Instruments. A
PCMCIA card interfaces the computer with the hardware.
The fruit sample holder shown in Fig. 4 is a new design that uses three
balls to support the product rather than three cups as in the first prototype. The
Methods
Firrnness-Measurement
263
Fig. 4 The improved prototypeof the laser air-puffuses anotebook computer to control
the measurements and to analyze and store the data.
cups had allowed some movement, which was only partially compensated by
for
calculating maximum displacement from the final rather than the initial displaceof movements. The purpose of using the balls is to allow a repeatable amount
ment rather than trying to eliminate all movement of the product.
The advantages of the laser air-puff approach include no contactwith the
product, high speed, and good accuracy. However, it is limited
by its cost (about
$lO,OOO) and the need for an improved sample holder.
IV. TOOLSFOREVALUATINGFIRMNESSMEASUREMENT
Nondestructive methods for detecting firmness are typically evaluated
by comparing results with the destructive MT test. For over 75 years the MT test has been
the standard for measuring firmness. One of two plungers (7.9 or 1 1.1 mm in
diameter) with a specific convex tip is inserted into the product to a depth 7.9
of
mm. The maximum penetration force is the value that represents the firmness.
Results should specify the plunger to be used and the maximum force (not pressure).
Hung et al.
264
A.
Modulus of Elasticity
where
EASAE
= modulus of elasticity, MPa
R , = maximum radius of the tested spot, m
R; = minimum radius of the tested spot, m
d
F
D
p
=
=
F, m
Firmness-Measurement Methods
265
The value for E found withthe ASAE procedure can be checked with other
methods such as the removal of a specimen of known shape, which allows direct
calculation of E from cross-sectional area, force, and deformation data.
A similar equation for E was developed by Fan (68) for the laser air-puff
method as
where
ELASER= modulus of elasticity, MPa
p = Poissons ratio
P = pressure in air tank, MPa
V = maximum voltage reading from laser sensor,
mV
2.5
1.5
y 1 . 0 8 ~+ 0.241
R z = 0.Q2lS
0.5
1.5
2.5
EASAE (MPa)
Fig. 5 Correlation of modulus of elasticity calculated from the laser air-puff firmnesssensing data (ELASER) versus modulus of elasticity calculated from the deformation data
using a spherical indenter (EASAE).
(unpublished data)
266
Hung et ai.
A similar study with apples by Fan (68) also had a high correlation between the
two methods for calculating E (R = 0.91).
New Zealand researchers (65) also had extremely high correlations (R? =
0.94) for kiwifruit between E values calculated from parallel plate compression
and values for E calculated from an equation using drop height and dwell time
when the fruit impacted their SoftSense instrument. The New Zealand researchers
(65) expressed the whole fruit stiffness (E) based on the parallel plate method
(ASAE Standard S368.1) with the Instron Universal Testing machine as
6. RegressionAnalysis
Resultsfrom a nondestructive test areusuallycomparedwiththe
MT test or
MT or other tests on
another method by regression analysis. Results from the
products with a range of firmness values are typically plottedon the independent
axis and values from the new method on the dependent axis.A correlation coefficient is used to judge the performanceof the new method. For biological products
such as fruit, a correlation coefficient above 0.70 is considered good.
However, correlation coefficients are affected
by the number of data points,
the range of the data, and the distribution of data over its range. A computer
spread sheet program was developed for simulating correlations between the outa hypothetical new
put of a hypothetical standard instrument and the output of
instrument (71). The initial simulation had 100 data points uniformly distributed
with a range of 12 N (4-16 N) on both the x and y axes. Each data point was
given a random variation on each axis with a normal distribution having a mean
atthepointandastandarddeviation
of 1 N. Linear regression analyses gave
differentresultsforslope,intercept,andcorrelationcoefficienteachtimethe
simulation was run (much like comparisons of real instruments).
Firmness-Measurement Methods
267
Typical values for R' increased from 0.9 to 0.95 when the range of the
to 16 N (4-20
uniformly distributed points was increased from the original 12
N). The typical values for R? decreased from 0.9 to about 0.8 when the ranges
in the data points were determined by using 100 normally distributed points with
a certain mean ( I O N) and standard deviation (23 N) (Fig. 6). There was very
little change in R' values (typically 0.8) when the number of normally distributed
data points was increased from 100 to 200. These results demonstratethat values
to very firm fruit in a test. The
for R' can be increased by including very soft
range used for evaluating an instrument should be the same as the range that is
important to the user.
Regression analyses do provide a useful tool for making general comparisons between two firmness-measurement methods. However, rather than summary statistics, commercial operations such as sorting fruit at packing houses
20 2 18 4 16
614
012
10
268
Hung et al.
C.DiscriminateAnalysis
The following description of discriminate analysis shows how individual items
are evaluated for a quality characteristic. A firmness-sorting operationat a packinghouse has the purposeof removing items that are softer or firmerthan a specified level. There are only four possible outcomes for such an operation with either
accept or reject decisions (72):
Hit
Miss
False alarm
Correct acceptance
or too firm)
Rates are calculated by dividing the four outcomes by the maximum number of
hit rate is the
itemsavailable in thecategorybeingsorted.Forexample,the
number of defective items correctly removed dividedby the total number of defective items in the lot. The miss rate can be obtained as the complement of the
hit rate or calculated by dividing the number
of misses by the total number of
defective items. Similarly, the false alarm rate is calculated
by dividing the number of good items discarded by the total number of good items in the lot. Again,
correct acceptance rate is the complement of the false alarm rate or the number
of good items allowed to pass divided by the total number of good items.
For a sorting operation the miss and false alarm rates are more important
than the hit and correct acceptance rates. The miss rate indicates the percentage
firm), while the false alarm
of items in the final pack that are too soft (or too
rate represents the percentage of good product that could have been sold at full
price but was discarded.
The calculations shownin Fig. 6 are examples of the simulated comparison
of the output of a new instrument with thatof an existing (standard) method with
an arbitrary firmness cut-off value of 6 N set for soft fruit. The comparison was
made by plotting 100 normally distributed points (mean = 10 N; standard deviation = kc3 N). The precision of both the standard instrument and the new instrument was also simulated by adjusting the position
of each point in both the x
and y directions according to a normal distribution (mean at original point with
a standard deviation of -C I N). The I O points to the left of the vertical line at 6
N represent soft fruit used for calculating the
hit and miss rates. The 6 points
below the horizontal line at 6 N and to the left of the vertical line represent hits.
The hit rate of 60% was calculated by dividing the number of hits, 6, by the total
number of soft fruit, IO. The miss rate of 40% is calculated by dividing the 4
Firmness-Measurement Methods
269
misses by the total number possible, 10. The number of false alarms was determined by counting the points to the right of the vertical line and below the horizontal line. The false alarm rate of 3.3% was calculated by dividing 3 by the 90
goodfruit (100 lessthe 10 softones).Similarly,thecorrectacceptancerate
(96.7%) was calculated by dividing the number of pointsto the right of the vertical line and above the horizontal line, 87, by the total number of points to the
right of the vertical line than, 90.
The R value of 0.81 1 shown in Fig. 6 is reasonably high compared to
or other biological products. Similar values
many correlations involving food
were obtained each time the simulation was run. However, the miss rates ranged
2 to
from 10 to 30% for the same runs, while the false alarm rates ranged from
8%, indicating that high R2 values do not assure low miss and false alarm rates.
The high miss rates indicate that poor sorting results can occur even when
an
instrument has high values of R compared with a standard, which also has vari2 1.0 N; Fig. 6).
ability (such as MT-simulated with a standard deviation of
Thus, it is understandable why a packinghouse manager is more interested
in the number of soft fruit in a shipment than the R2 values from a comparison
of two instruments. Discriminate analysis provides practical results for evaluating
the performanceof a firmness-measurement method. However,it is still necessary
to have a standard of comparison to determine the miss and false alarm rates.
V.
PERFORMANCEEVALUATIONOFDIFFERENT
METHODS
Consumers often base their choice of product on their perception of the food
quality assessed by their senseof smell, touch, and sight. Therefore, the relationship of objective (nondestructive) methods of quality evaluation need to also
of this
closely mirror subjective evaluations made by the human. In the remainder
section. different methods usedby researchers recently to measure the firmness
of
fruits and vegetables are summarized. The information is presented in Tables I 3 showing the principle involved for each
method reported and the typeof sensor
system used to record the response. In addition, the products evaluated, measurement conditions, and performance factorsof each method are presented as well as
the stageof development of the technique, along with the sourceof the reference.
A.
DestructiveFirmnessTestingMethods
Results of firmness measurements with the MT and Effe-Gi tests were shown by
Abbott et al. (73) not to be entirely interchangeable, even though the probes and
indicated force ranges were essentially the same. Different sizes and shapes
of
Table 1 Firmness Measurement by Force-Deformation Methods (with and without mechanical contact)
Principle
Force deformation
Measurement
Deformation
Plunger
Product
Tomato
Tomato
Measurement condition
Test 4 - 5 T
92-99% RH
Test ambient
Oranges
Deformation
Parallel plate
Moment
Deformation
Puncture force
Crushing
strength
Kiwifruit
Test 0C
Kiwifruit
Melon
Blueberry
Cherries
Blueberry
Strawberry
Store 2 1 "C. 57% RH Test room
Apple
temp.
Cut specimen
Pear
Apple
Kiwifruit
Store 2C
Test 2C
Store 12C Test room temp.
Store 0C. 95% RH. Test 24'C
Peach
Apple
Store 1C
Test room temp.
Test room temp.
Test room temp.
h)
Performance of device
R' = 0.903 (device vs. penetrometer) cv. 0.17-0.28
Accuracy 100% (separation red from
green tomatoes)
Status
Ref.
Research
83
Research, on-line
prototype ( I 0
fruitds: 3.5
fruitds)
22
Research
25
Research and
commercial
23
Commercial
27
Research
21
Research
74
Research
17
Commercial prototype
Patent research
prototype
7
68
-l
0
RH = relative humidity (5%); Store = stored at indicated temperatudcondition: Test = temperature during test: temp. = temperature.
e
a
Methods
Firmness-Measurement
271
the two instruments as well as spring characteristics were thoughtto be responsible for the differences observed. In addition, operators reacted differently to the
machines in anticipation of sudden failure. Lack of sphericity or roundness of
in alignment of the fruit, the tester, and
most fruits also presented difficulties
compression surface. Oftenthe variability in measured results occurred by removing the skinto unrepeatable depths during sample preparation, since subcutaneous
cells are smaller than those nearer the fruit center. Penetrometers in general are
unable to assess flesh properties as a function of depth, and this becomes a problem for fruitsin which internal changes can occur, for example, columella softening in kiwifruit or central softening in mangoes.
B. NondestructiveForceMethods
Methods for measuring the relationships between the deformation resulting from
an applied force give a direct measure of firmness because the slope of a forcedeformation curve can be used to calculate the modulus of elasticity or stiffness
of the product (Table 1). Mehlschau et al. (20) found a curvilinear relationship
between firmness values andMT firmness. Typical characteristic curves for apple
and pear were analyzedby nonlinear rheological models by Wan et al. (74) using
apple and pear. Although the sorting rate was high (four fruitds), more accurate
results were obtained for softer fruits than firmer ones. This
is considered a major
limitation of this system according to the study of fruit classification with nondestructive firmness index based on either weighted grade purity or contamination
by Peleg (40).
Ozer et ai.(32) found that for each impact, the impact parameter correlated
well with flesh firmness by penetrometer (r = 0.86) and elastic modulus (r =
0.94). They also found that the same fruits can be subjected
to four multiple
impact tests without causing damage. Tissue damage after multiple drops at 40
mm height was evaluated to confirm that the test was nondestructive. They concluded that combination of multiple impact with a simple average gave better
results than using multiple regression. They also claimed that random multiple
impacts eliminate the need to specifically orient the fruit on a conveyor system
and is fast for real time sorting (0.2 s). However, it is only suitable for fruits that
do not bruise easily to ensure minimal damage during testing.
A common difficulty for all the firmness-measurement methods based on
nondestructive force is the need for mechanical contact between the product and
the sensor. Mechanical devices can limit the speed of operation and can limit
reliability. Speed is also limited in some of the approaches by the need to complete complicated analyses on the signal generated. There
is also a potential problem of cross-contamination from one product to another when touched by the
same probe.
h)
Princiole
Impact of fruit on
plate
Measurement
Force
Piezoelectric
force sensor
Product
Kiwifruit
Peach
Multiple impact of
fruit on surface
Impact rebound by
low-mass object
on fruit
Measurement condition
Performance of device
Store 4C
Test 20C
Drop height 10-50 mm
Test 23C
Drop height 50 mm
Research
65
r = 0.73-0.89 (device
vs. Effegi) cv =
0.057-0.062
R' = 0.47-0.83 (device
vs. penetrometer)
Reject 89 2 3% of soft
fruit (device)
Reject 96 2 1% of soft
fruit (human)
r = 0.86 (device vs.
Penetrometer)
r = 0.94 (device vs. parallel plate)
Accuracy = 7 8 4 9 %
(device vs. Instron)
Variability = 1.55%
(device)
S.D. = 12.9% (device)
S.D. = 15.3% (Instron)
R? = 0.78-0.84 (device
vs. MT)
No damage (20 g impactor)
32% damage for 59 g
impactor at 2 cm
Research
84
Research
33
Research
32
Research
85
Research
86
Research
87
Kiwifruit
Cantaloupe
melons
Ambient
Drop height 2 crn
Peaches
Acceleration
(mass impactor)
Cherry
Force
Piezoelectric transducer
Pears
Store 0C
Test 2OoC, Drop height
2-4 cm
Impact mass 10-50g
Status
Ref.
h,
3
u)
2
'u
00
00
o \ D
Firmness-Measurement Methods
!z
I
.
e,
3
E
Q
273
274
Hung et at.
Acoustic resonance
Sound velocity
Microphone
Melon
Store 2C
Test 20C
2 rnic. 1.5 cm apan
Kiwifruit Store 5C
Test 2 0 T
Ethylene-treated
Store 7 2 2C. 4 0 4 0 % RH
Peach
Test 20C I rnic
Store 1C. 96% RH
Apple
Test 20C
Tomato
Store and test 20C. I mic
Store 1C
Apples
Test 1C
I mic
Store 20C. 65% RH. Test 20C
Refleaion spectra IOOO-1650 nm
Apple
Vibration
Microphone
Vibration
Microphone
NIR spectroscopy
Microwave (0.2-20
GHz)
Laser doppler
Avocado
Tomato
Peach
Test r w m temp
Test 23 f I T
Kiwifruit Store 5C
Test 20C
Store 10C
Peach
Test 20C
Store 10C
Pea
Test 20C
research
98
Research
87
Research 43
R'
Research 93
n
-'
(D
u)
'p
u)
23
(D
=
=
Research 99
Research
100
u)
Research 59
Research
R:
Research
!2
R'
101
Research 55
Research 102
53
Store = Stored at indicated temperature/condition; Test = temperature during test; Freq = frequency; mic = microphone; temp. = temperature: NMR =
nuclear magnetic resonance; PMR = proton magnetic resonance; NIR = near-infrared
h)
-I
vl
276
Hung et al.
C. MethodsBasedonSecondaryProperties
A number of methods basedon indirect measurement of fruit firmness have been
developed and investigatedby several researchers. These methods, most of which
are experimental, are based on the secondary properties of fruits, which could
be calibrated with a direct firmness-sensing method such as the
MT test.
Consumer studies by Hung et al. (19) showed that an experts ranking correlated well with the consumer panel ranking (r= 0.92) and that firmness among
all five firmness groups were significantly different. Sorting accuracies
of the
experts ranking comparedto the consumer results obtainedby discriminate analysis were 76, 73, 74, 66, and 87% for most firm, very firm, firm, less firm, and
least firm, respectively. This indicates that although human firmness sorting may
be subjective, it is still a reliable method.
Abbott (75) found that sonic measurements of Delicious apples correlated
not as
significantly with other destructive measurements but correlations were
good as the ripeness scores obtained
by the USDA inspectors. Mechanical impulse excitation and piezoelectric sensors were used
by Galili et al. (52)to nondestructively measure the firmnessof avocado fruits. They foundthat the correlation
coefficient, r, betweentheacousticparametersandthemaximumpenetration
force ranged from 0.66 to 0.8 1.
Kiwifruit, peach, and pea firmness was investigated by Muramatsu
et al.
(53, using a laser doppler vibrometer(LDV), with which there was no mechanical
contact with the product. They found that the phase shift between the received and
emitted wave by the fruit gave good correlations (R 5 0.9) with fruit firmness.
of deMuramatsu et al. (53) also reported that the LDV technique was capable
tecting citrus fruits afflicted with internal disorders.
Using NMR, fairly good correlations(r = 0.7 I ) were obtained by Stroshine
et al. (76) for cherries between elasticity and T? time as measured in a magnetic
resonance process analyzer. Kim et al. ( 5 5 ) found the signal decay rate from the
magnetic resonance was highly correlated with the firmness
of tomatoes. Laser
vision imagery employing an He-Ne laser at 632.8 nm wavelength and two diode
laser modules at 670 and 685 nm as light sources were used by Lee et al. (77)
to nondestructively measure the firmness of Golden Delicious, Fuji, and Rome
apples. They compared the firmness values with those obtained destructively by
the MT method(withoutpeeling theappleskin).Factorsthatinfluencedthe
firmness measurements included light type (monochromeor color), skin removal,
laser optical power, and laser wavelength.
The performance data for these and
other instrumental firmness measuring methods are summarized in Table 3.
D. NondestructiveMethodswith No MechanicalContact
Several direct comparisons of the laser air-puff deformations with MT measurements have been made for peaches yielding R? values of 0.79 (78), 0.75 (7), and
Methods
Firmness-Measurement
277
Laser
Predicted
soft
Predicted
firm
Confirmed 21%
79%
(miss)
soft
(hit)
Confirmed (correct
73%
27%
firmacceptance)
(false
(false firmacceptance)
alarm)
Magness-Taylor
detector air-puff
ceptance)
(false
Packinghouse Predicted
soft
manager
Confirmed
soft
Confirmed
Predicted
firm
75%
(hit)
25% (miss)
15%
85% (correct
alarm)
Laser
Predicted
firm
USDA
inspector
Predicted
soft
Confirmed
soft
Confirmed
firm
100%
0% (miss)
(hit)
(hit)
37% &
63% (correct
alaI?Tl)
USDA
inspector
Confirmed
soft
Confirmed
Predicted
soft
Predicted
firm
100%
0% (miss)
26%
(false
alarm)
74% (correct
acceptance)
VI.
APPLICATIONS
Nondestructive textural quality evaluations are made over a wide range of applications. Produce growers manually
feel products as they matureto help determine
278
Hung et al.
harvest date (7). Workers who harvest products also use tactual information to
evaluate maturity. Sorters on packing lines manually remove items that are too
soft or too fitm. Government inspectors use tactual input to verify that grade
standards are satisfied. Buyers judge quality by feeling the product. Consumers
predict eating quality of products based on firmness when purchasing products.
Mouthfeel judged by the consumer is the final evaluation of firmness.
A primary application for a nondestructive firmness sensor is
to replace
subjective human evaluations with a quantitative measurement. Objective measurements of fruit firmness would enable precise descriptionsof the condition of
shipments as they move from the grower
to the final consumer. Firmness data
from a calibrated instrument fora sample of product from a shipment would help
resolve differences in opinion about the condition of the product.
A relatively small sample size
is collected from shipments for firmness
evaluations when a destructive test is used because all of the product examined
must be discarded after the test. When usinga nondestructive instrument a much
larger sample size can be measured and returned to the containers. The increase
in sample size provides a stronger inference for the actual condition
of the lot
(shipment).
Researchers will benefit in a similar way as the private sector from equipment for making nondestructive firmness measurementson samples from storage
of productare
studies.Whenusingdestructivemeasurements,largeamounts
A nonneeded so that small samples can be removed periodically for evaluation.
to be returned to the study.
destructive measurement allows the product tested
Periodically following changes in the firmness of each item in the study also has
the advantage of providing the opportunities of modeling changes to each item
rather than modeling average changes.
The ultimate goal of nondestructive firmness sensing is detection of firmness on fruit and vegetable packing lines. On-line firmness sorting of each item
flowing through a packinghouse would improve the consistency
of shipments
and reduce the amount of good product discarded. Removal of the few overripe
of decay spreading to good
(soft) items in a container reduces the possibility
items. The end result is higher economic returns not only for the shipper but for
each business in the marketing chain. Finally, consumers benefit from improved
firmness sorting by having product at the desired firmness
at the point of purchase
and when consumed.
Firmness-Measurement Methods
279
Hung et al.
280
firmness include puncture and laser air-puff. On the other hand, gross firmness
is determined when the whole food product receives the input signal and responds
with a measurable output response. Examples of gross firmness would be results
obtained from acoustic, NMR, and NIR measurements.
in this section indicate continuing
The many recent dates for work cited
interest in new approaches for measuring product firmness. However, there are
difficulties with all the attempts described above in all three general approaches.
of the need to extrapolate
Destructive methods have the inherent disadvantage
to represent the firmness of the batch or shipment.
the results from a sample
Methods that apply a controlled force to the product often have limited capacity
due to speed restrictions from inertial responses of moving masses. Methods that
measure a secondary property suffer from difficulties
in relating the results to
the firmness property. The Washington Fruit Research Commission conducted
evaluations of several nondestructive firmness-measuring devices in 1994. The
devices included an impact system in which a rod strikes the fruit with the force
of the impact converted to a firmness reading, measuring sound resonance when
the fruit is tapped by a plastic hammer, and by ultrasound (79). The results of
all the devices had very weak relationships with readings from Magness-Taylor
tests. Thus, a need is evident for a new solution
to the problem of measuring
firmness. However, Warner (80) contended that the fruit industry is searching
for firmness-sorting devices that need not be compared with the Magness-Taylor
method, which has its limitations, but devices that are themselves capable of
demarcating soft from firm fruit on an absolute basis.
To improve the accuracy and dependability of on-line firmness measurements, a concept of sensor fusion, that is, combining two or more sensing methods
(8 1 ) attempted to automate fruit sorting
is currently being investigated. Ozer et al.
by fusion of data acquired from selected sensors. The information obtained was
used to classify cantaloupe fruits into four maturity stages. Also, Zhang et al.
(82) presented a method based on fuzzy modeling
of the overall quality of a
product based on a setof quality factors, F (which included firmness), and another
p. Because each individset of grading factors,G, to obtain a single grading factor
ual method for firmness evaluation hasits own limitations, sensor fusion can take
the advantage of several sensing methods based on different sensing principles
to obtain the best prediction/measurement of firmness. We believe that Sensor
fusion (Fig. 1 ) is the correct direction for future firmness sensing.
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1.
INTRODUCTION
288
Ak and Gunasekaran
289
II.
VISCOELASTICITY
A.
IdealElasticandViscousBehaviors
Gy
It should be noted thatG is more accurately called shear modulus (or modulus of rigidity) because it is obtained via a shear test. However, similar relationa symbol E is used to
ships hold good for tension and compression tests, and
represent Youngs modulus obtained via these tests. Since shear
is the favored
in this
mode of deformation in linear viscoelastic studies, we limit our discussion
chapter only to shear tests unless stated otherwise. For a perfectly elastic solid
E and G are related as E = 3G (1 7).
An ideal fluid will deform and continue to deform as long as the load is
applied. The material will not recover from its deformation when the load
is
removed. This responseis called viscous. Theflow of simple viscous materials
is described by Newtons law, which constitutesa direct proportionality between
i.e.,
the shear stress and the shear rate
(v),
B. TimeFactor in Viscoelasticity
An important feature of viscoelastic materials, in contrast to the ideal materials,
is that their response depends on the time-scale of observation. A viscoelastic
material may behave more like viscous liquid with some elastic effects, or like
on how it is studied. Generally,
elastic solid with some viscous effects, depending
the faster the deformation, the closer the response
is to being elastic, and the
slower the deformation, the closer the response
is to being viscous. Such behavior
results from the molecular structure
of the material: stretching intermolecular
bonds, which causes an elastic response, is very quick; the motion of molecules
past one another, which causes a viscous response, takes much more time ( 1 8).
The time scaleof experiments should be considered
in relation to the characteristic time, denoted by h, of materials (e.g., relaxation time).The character-
Ak and Gunasekaran
290
istic time of the materials varies widely-infinite for ideal elastic solids and zero
for ideal viscous liquids. A dimensionless number, Deborah number (De), is defined to relate h with the time-scale of observation (t) (19,20).
D, = hlt
A high Deborah number (De >> 1) corresponds to solid-like behavior (i.e., relaxation not observed), whereas a low Deborah number (D, << I ) corresponds to
a liquid-like response (i.e., fast relaxation observed). The viscoelastic behavior
(i.e., finite relaxation observed) will dominate when De is on the order of I .
From the definition of De, a material can exhibit solid-like behavior due
either to the long characteristic time orto rapid deformation processes (e.g., high10
speed testing). For instance, water has practically zero relaxation time,
s, and consequently is perceived as liquidin most cases (20). However, water can
respond as a solid material if the time of testingis very short (or equivalentlyif
the frequency of testingis very high). During production, handling, and consumption, food materials are subjected
to a wide variety of deformation processes,
in Fig. 1, the range of shear
each with a different time scale. As can be seen
rates, or time scales, in various processes (not all food-related) differs greatly.
In general,however,foodsarecomplexsystemsand
do notpossess a single
relaxation time but rather a broad range of relaxation times (21). Therefore, in
a given process of finite duration, foods are more likelyto behave as viscoelastic
materials. The significance of viscoelastic behavior and associated rheological
properties in extrusion processes, for example, is discussed in several articles in
Kokini et al. (22).
At this stage,it is interesting to mention that rheologists have recently been
deprived of a delightful and mythical example often usedto emphasize the effect
of the time scale of observation on the material response.
To illustrate liquidto an observation
like behavior of a solid material, rheologists sometimes referred
that windowpanes of old cathedrals are thicker at the bottom than at the top.
The
explanation was that the glasses of these ancient windowpanes slowly flowed at
ambient temperatures under the influence of gravity. Given that the time scale
as a plausible explanation
of flow was several hundred years, this was regarded
(23,24). However, earlier, Ernsberger (25) pointed out that at ordinary temperatures, glasses of commercially useful compositions are rigid solids and observation of thicker ancient windowpanes cannot be related
to flow due to gravity.
Plumb (26) pointed out that the correct explanation for thicker windowpanes at
the bottom liesin the glass processingof the ancient times. Recently, the explanation based on sagging of glass due to gravity was disproved by Zanotto (27),
who calculated that it would require more than the age
of the universe for a
typical window glass to flow appreciably so that the bottom would be thicker.
Viscoelastic materials are characterized by various phenomena observable
under different conditions. Some of these are (28):
astic
Linear
291
TIME (s)
mm
m
Fig. 1 Shear rate ranges (and time scale of operation) for various food and industrial
processes. A: Sedimentation of fine particles in a suspending liquid (spices in salad dressing, medicines, paints); B: leveling due to surface tension (frosting, paints, printing inks);
C: draining under gravity (vats, small food containers, painting, and coating);
D: extrusion
(snackandpetfoods,cereals,pasta,polymers);
E: calendaring(doughsheeting);
F:
chewing and swallowing (foods); G: dip coating (paints, confectionery); H: mixing and
stirring (food processing); J: pipeflow(foodprocessing,blood
flow); K: spraying and
brushing (spray-drying, painting, fuel atomization);L: rubbing (applicationof creams and
lotions to theskin); M: high-speedcoating(paper); N: lubrication(bearings,gasoline
engines.)
If the stress is held constant, the strain increases with time (creep).
If the strain is held constant, the stress decreases with time (relaxation).
Effective stiffness depends on the rate of application of the load.
If cyclic loading is applied, hysteresis (or phase lag) occurs, leading to
dissipation of mechanical energy.
Acoustic waves experience attenuation.
Rebound of an object following an impact is less than 100%.
Frictional resistance occurs during rolling.
Ak and Gunasekaran
292
C.
Ideal elastic and ideal viscous behaviors present two extreme responses
of materials to external stresses. As the terms imply, these are only applicable for ideal
materials. Real materials, however, exhibit a wide array
of responses between
ideal viscous and elastic behaviors. Most materials exhibit both viscous and elastic properties simultaneously and, therefore, are characterizedas viscoelastic. Almost all foods belong to this group.
Ideal elastic and ideal viscous behavior is represented
by a spring and a
2). Thus,a
dashpot(a fluid-filled, piston-cylindersystem),respectively(Fig.
spring by itself can adequately represent a Hookean deformation, and the dashpot,
the Newtonian flow. Since viscoelastic materials have both viscous and elastic
properties, they are simply represented by a combination of spring(s) and dashpot(s).
The series combination of a spring and a dashpot
is called a Maxwell model
(Fig. 2). In this, both the spring and dashpot experience the same total stress ((3)
but share the total strain (y).
(3
ch=
(3d
(4)
y = 1: + yd
The subscripts s and d represent spring and dashpot, respectively. From the above,
the following Maxwell equation is obtained:
293
Output
nseFunction
Input Model
Mechanical
Element@)
Spring
Hookean
YC
Newtonian
Dashpot
Maxwell
Relaxation
Stress
u = ooexp(-fflR)
:Lh_
Kelvin
Y =ro[l-ex~(-ff~~)l
Fig. 2 Some simple mechanical elements used to model viscoelastic behavior of foods
and a set of their input-output relations.
where, T~ is calledtherelaxationtime.Stressrelaxationresultsareoftenexpressed in terms of a material function called the shear relaxation modulus, G(t):
Gunasekaran
294
Ak and
The Maxwell model with a single exponential term is generally not sufficient to describe relaxation behavior of foods. Therefore, it is often modified to
include multiple exponential terms, each with a different relaxation time, representing an array of Maxwell elements in parallel (29). As can be seen in Eq. (7),
the Maxwell model predicts a zero stress at very long times>>
(t T ~ ) .That means
the model cannot represent a truly viscoelastic solid, which would have residual
stress after relaxation, as observed in many food products (30). To account for
the residual stress, the Maxwell model is modified
by adding a constant term
(31).
The parallel combination of a spring and a dashpot is called a Kelvin (or
Voigt or Kelvin-Voigt) model (Fig. 2).In this, both the spring and dashpot experience the same total strain (y) but share the applied stress (0).
d =
os+ dd
=
yd
(9)
(10)
Gy
+ qj
(1 1)
where T~ is the retardation time. Creep results are often expressed in terms of a
material function called the shear creep compliance, J(t):
The Kelvin model can be generalized by combining many elements in series, each with a different retardation time (29). As can be seen in Eq. (12), the
Kelvin model predicts an asymptotically reached strain (y(J at very long times (t
>> 7,). That means the model cannot represent viscoelastic liquids showing continual flow. A viscous term is then included in the Kelvin model to account for
flow at long times (32).
The Maxwell and Kelvin elements are the simplest
of many mechanical
models. Many combinationsof several springs and dashpots (and other elements)
are used to fully describe the viscoelastic nature ofreal materials (33). For exam-
295
Gunasekaran
296
Ak and
these, data points (for, e.g., a, b, c, d in Fig. 3) are gathered at different times
o versus y plot is constructedfor
(e.g., t,and t2). Thenthecorresponding
each of the times at which the stress response is measured (Fig. 3C). The strain
by dotted
value at which the isochronal beginsto deviate from linearity (indicated
line in Fig. 3C) is the upper limitof the linear viscoelastic region for the material.
Thehatchedregion in Fig.3Cindicatesthenonlinearrangeofthematerial
studied.
Performing experiments within the linear range is helpful because the data
can be analyzed using relatively simple linear viscoelastic models discussed previously. Within the linear range, the material functions (modulus and compliance)
are independent of the strain or stress level used in the experiment. Conversely,
once the modulus G(t) is known, the stress o(t) for
any strain y and time t is
= o(t)/y. Most polymers exhibit linearity
known through the relationship G(t)
at low stressesor strains (e.g., y z 0.5%) (36). In the case of food materials, strain
levels less than 3% are considered acceptable
to assure linearity(12). However, it
is important to examine the linearity over the entire range
of experimental condiso that the entire data set can be analyzed
tions and select the smallest strain level
uniformly.
D. ViscoelasticBehavior in DynamicTests
Although it is possible to conduct small-amplitude oscillatory tests i n tension
and compression, and there are commercial rheometers for such configurations
(37,38),thesheardeformationhasbeen
thedominantmode
of deformation.
Hence, the abbreviation SAOS, for small-amplitude oscillatory shear, is commonly used to represent dynamic viscoelastic tests. Concurrent with the developSAOS methodhasgainedmuchpopularity
ment of modernrheometers,the
among food researchers.
The SAOS test is based on applying a sinusoidal strain and measuring the
resulting stress. Of course, one can apply sinusoidal stress and measure the resulting strain. Either method,in principle, should produce the same material propertiesprovidedthattheimposeddeformation
is withinthelinearviscoelastic
region.
Let us consider Fig. 4, where a thin disk of a Hookean solid is subjected
to a sinusoidal shear strain r(t) between parallel plates
of the instrument:
r(t) = yo sin(ot)
(14)
where yo is the shear strain amplitude, o is the angular frequency, and t is the
time. Inserting Eq. (14) into Eq. ( I ) , the constitutive equation for a Hookean
solid, for y will give the resultant stress o (t), which is also sinusoidal:
o(t) = oosin(mt)
(15)
297
Hookean Solid
T
...................
....__ .__.
.......
/=+
,
ot
1 Stress output
Fig. 4 Illustration of parallel plate geometry in a dynamic rheometer (top) and typical
stress-strain data for a Hookean solid (bottom). Stress and strain are in phase.
where o0is the shear stress amplitude. Therefore, for Hookean solids the resultant
stress wave is exactly in phasewith the input strain wave. This means that when
the strain is at maximum, so is the resultant stress, as shown in Fig. 4.
Let us now apply the same strain input [Eq.(14)], this time using a concentric cylinder configuration (Fig. 5 ) , to a Newtonian liquid whose constitutive equation is given, in simple form, by Eq. (2). Taking the time derivative
of the input strain function [Eq. (14)] and inserting the result for p in Q. (2)
yields:
o(t) = qmy, cos(0t)
(16)
298
Ak and Gunasekaran
Newtonian Liquid
+ yoG"(o) cos(ot)
(17)
where two frequency-dependent functions,G' and G", represent the shear elastic
(storage) modulus and the shear viscous (loss) modulus, respectively. The term
in Eq. 17 having sin (cot) is in phase with the strain input, and the term having
cos(ot) is 90" out of phase with the strain input. Relative magnitudes of these
299
Viscoelastic Material
'
Stress output
terms will determine the degree of viscoelastic behavior. Therefore, we can rewrite the stress response of a linear viscoelastic material in a format similar to
the strain input as:
o(t) =
(so
sin(wt
+ 6)
(18)
+ (o0cos(wt)
sin6)
(19)
and
G' is a measure of the energy stored and subsequently released per cycle
of deformation per unit volume. It is the property that relates to the molecular
events of elastic nature. G is a measure of the energy dissipated as heat per
cycle of deformation per unit volume. G" is the property that relatesto the molecular events of viscous nature.
300
Ak and Gunasekaran
From the last two equations, we obtain another commonly used dynamic
viscoelastic property, the loss tangent (tan 6):
Hence, tan 6 denotes relative effectsof viscous and elastic componentsin a viscoelastic behavior.
Recall that for a Hookean solid 6 = 0; hence in-phase shear storage modulus is G = o&, and out-of-phase shear loss modulus is G = 0, as expected
6 = d 2 ; hence G =
and required. On the other hand, for a Newtonian liquid
0 and G = o,,/y,,, as expected and required.
Another way to present results of dynamic measurements is to use complex
modulus G*, the magnitudeof which is related to G and G through the following
equations (39):
where,
tic
Linear
301
Shear
parameter Rheological
Shear strain
Shear modulus (modulus of rigidity)
Shear relaxation modulus
Shear compliance
Shear creep compliance
Equilibrium shear compliance
Steady-state shcar compliancc
Complex viscosity
Dynamic viscosity
Out-of-phase component of q*
Complex shear modulus
Shear storage modulus
Shear loss modulus
Complex shear compliance
Shear storage compliance
Shear loss compliance
SI units
-
Pa
Pa
Pa '
Pa- '
Pa- I
Pa- I
Pa . s
Pa . s
Pa s
Pa
Pa
Pa
Pa- '
Pa I
Pa- '
Ak and Gunasekaran
302
oKj
'r\A
0 05
0.01
0
O
'
K
; J
-0.01
-0.05
-0.02
3.14
Time (s)
6.28
3.14
Time (s)
6.28
3.14
Time (s)
6.28
A
G',G
"*
4 -
,
0
k4
LINEAR
VISCOELASTIC
REGION
.*
>'
b
NONLINEAR
VISCOELASTIC
REGION
STRAIN AMPLITUDE
Fig. 7 Typical strain sweep test is conducted by applying increasing strain amplitudes
(top) and the resultant moduli (bottom) values are examined
for their departurefrom being
horizontal.
tic
Linear
Methods
303
withfurtherexperiments
(e.g., frequency-sweep)performedatstrains
(or
stresses) smaller thanthe limit. It is highly recommended to repeat the strain
sweep test at extremes of experimental variables, for there are data indicating
that the linear viscoelastic region can vary with test frequency, temperature, and
sample age (42.43). Variation of the linear viscoelastic region with test temperature is shown for mozzarella cheese in Fig. 8.
I.
Shear Strain
Fig. 8 Linear viscoelastic region for mozzarella cheese as a function of cheese temperature. (Data from Ref. 43.)
304
Ak and Gunasekaran
2.
3.
4.
5.
6.
7.
8.
astic
Linear
Methods
305
111.
APPLICATIONS
A. Transient Tests
In terms of experimentalmethodology,compressiontesting
is anexpedient
it provides
technique to distinguishgoodandbadsamples.Butbeingstatic,
limited information on the mechanism(s)of the process(es) involved, understanding of which is needed for the development of newer products and/or textures.
Transienttestsareinherentlyusefulforevaluatingmaterialstructureand/or
structure development during processing. This has been the primary application
of transient tests. Nonetheless, there have been many investigations
to develop
empirical correlations between the transient test parameters (relaxation time. retardation time, equilibrium modulus, etc.) and some food quality/texture attributes.
Transient tests have been very popular among food researchers because of
their simple test configuration. Stress relaxation tests can be readily performed
using universal testing machines
(e.g., Instron, Texture Analyzer). Creep
tests
can be performed by means of a loading device with a provision to continuously
monitor (and record) the sample deformation. However, some practical problems
exist. In the case of stress relaxation tests, it is virtually impossible to impose
an instantaneous strain, andin the case of creep tests, dueto continually changing
sample cross-sectional area, the applied stressis often not a constant, as generally
required by the models used for data analysis.
Evageliou et al. (52) described developing a new fat mimetic by carefully
a
evaluating the structure of the material by creep tests. A model combining
Maxwell element in series with one or more Voigt elements is used to describe
a s protein
time dependency of the linear creep compliance. Biopolymers such
(from egg, milk. gelatin), intact and modified starch, and/or gums
(e.g.. carrageenan, alginate, pectin) are used to provide structure to fat mimetics. Since no
306
Ak and Gunasekaran
biopolymer by itself can provide the required structure and a plastic flow,
mixtures are used(e.g., gelatin and maltodextrin)in an attempt to impart plasticity. However, if incompatible polymers are mixed, phase separation leads to
an unacceptable product (52).
Ingel systems,networksareformedbyphysicalcross-linkswith
finite
lifetimes, which will stretch and eventually relax when under mechanical stress,
thus allowing the system to flow. Creep curves of full-fat products suchas butter
and margarine exhibit lower initial strain (about two orders of magnitude) than
those of biopolymer networks (37,52,53).The difference is related to permanency
of cross-links. The fat lattice is formed by the short-range van der Waals forces
of attraction between glyceride crystals as opposed to the long junction zonesof
helical strands in a biopolymer network.
Comparing the creep curves of margarine and gelatin gels, the fat crystals
were reported to flow more rapidly (larger compliance) than did the mimetic
made of gelatin triple helix. Much of the creep was observed to occur beyond
the instantaneous deformation. The instantaneous compliance is associated with
of a network, and the higher proportion
of the
the initial elastic deformation
total rearrangement in gelatin gels reflects the elastic character of gelatin helical
associations as opposed to the plasticity of a liquid/solid fat body (54).
Sakurai and Nevins (55) recommended use of stress relaxation measurements to study fruit and vegetables since physiconlechanical parameters derived
through application of the Maxwell model were related to time-dependent biochemical and microstructural changes associated with softening (56). The minimum stress relaxation time for tomatoes changed with ripening as a function of
(56) suggested that
softening and position within the fruit. Sakurai and Nevins
stress relaxation tests might be able to distinguish differences in fruit tissue due
to ripening that are not observable by means of large deformation tests.
Creep tests modeled as per the Burgers model (series combination of the
MaxwellandKelvinmodels)mayprovidemoreinformationthanthesimple
stress-relaxation model (Maxwell). Creep tests allow prediction of elastic, viscoelastic, and viscous flow characteristics separately. The creep test is perhaps the
most appropriate means to determine the elastic parameter (instantaneous compliance) of a material (57). Model parameters may be associated with discrete components or constituents of the material being tested (14,15,58). This facilitates
of the physicomechanigreater understanding of the microstructural determinants
cal behavior or texture exhibited
(1 2). A six-element model is found to best define
in terms of physicomechanical
the viscoelastic behavior of tomato pericarp tissue
interpretation of the tissue softening mechanisms (12). The instantaneous elastic
properties of tissue are attributed to the combination of cell turgor pressure and
primary cell wall strength as dictatedby cellulose. The retarded viscoelastic properties are related to the independent changesin hemicelluloses and polyuronides.
The steady-state viscous flow properties are related to higher cell wall fluidity
307
where J(t) is the total creep compliance at time t, J,, is the instantaneous rigidity
compliance, J , and J2 are the retarded compliances, T~ and zz are the retardation
times, and vv is the Newtonian viscosity.
Thevaluesoftheviscoelasticparameterscalculatedfor
bothfull-and
reduced-fat cheeses are given in Table 2 . The reduced-fat cheese had a lower
instantaneous compliance (J,,) than the full-fat cheese. This suggeststhat a reduction in fat resulted in an increase in the elastic (or solid-like) character of the
cheese. The instantaneous compliance may be related to the undisturbed protein
(q,)of reduced-fat cheese
network structure (63). The higher Newtonian viscosity
suggested a greater resistance to flow at longer time. Newtonian viscosity may
be attributed to the breakdown of protein network (66). Thus, the reduced-fat
cheese could be considered to retain more of its solid-like viscoelastic structure
than the full-fat cheese.
ters
308
Ak and Gunasekaran
Cheddar cheese"
Six-element Kelvin
model
Jo( 1 /kPa)b
J , (1 /kPa)'
Jz( 1/kPa)'
0. I 4x
0.27"
0.55'
3.35"
28.04'
137.9'
2, (S)d
T?(s)d
qv(kPa.s)'
0.1 1"
0. 19x
0.46x
3.25"
23.89"
149.6"
( p
.........
0.9
"=
:,-:
........ ..~"."."."._..
Wl")
J,+J,
...........................................
" ' . ,
50
100
Jo
150
Time (s)
Fig. 9 Six-elementKelvinmodelusedtocharacterizecheesemeltability.(Datafrom
Ref. 65.)
astic
Linear
309
in Cheddar cheese under external loading. The instantaneous slope of the creep
curve is calculated by taking the first derivative of Eq. (31) at time zero. This
instantaneous slope is defined as the viscoelasticity index (VEI), which is computed as follows:
B. Dynamic Tests
1. Gelation and Properties of Dairy Products
One commonuse of the SAOS technique is to measure linear viscoelastic properties of materials during gelation and aging (or curing). Here, we consider examples of milk gels formed by rennet or acid addition, and other dairy products (e.g.,
yogurt, cheese). Studies on gel development usually employ concentric cylinder
system, whereas those on mature gels and cheese use parallel plate or cone and
plate configuration. The cone and plate system
is similar to theparallelplate
configuration (Fig. 4) except that the top plate is replaced with a cone of small
angle.
The SAOS method has been used to follow gelation kinetics of various
proteins and polysaccharides. In these studies, the technique is used to monitor
the change in the dynamic moduli G and G with time, and in some cases with
temperature, at some selected frequency (usually 1 Hz). In a gelling system we
usually observe sudden increases in G and G after an initial lag period. This
is often followed by a gradual decrease in rate of increase of G and G, which
eventually levels off in most cases. Moreover, once the mature (or fully cured)
gel is obtained, it is common practice to make further measurements such as (a)
Ak and Gunasekaran
310
strain sweep (G and G vs. y) to determine the linear viscoelastic region, (b)
frequency sweep (G and G vs. O ) to determine the elastic character of the gel,
and (c) temperature sweep (G and G vs. T) to evaluate thermal characteristics.
Oneimmediateconcernariseswhen
we want to determineviscoelastic
to elastic gel. This is whether
properties of a system going from viscous milk
the applied strain or stress amplitude affects the aggregation process. Dejmek
(70) measured G and G during rennet coagulation of milk. When the applied
shear strain was high (i.e.,yo = 0.2), the resultingG* from the continuous oscillationmodewassignificantlygreaterthan
that from the intermittent oscillation
mode. Apparently, continuous oscillation affects the aggregation process. Dejmek
also reported that the coagulation process was not affected by continuous shear
strains below 0.05 at 1 Hz (70). To avoid disturbing the skim milk gel network,
Zoon et al. (71) started oscillatory shear measurements
at 1 rad/s (-0.16 Hz)
only after a weak gel formed (i.e. G = 2 Pa).
Dejmek (70) reported that the linear viscoelastic strain limit for mature
rennet gels is 0.05. The corresponding limit for rennet-induced skim milk gels
is reported to be 0.03 (70). Paulsson et al. (72) applied strain amplitude of 0.02
of
at 1 Hz to stay in the linear viscoelastic region while monitoring formation
heat-induced P-lactoglobulin gels. Most studies on dairy gels seem to use strain
amplitudes less than 0.05 and a frequency of I Hz.
A typical curve obtained during gelation (or curing) is schematically illustrated in Fig. 10. The beginning of time axis (t = 0) usually refers to the addition
Time
311
of rennet or acid to the milk. There is generally a lag period before dynamic
properties attain values greater than the minimum torque of the rheometer. After
the initial lag period, both G and G increase with a rate that depends on the
experimental conditions. Whether a dynamic property reach a plateau value
or
not also depends upon experimental conditions such as the temperature at which
the gel is formed and aged (73).
Bohlin et al. (74) designed and developed a dynamic testing instrument
with coaxial cylinder geometry and used it to monitor coagulation of milk under
conditions similar to thosein cheesemaking. They investigated effectsof calcium
on coagulation of
chlorideandrennetconcentrationsalongwithtemperature
milk. Their data show that higher concentrations of rennet and calcium chloride
and elevated temperatures result in an earlier start of coagulation and a faster
gelation rate, leading to stiffer gels (i.e., higher G* values). The start of coagulation is identitied as the time at which G and G began to deviate from zero (see
Fig. IO). Nakamura and Niki (75) studied the influence of calcium concentration
on rheological properties of casein micelles during gelation. They concludedthat
of
changing calcium concentration affects gelation rate but not the mechanism
gelation.
Several quantitative coagulation parameters may be obtained from gel development curves. For instance, Dejmek (70) presenteda method based on threeto derive coagulation parameters such as
parameter Scott Blair-Burnett model
of incipient gelation from
the time constant of gel build-up and time constant
gel development curves. Zoon et al. (73) obtained clotting time as the time from
rennet addition to the formation of visible clots; it generally decreased with increasing gelation temperature. On the other hand, van Hekken and Strange (76)
determined coagulation time as the time from rennet addition to the point where
et al. (77) conducted
G exceeded G, or tan 6 became less than one. Guinee
SAOS tests to investigate the effect of heat treatments of milk before ultrafiltration on the coagulation characteristics of retentates with different protein levels.
They also used the Scott Blair-Burnett model to extract several coagulation parameters, which enabled them to compare the effects quantitatively.
SAOS measurements are also used to characterize behavior of dairy products (e.g., yogurt, cheese, whey gels) as well toasevaluate effectsof many factors
on viscoelastic properties of these products. Skriver (78) conducted SAOS tests
on stirred yogurt to assess the effect of technological parameters such as culture
type, fermentation temperature, and dry matter content on rheological characteristics of the product. Based on dynamic shear data, stirred yogurt is characterized
as a weak gel. The main features of weak gels are given by Ross-Murphy (79)
as G > G. both G and G are largely independent of frequency, and the linear
viscoelastic strain limit is small (y < 0.05). Moreover, Skriver (78) found that
exo-polysaccharide produced by the ropy culture did not contribute
to the dyin contrast to the viscometry
namic gel stiffnessof stirred yogurt. This tinding was
312
Gunasekaran
Ak and
Viscoelastic Linear
313
differencesamonglabnehsamplespreparedafterdifferenttreatments.However, oscillatory shear tests clearly differentiated between the samples and, therefore, are considered to be more reliable to determine rheological properties of
labneh.
the qualityof prepared foods
Melting characteristicsof cheese are critical to
containing cheese (e.g., pizza). Several methods have been developedto measure
meltability of cheese (87). There is, however, a lack of correlation among traditional methods (88), and thus new methods are needed. Riiegg et al. (87) stated
6, could be potentially used as a predictor for cheese
that the loss tangent, tan
meltability. Indeed, the use of dynamic rheometry to study cheese properties at
high temperatures has been increasing (89-91).
(G', G",
Ustunol et al. (89) correlated the dynamic rheological properties
and G*) of Cheddar cheese up to 90C with the meltability data from the traditional Arnott test. The minimum complex modulus G* is suggested as a possible
meltability indicator as it showed a significant correlation ( I = -0.80) with the
empirical meltability results. A recent study on the melt characteristics of imitation cheese reports a higher correlation coefficient ( r = 0.96) between the maximum tan 6 values and meltability results obtained by the empirical Olson-Price
tube method (90).
Meltability of process Cheddar cheese made with different emulsifying
salts has been measured by dynamic stress rheometry (91). In this study, the
parameter to compare behavior of cheeses was the so-called transition temperature, which is defined as the lowest temperature at which tan 6 = 1. The change
from more elastic to more viscous behavior occurs at a lower temperature for
process cheese containing trisodium citrate (56.5"C) than that containing disodium phosphate (64.6"C) (91).
A significant concern in dynamic rheometry at high temperatures
is the
slippage (92), which results in inconsistency in the rheological data (93). Howto the plates and/or using serrated
ever, remedies such as bonding the sample
plates or compressing the specimen slightly have
been applied to reduce the slippage problem (89-91,94-96). Another technique
to study properties of melted
cheese, which does not suffer from slippage problem, is the lubricated squeezing
flow. This technique is a simple but fundamental alternative to the traditional
empirical meltability tests and has been frequently usedto study cheese meltability (67,97-99). The sample and test geometry of the squeeze flow method makes
it also suitable for performing creep and stress relaxation tests within linear viscoelastic region (65).
2. Determining Gel Point in Polymeric Systems
314
Ak and Gunasekaran
315
Viscoelastic Linear
In the study by Tung and Dynes (106), it was suggested that the time at
which G and G curves cross each other can be used for determining the gel
point. This studyalso reported that the gel time determined as such was a function
of frequency of the oscillatory test. Recognizing that the gelation of a material
must not depend on the frequency of the rheological test, Winter and Chambon
(100) havedeveloped a newmethodforlocatinggelpoint.According
to the
Winter-Chambon criterion, tan 6 (= G/G) at the gel point becomes independent
of frequency (104) (Fig. 12). Moreover, the cross-over method is a special case
of the Winter-Chambon method (102,104).
Lopes da Silva and Gonqalves (107) studied rheological properties of curing high-methoxyl pectin/sucrose gels at different temperatures using SAOS experiments. The G-G cross-over method could not be used asa criterion to identify the gel point. They instead applied the Winter-Chambon criterion. Michon
et al.( 1 08) applied the Winter-Chambon criterion to determine critical parameters
of gelation (e.g., time, temperature) at different polymer concentrations for systems involving iota-carrageenan and gelatin. When gelatin was cooled from
60C
the gelling time was found to be 44 minutes. In another set of experiments, the
gelation temperature of iota-carrageenan was determined to be 53.5OC based on
the same criterion. These researchers provided phase diagrams where critical temperature of sol/gel transition was charted against concentration of the polymers.
These graphs showed that at a given temperature, a gelatin with mean molecular
mass of 70.000 daltons would require a much higher concentration for the sol/
gel transition than that with a mean molecular mass of 182,000 daltons.
.
4
c
(TI
.
I
-
0.1
Gunasekaran
316
Ak and
Labropoulos and Hsu (109) have used the SAOS method to investigate gelforming ability of whey protein isolate (WPI) dispersions subjected to different
heattreatmentsandotherprocessingvariables(e.g.,pH,concentration).Employing the Winter-Chambon criterion for gel point detection, a wide range of
gelation times from 12 to 164 minutes is obtained depending on the experimental
conditions. Information from such studies is expected to allow processors to obtain desired gel properties by controlling the variables during gelation of WPI
dispersions.
Emulsion droDlets
may
Which
mav:
Cream
lead to:
A
Creaming
01
Flocculate
(seml-reversibly)
0
01
Aggregate
(irreversibly)
andlor
or
Coalescence
flocculation
or
Q0
Flocculate
by bridging
~~~~~
.
"
+
@-0
317
Rheological properties of food emulsions play important roles in the stability as well as texture and mouthfeel of these products. In this section we present
examples on the use of SAOS method to characterize food emulsion gels, to
understand stability mechanisms, and to determine the roles of various factors
contributing to the emulsion stability.
Dickinson and Golding (1 12) used the SAOS method (0.04 Pa, 0.1 Hz) to
examine viscoelastic changes due to development of a particle network in emulsions prepared at pH 6.8 with sodium caseinate as the only emulsifier( I -6% by
mass) and n-tetradecane as the dispersed phase (10, 35, or 45% by volume). The
2% (by mass) caseinate emulsion (35% by volume n-tetradecane, pH 6.8, 30C)
behaved like a viscous liquid with G > G at all times. This behavior is associated with a nonflocculated system.
In contrast, for the 6% by mass caseinate
emulsion (35% volume n-tetradecane, pH 6.S,3O0C), there was an initial decrease
in the dynamic moduli followed
by a steady-state region, which was followed
by a steady increase of both quantities (G faster thanG) until a cross-over point
(G = G). Thisbehavior is associatedwiththe
floc reorganizationtomore
closely packed structures and the formation of a gel network by the flocculated
particles.
Muiioz and Sherman( 1 13) measured the viscoelastic properties of commerSAOS tests
cial mayonnaise, reduced-calorie mayonnaise, and salad creams using
with a controlled stress rheometer (1 Hz, 8.96-1 8.51 Pa). They have explained
the observed differences in viscoelastic properties by variations in the ingredients
of these emulsions. For instance, the lower
G value of one mayonnaise compared
to another is related to the presence of sugar in the former, as sugar molecules
exerted a shielding effect on protein groups involved in interaction and network
formation among the oil droplets. The lowest G values for salad creams are
attributed to the lower oil content, yielding a lower concentration of oil droplets
in the emulsion. This is in accord with the results of Dickinson and Golding
( 1 12) in that even when protein concentrationwas high (>8% by mass), emulsion
made with lower volume fraction of dispersed phase (oil) had a weaker structure.
Other studies by Gallegos et al. ( 1 14) and Ma and Barbosa-Cinovas ( I 15) confirm that higher oil contents result in higher dynamic moduli for commercial and
modelmayonnaisesamples.Moreover,thestrainsweepmeasurements
at I O
rad/s on model mayonnaise samples indicated a narrower linear viscoelastic region for samples with lower oil content ( 1 14).
Past work on whey proteinshas shown that co-homogenizationor preemulsification of the oil with lecithin reduces significantly the strength of heat-set
emulsion gels ( 1 16). Consequently, Dickinson and Yamamoto ( I 17) used the
SAOS method (1 Hz, maximum strain amplitude of0.5%) to examine thoroughly
the influence of lecithin added after emulsion formation on the properties of heatset P-lactoglobulin emulsion gels. The dynamic data(G and G) clearly showed
that the addition of lecithin at a concentration of 4.4% by mass increased G of
31 8
Ak and Gunasekaran
4. DeterminingSensoryPerception
Linear viscoelastic tests have also been applied to study the interaction between
texture, flavor, and taste. It has been shown that complex viscosities q* from
dynamic tests rather than steady shear viscosities give better correlation with
sensory thickness for weak gel systems such as liquid and semisolid foods( 1 19121). Hill et al. (122) investigated the relationship between the perceived (sensory) thickness, taste and flavor and rheological parameters
of a lemon pie tilling.
They established a general linear relationship between perceived thickness and
q* over theviscosityrange of 1,000-70,000mPa.s. (Fig. 14). Richardsonet
4.0
log (q, rnPa.s)
4.5
5.0
Fig. 14 Linear relationship between perceived sensory thickness and complex viscosity.
(Data fromRef. 122.)
Viscoelastic Linear
319
al. (1 19) covered the viscosity range of 10-10,000 mPa.s using model systems
thickened by xanthan, guar, and starch. These results indicate that q* measured
at a frequency of 50 rad/s can be considered a suitable criterion for predicting
perceived thickness in widely differing systems (122).
The relationship between taste and flavor and rheological properties seem
to depend on the structure of the solution and the concentration. It is well established that the intensity of perceived taste and flavor decreases as the product
thickness increases ( I 23). In model solutions containing random coil polysaccharides, suppression of taste and flavor starts when the polysaccharide concentration
exceeds the critical concentration where the polymer coil starts to entangle. The
exception to this is xanthan gum where good flavor release is achieved at a concentration in excess of the critical concentration ( 120,121,124,125). At comparable viscosities, cornstarch suppresses sweetness far less than random coil polysaccharide, which is thought to be a consequence of a weak gel structure ( 1 22).
Windhab et al. (126) established stability domains for emulsions processed
under various conditions using different rotor/stator gap geometries. They reported a linear relation between the sensory thickness impression from the spoon
test and the ratio of G/G from the dynamic rheometry for optimally processed
and stable emulsion samples.
R@nnet al. (127) recently reported on the predictionof sensory properties
from dynamic rheological measurements for low-fat spreads. Ten commercial
low-fat spreads are evaluated bothby a trained sensory panel and by SAOS measurements. Two of the I 3 properties evaluated by the panel, namely, meltability
of the temperature sweeps
and graininess, are successfully predicted from features
(G* vs. T) from the rheological measurements.
5. Determining GlassTransitionTemperature
Materials with amorphous or partially amorphous structures undergo a transition
from a glassy solid state to a rubbery viscous state at a material-specific temperature called the glass transition temperature,
Tg (29,38,44). Glass transition in
amorphous food materials generally occurs over a range
of temperature rather
than at a single temperature (44,128,129).
The technological importanceof the glass transition phenomenon for different types of foods has been discussed in detail in the literature (44,130-136).
The glass transition or the associated parameter Tg has a great effect
on the
processing,properties,quality,safety,andstabilityoffoods(137).
Tg or the
difference between the temperature of a material and
its Tg (Le., T-Tg) affects
the physical and textural properties of foods (e.g., stickiness, viscosity, brittleness, crispness, or crunchiness), the rates of deteriorative changes (e.g., enzymatic
reactions, nonenzymatic browning, oxidation), and the success of many processes
(e.g., flavor encapsulation, crystallization). Hence, the knowledge of Tg is essen-
320
Gunasekaran
Ak and
tial in assuring the quality, stability, and safety of various foods such as confectionery products, breakfast cereals, baked goods, coated flavors, frozen products,
and food powders (128,130,133,134,137-146).
At the glass transition temperature,* properties such as the thermal expansion coefficient, the dielectric constant (for polar materials), and the heat capacity
exhibit a discontinuity. Thus, techniques measuring such property changes have
of Tg (e.g., dilatometry, calorimebeen developed for experimental determination
try) (29,39). Differential scanning calorimetry (DSC) is probably the most commonly used technique for determining Tg. Besides DSC, the
utility and advantages of other techniques such as nuclear magnetic resonance (NMR), electron
spin resonance spectroscopy (ESR), and dynamic mechanical (thermal) analysis
(DMA or DMTA) have been increasingly appreciated (148-153). Kalichevsky
et al. (151) stated that the dynamic mechanical techniques are more sensitive
than DSC to the molecular motions around Tg and the relaxations below Tg.
When dynamic mechanical spectroscopyis employed within the linear viscoelastic regime to determine Tg, the storage and loss moduli (G' and G" or E'
and E"), and loss tangent (tan6 = G"/G' or = E"/E') are measured as a function
of temperature at a constant frequency and a selected heating or cooling rate. In
of biological and synthetic polymers exhibits
glass transition, the storage modulus
a sharp drop with temperature. whereas the
loss modulus or tan 6 shows a characteristic peak (29,39,152,154-157). The decrease observed in modulus of amorphous synthetic polymers is typically about 3 orders of magnitude (29), whereas
that observed in biopolymers is about one order of magnitude (148,151-153).
Moreover, as Peleg (158) demonstrated, the plot of stiffness or rigidity (E', G')
versus temperature in the transition region of biomaterials has a downward concavity, which cannot be described by conventional models, such as WLF ( 1 59),
developed for synthetic polymers. Peleg, therefore, suggested another model and
demonstrated its applicability to describe the stiffnessor rigidity versus temperature relationship of biopolymers at the transition region ( I 29,155,158,160- 162).
Different definitions have been used to facilitate experimental determination of Tg from the dynamic mechanical data: (a) temperature corresponding to
the onset of drop in storage modulus T,,,,,, (b) temperature corresponding to the
(c) temperamidpoint of the glass transition region for storage modulus Tlllldpolnl,
turewheretheextrapolatedline
of theinitialmodulusintersectsthat
of the
(d) temperature corresponding to the loss modulusG" peak
steepest slope TInlerrec,,
6 peak Tlans.These are schematiTG",and (e) temperature corresponding to the tan
of Tg for the same material
cally illustrated in Fig. 15. It must be noted that values
may differ slightly or significantly depending on the definition, the experimental
conditions (e.g., heating/coolingrate in DSC and test frequency in DMA or
321
Steepest slope
Initial slope
T
.,
'tan
Temperature
322
Gunasekaran
Ak and
DMTA), and the technique (e.g., DSCvs. DMA) (29,153,154). Hagen et al. (163)
reported that Tg values, for instance, of an unfilled natural rubber differby about
10Cwhen determined by using two commercial dynamic mechanical instruments (DMA vs. DMTA).
Frequently the temperature corresponding to G or tan 6 peak is used as
a marker ofTg (29,148,149,153,164). However, Peleg (157) showed
by computer
6 peak location does
simulations and published experimental data that the tan
not always correspond to the transition zone even for the same material with
different moisture contents. Peleg (157) has instead suggested, as a more meaningful index of Tg, to use the temperature at which 50% of the initial stiffness
(i.e., storage modulus, G) is lost.
Several factors, such as composition,* molecular weight, and features
of
chemical structure (e.g., cross-links, side groups) can alter
the glass transition
temperature of materials. Among the constituents of foods, water is an effective
andubiquitousplasticizer,whichlowersthe
Tg of mostbiologicalmaterials
(134). Plasticizers are relatively low molecular weight materials
that, when added
it
to amorphous polymers, lead to a large increase
in mobility and thus make
easier for changes in molecular conformation to take place.
Cocero and Kokini (148) demonstrated the plasticizing (or softening) effect
of water, as measuredby the storage modulusG, on the major protein component
(i.e.. glutenin) in wheat Hour. Hallberg and Chinachoti (166) used DMAto study
phase transitions in shelf-stable MRE (meal, ready-to-eat) bread. Three distinct transitions are reportedin fresh MREbread and among those the main transition temperature decreased from about 160
to - 1 1C as the moisture content
increased from about 2.6 to 28.8%. It is also found that transition temperatures
( 166). Gontard et al.
( 167)
remained nearly constant throughout 3 years of storage
on mechanical
reported on the strong plasticizing effect of water and glycerol
and barrier properties of edible wheat gluten films. Kalichevsky and Blanshard
of amylopec(164) studied the effectof fructose and water on the glass transition
tin to find that the effect of fructose on the Tg of amylopectin is greater at lower
water contents.
The depression of Tg, due to plasticization of amorphous components by
water or other plasticizers, to the vicinity
of ambient temperatures may have a
significant effect on the shelflife and stability
of foods (128,168).The importance
of plasticization by water becomes more evident when one considers the hygroof water plasticiscopic nature of most dehydrated foods. A typical manifestation
zation is the loss of crunchiness or crispness in snack foods and breakfast cereals
(161,169,170). It is important to note that the loss of crispness in the corn Hour
* It
is interesting to note here that Matveev et al. (165) calculated Tg of proteins from the amino
acid composition and reported good agreement with the experimental results.
lastic
Linear
extrudates and in the corn cakes occurred within the glassy state, meaning
that
the knowledge of Tg alone may not be sufficient to predict crispness (142,171).
in thelowWater can also exertanantiplasticizingeffectparticularly
moisture or low-a, region by causing an increase in puncture strength, modulus,
and brittleness (167,172). Moreover, addition of low molecular weight diluents
other than water (e.g., fructose, glycerol) to glassy polymers (e.g., amylopectin,
starch) on the one hand lowers Tg but at the same time exerts an antiplasticizing
effect on the mechanical properties of the polymer ( 172- 175).
IV. SUMMARYANDFUTURETRENDS
Evaluation of food quality parameters has been traditionally performed via sensory panel tests and/or destructive mechanical tests suchas TPA and the uniaxial
compression and tension. Such methodsstill remain popular as they relate reasonablywell to the consumer perception of macroscopic food quality. However,
increasing consumer demand for consistently high-quality products and the comto obtain fundamental information
petitive marketplace have led the food industry
to improve quality assurance and/or control operations. Linear viscoelastic methods arewidely used for obtaining information regarding structural organization
of
the food materials. Since food texture
is largely a manifestation of microstructural
organization, viscoelastic methods provide the basic information needed
to understand the factors that influence quality.
The transient methods such as stress relaxation and creep have been the
most commonly used viscoelastic methods. By appropriate mechanical models,
transient test data can be used to obtain structural information and determine the
viscoelastic properties of the materials. The theory of dynamic viscoelastic tests
has long been known (176) and has been used in the polymer industry (46,177).
However, the application of dynamic tests for studying food materials have been
to eitherlack of properinstrumentation
delayed until relativelyrecentlydue
and/or highcost. The advances in computerandinstrumentationtechnology
have led to improved rheometer design at an affordable cost. This has promoted
widespread use of dynamic rheological testing of foods. While the transient tests
are relatively easier to perform, they may take a long time, whereas the dynamic
a
tests allow us to obtain viscoelastic parameters fairly accurately and within
shorttime (at frequenciesabove 1 Hz). In addition,theability
of dynamic
rheometers to perform tests at a wide range of frequencies provides information
not easily obtainable by other methods.
In the case of high-speed processing
of foods, for example, viscoelastic characterization at a high frequency is very
critical.
Determining the fundamental structural characteristics of foods is still the
main focus of linear viscoelastic tests. Nevertheless, new research efforts have
324
Gunasekaran
Ak and
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141. D Torreggiani, E Forni,I Guercilena, A Maestrelli,G Bertolo, GP Archer,CJ Kennedy, S Bone, G Blond, E Contreras-Lopez, D Champion. Modifications of glass
transition temperature through carbohydrates additions effect upon colour and anthocyanin pigment stability in frozen strawberry juices. Food Res Int 32:44 1-446,
1999.
142 Y Li, KM Kloeppel, F Hsieh. Texture of glassy corn cakesas function of moisture
content. J Food Sci 63(5):869-872, 1998.
in relation to
I43 WLKerr, MH Lim, DS Reid, H Chen. Chemical reaction kinetics
332
Gunasekaran
Ak and
333
Biosensors in Food
Quality Evaluation
Sudhir S. Deshpande
Signature Bioscience, Inc., Burlingame, California
1.
INTRODUCTION
Quantitative determination of the composition and propertiesof both raw materials and processed food products is important from the viewpoint of quality and
safety of the food. Quality depends on color, flavor, aroma, texture, nutritional
attributes, and microbial content. Food safety relatesto the content of pathogenic
microorganisms and toxic compounds, including microbially produced toxins,
pesticides, and naturally occurring toxic and antinutritional compounds, as well
as compounds produced by processing. Food analysis thus presents several chalof foods available,
lenges, in large part because each analyte in the wide range
from fluid products such as milk and fruit juices to processed meats, is complex
and nonhomogeneous. Quality control testingin the food industry has been based
upon traditional analytical methods that require hours, if not days, to complete.
In recent years, the food industry is increasingly adopting food safety and
quality management systems that are more proactive and preventive than those
to rely on end-product testing and visual
used in the past, which have tended
inspection. The regulatory agencies in many countries are promoting one such
management tool, Hazard Analysis Critical Control Point (HACCP), as a way
to achieve a safer food supply and also as a basis for international harmonization
of trading standards. For HACCP to be effective and successful, rapid methods
for monitoring and verifying performance need to be available. This, in turn, has
provided an impetus for developing newer and improved detection methods and
technologies that are cheaper, better, and faster.
Until recently, rapid quality-monitoring methods for on- and near-line use
were considered not to be very accurate or very reliable. The consensus was that
335
336
Deshpande
Table 1 Factors to Consider in Using On-line Sensor Technologies for Food Process
Accuracy
Biosensors
337
II. WHATAREBIOSENSORS?
Deshpande
338
the physical stimulus; the transducer, which converts the stimulus into a suitable
signal; and the output system itself, which involves amplification, analysis, processing, and display that is usually electrical.
The term biosensor is now generally applied to those devices that employ a biologicaUbiochemica1 detection system. The reactive surface (e.g., the
biochemical or signal transducer) generates a measured response upon binding
with an analyte. The device and associated instrumentation (the physical transducer component) convert analyte-binding events into qualitative or quantitative
units of analyte concentration. Thus, the conversion of the analyte by the biochemical transducer into another chemical species and/or physical property is
sensed and converted into a measurable signalby the physical transducer. Immunosensors are essentially biosensors that use antibodies as signal transducers.
Molecular recognition of a specific sample analyte by the respective receptor is realized by the lock-and-key principle, which is the biocomponent or
the biocatalyst that gives the biosensor its specificity and selectivity ( 5 - 8 ) . It is
also involved in rapid recognition and detectionof specific molecules or chemical
reactions. This molecular recognition and detection mechanism, in general, creates a chemical change in or by the biological element proportional to the reaction, which can then be monitored by the physical transducer. The biocomponent
must, therefore, have intimate contact with a suitable transducing system. Enzymes, receptors, organelles, tissues, nucleic acids, antigens,
or antibodies, as
well as whole cells of bacterial, fungal, plant, or animal origin, can be used as
a biological detection system.
The physical transducers or measuring principles generally vary with the
type of biological detection system used in the biosensor. The transduction systems commonly used in biosensors are summarized in Table 2 . For example,
Table 2 CommonlyUsedBiosensorTransductionSystems
Transduction
system
Options
ElectrochemicalPotentiometry,amperometry,voltametry,ion-sensitiveelectrode
(ISE), ion-sensitive field effect transistor (ISFET), immuno-field effect transistor (IMFET)
Electrical
Surface
conductivity,
conductivity,
capacitance
Thermal
Calorimetry,
enzyme
thermistor
(heat
of
reaction
or
absorption;
TELISA or thermistor ELISA)
Paramagnetism
Magnetic
Optical
Fluorescence,
luminescence,
reflection,
absorption,
surface
plasmon
resonance, scattering, evanescent waves
PiezoelectricQCM.SAQ,
SH/APM, Lambwave,Lovewave
Biosensors
339
340
Deshpande
Blologlcal
element
Semipermeable
membrane
medium
Electrolyte
Signal
/
Transducer
Internal
membrane
111.
Biosensors
341
analytical system that doesnot need trained specialists to produce reliable results.
The aim isto develop a small, portable unit into which the sample canbe inserted
without preprocessing. The result should be obtained within seconds,
and the
answer should not be subject to interference or modification by the matrix, the
user, or environmental factors.
The optimum design of a biosensor is dictated by several basic physical
properties of the measuring system, as well as those of the media in which the
measurement is made (10,l I). Some of the most pertinent properties and characteristic behaviors of an ideal biosensor are described below.
A.
Sensitivity
B. Calibration
An ideal biosensor should be easily calibrated
by exposing it to prepared standard
solutions or gases containing different known concentrations
of the target analyte.
It is preferable to perform a calibration procedure only once to determine the
sensitivity of the biosensor for subsequent measurements. However, in practice
it is usually necessary to make periodic calibrations at regular intervals to characterize changes in the sensitivity with time.
Linearity
C.
A biosensor need not be linear to be practically useful, as long as the calibration
curve can be obtained with sufficient accuracy to interpret the biosensor signal.
Ideally, the measurements should be made in the linear range of the calibration
curve.
342
Deshpande
D.DetectionLimit
Ideally, the range of expected analyte concentrations for an intended application
be useful. Theoretically, the
mustbe detectable in order for the biosensor to
lowest limit of detection is dependent upon the resolution of the electronic instrumentation used for the measurement.In practice, other considerationsmay often
cause the lower limit of detection to be higher. For example, electrochemical
transducers using potentiometric measurements
may have interference from other
ions and surface reactions that limit the measurement.
E. BackgroundSignal
The background signal may make the determination of the lower limit of detection for a biosensor difficult to determine accurately. Current leakage, small potential differences in the electronic instrumentation or due to dissimilar metalto-metal contacts in the wire leads of the biosensor, or electrochemical factors
are often the major causes for the background signal.
F. Hysteresis
If the outputof a sensor follows different paths during increasing and subsequent
decreasing inputs,it is said to suffer from hysteresis.In essence, it can be considered as an effect of measurement history. An ideal biosensor should not be affected by its past history of measurements and would have zero hysteresis. Hysteresis could also affect the transient responses of the biosensor. In some cases
it can be minimized by making slow changes.
G. DriftandLong-TermStability
Drift is a slow change in output without any change
in input quantity, and it
directly relates to the long-term stability
of the measurements takenwith a biosensor. An ideal biosensor should have zero
driftand constant sensitivity for its
It
entire lifetime, or at least during the time the measurements are being made.
is usually necessary to recalibrate the biosensor at frequent intervals during its
for thedrift in sensitivity with time.
use, so thatthesignalcanbecorrected
Generally, drift is approximately linear with time between calibrations (10).
H. Specificity(Interference)
The response of a biosensor should be specific to only the changes in concentration of the target analyte andnot be influenced by the presence of other chemical
Biosensors
343
1.
DynamicResponse
The physical properties and relative size of a biosensor determine how quickly
it will respond to a change in concentration of the target analyte it measures
(10,ll). The principal mechanism that affects the dynamic response
is usually
simple diffusion of the chemical species from the sample to the active surface
of the transducer. Itis desirable that the response be as fast as possible (but
comparable to the speed of data acquisition).
J.
Flow Sensitivity
K. TemperatureDependence
Since all the physical properties are dependentupon temperature, biosensor measurements should ideally be made under isothermal conditions. Special efforts
should be made to minimize effect of varying temperatures on the output.
L.Signal-to-NoiseRatio
A large signal-to-noise ratio is an indication that the biosensor is responding
strongly to the analyteof interest, and the other extraneous effects are
kept rather
small. The signal-to-noise ratioof the biosensor measurements can be improved
by using digitalfiltering techniques to clean up the signal,
by ensemble averaging
that is synchronizedwith the repetitive stimulus,by reducing the cutoff frequency
of the amplifier, or by passing the output through analog filters.
M. Lifetime
The biological elementsused in a biosensor are generally the least stable components of the system. The lifetime of a biosensor may be dependent on the total
number of measurements made or may depend on the magnitude of the analyte
concentrations measured. Higher concentrations may lead to more rapid losses
in sensitivity. It may also be necessary to store the biosensor under refrigeration,
Deshpande
344
or the biological element may need to be supplied with a specific chemical environment to maintain its bioactive properties.
also need to be
In addition to the above properties, several other factors
considered during the design and development of biosensor products, including
the following:
Instruments should be small, self-contained, cheap and robust, capable of
interfacing with existing central laboratory systems.
The user interface should be simple for use by even unskilled operators.
No volumetric measurement of the specimen, e.g., by pipette, should be
necessary.
The test specimen should be the only addition; no further reagent addition
should be required.
Results should be unaffected by the test specimen matrix.
The time between presentation of the specimen and final result should be
less than 5 minutes.
Built-in standardization and controls are required.
Results obtained need to be quantitative, with a printed copy of the results
available.
The detection limit should be subnanomolar.
A wide analytical range is required, with
a capability for immunochemistry,
clinical chemistry, enzymology, DNA probe measurements, and a variety of other applications.
The potential for simultaneous measurement of multiple analytes should
be considered.
There should be a good correlation of results with existing test methods.
Biosensor consumables must be cheap to manufacture in bulk.
IV.
BIOLOGICALTRANSDUCERS
Biosensors
345
Deshpande
346
method, however, is that the binding forces between the biocomponent and the
in matrices
support cannot be controlled easily. Entrapping the biocomponent
such as gels, polymers, pastes, or inks considerably improves its stability. However, to improve the performance of the biosensor, it may be essential to covalently link the biological transducer to the solid support. A prerequisite for these
immobilization procedures is the availability of functional groups on the protein
as well as on the solid support. Covalent binding
of the biocomponent to the
solid phase often results in greater precision and accuracy of the assay by eliminating within- and between-assay variation.
V.
PHYSICALTRANSDUCERS
Almost all biosensor technologies fall into four methods of signal transduction:
optical, mass, electrical, and thermal. The nature of the sensor surface, the sensors ability to measure in real time, and whether the sensor can be reused
tend to be distinctive in each method. Similarly, the merits of each method are
dictated by the application and breadth that it brings to the diagnostics industry
compared to technologies and products currently in the marketplace. The basic
characteristics of these four classesof signal transduction technologies are briefly
described below.
A. Optical Transducers
Signal transduction by monitoring changes in optical properties can be measured
by fluorescence, absorbance, refractive index, interferometry, diffraction,or polarization. Optical transducers can
be broadly divided into two further categories:
extrinsic and intrinsic. In the extrinsic mode, the incident light passes through
the sample phase and interacts directly with the sample. In the intrinsic mode,
the incident wave is not directed through the bulk sample but propagates along
a wave guide. It thus interacts with the sample only at the surface within the
evanescent field.
6. MassTransducers
The surface acoustic and piezoelectric methods of mass signal transduction are
primarily based on the measurement of interdigitating arrays of the planar devices. This type of biosensor is sensitive to changes in density, elasticity, or
electrical conductivity of the surface on whichthe acoustic wave propagates.
Piezoelectricsystemsareessentiallymicrobalancesbased
on quartzcrystals.
These crystals are mechanically distortedwhen subjected to an electric potential.
347
Biosensors
C.ElectricalTransducers
There are six typesof electrical transduction: potentiometric, amperometric, conductimetric, capacitance, impedance, and oxidation state. Of these methods, potentiometric (monitoring pH changesof the medium) and amperometric (monitoring the redox potential) have found the widest applications in the biosensor field.
D. ThermalTransducers
Since most enzyme- or microorganism-catalyzed reactions are accompanied by
considerableheatevolution,thermaltransducers,whichmonitortemperature
changes by thermistor devices, may find broader applications than other physical
transducers in the food industry.
A summary of various transducers used
in biosensor construction, with
their potential advantages and disadvantages, is presented in Table 3.
Table 3 AComparisonofTransducersUsedinBiosensorConstruction
Transducer
Sluggish response, requires a
stable reference electrode.
susceptible to electronic
noise
Simple, high selectivity
Low sensitivity
Simple, high selectivity
Low sensitivity
Remote sensing. low cost.
Interference from ambient
miniaturized, can be free
light, requires high-energy
from electrical interference
sources, only applicableto a
narrow concentration range
Fast response, simple stable
Low sensitivity in liquid apoutput signal, low cost for
plications, interference rereadout dcvicc. 110 spectal
sulting from nonspecific
sample handling
binding
Vcrsatility, free from optical
Expensive. cumbersome. reinterferences such a s color
quires a large amount of
and turbidity
enzyme
Low cost. mass production, sta-Temperature-sensitive, fabrible output. rcquires very
cation of different layers
small amount of biological
on the gate is not pcrfccted
material. can monitor several analytes simultaneously
Oxygen electrode
H 2 0 zelectrode
Optical systems
Piezoelectric
Calorlmetric
Ion-selective tieldeffect transistor (ISFET)
Deshpande
340
VI.
BIOSENSOR TYPES
Biosensors can be classified based on either the biocomponent used (e.g., enzymatic, immunological, or cellular) or the signal transducing system (e.g., potentiometric, amperometric, optical, thermal, etc.). The latter classification is used
in this discussion.
A.
Optical
Optical biosensors make use of the physical properties of light in various ways
to detect small changes in analyte concentration. Based on the principle used,
as surface plasmon resooptical biosensor devices could be broadly classified
nance (SPR), total internal reflection (TIR), or photon correlation spectroscopy
(PCS) instruments.
SPR instruments containing an antibody layer detect minute changesin the
refractive index on binding of the antigen (15). These changes are related to the
size of the antigen as well as the conformational change of the antibody-antigen
complex on binding. The latter involves solvent reorganization and protein unas a shift in the angle
folding. The change in the refractive index is then detected
of total absorption of incident light on a metal layer (usually gold or silver) carrying the antibodies. This system can be usedas a biosensor because the surface
plasmon resonance, which extends horizontally along the metal surface for about
100 nm, has an associated, exponentially decreasing, evanescent
field that extends
a few hundred nanometers. When
vertically into the surrounding medium for
molecules bind at the surface within the evanescent field (antibodies are about
10 nm in diameter), they perturb the field and hence the surface plasmon resonance, which therefore alters the angle of the light at which SPR occurs.
A schematic diagram of an SPR device consisting of a prism on a glass
slide carrying a thin metal layer is shown in Fig. 2. SPR-based instruments have
been successfully used for the characterization of dye monolayers (1 6), for the
measurement of antibody concentration in liquids (15), and for the binding of
the anti-human serum albumin without prior incubation or separation steps (17).
Inexpensive immunosensing devices based on SPR technology could be manufaca metalized diffraction grating on
a
tured using holographic techniques using
plastic support (18). These devices could be used for the detection of microbial
pathogens, toxins, and pesticide residues in plant and animal food products.
TIR-based biosensors detect internal reflections in a light guide. The guide
surface is coated with antibodies that come in contact with the analyte (7,s).TIR
immunosensors make use of the evanescent wave penetrating only a fraction of
a wavelength into the optically rarer medium when light coming from
an adjacent
denser medium is incident on the interface with an angle above the critical angle
(Fig. 3). Changes in the surface refractive index or absorptivity then reduce the
349
Biosensors
Reflected
light
Incident
light
- Glass substrate
- Metal
Sensitizing layer
( antibody )
""""""""
""""""""
""""""""
""""""""
""""""""
Sample solution
""""""""
""""""""
""""""""
( antigen )
""""""""
""""""""
""""""""
""""""""
Fig. 2 Biosensorsbasedonsurfaceplasmonresonance
(SPR) principle to measure
analyte/antigen concentrations in solution kinetically without prior incubation or separation steps. The device measures changes in the refractive index upon analyte-antibody
binding.
"_""""""_
_""""""""
""""""""
""""""""
""""""""
""""""""
""""""""
Light
~-guide
Incident
light
Sample solution
Sensitizing layer
-Filter
Detected
light
Fig. 3 A schematic diagram ofa biosensor based on total internal reflection (TIR) geometry. Based on the evanescent wave principle, it measures the effects of analyte binding
to its antibody or receptor immobilized on the surface.
350
Deshpande
B. Acoustic
Acoustic biosensors sensitive to changes in the density, elasticity, or electrical
conductivity of a surface on which the surface acoustic wave (SAW) propagates
have potential in agriculture and veterinary diagnostics for monitoring food quality. The variety and ingenuity of these
devices is extensive. Two extensive reviews cover the use of piezoelectric transducers in detection of mass (22). The
methods rely, in general, on measuring the changes in vibrational resonant frein mass on
quency of piezoelectric quartz oscillators that result from changes
oscillators surface.
A SAW immunosensor consists of a piezoelectric crystal such as quartz or
lithium niobate carrying thin-film interdigital electrode arrays. Radiofrequency
excitation of the electrode pair creates a synchronous mechanical surface wave
that is propagated on the surface of the piezoelectric substrate and recorded by
either another electrodepair on a SAW delay line or
by the same pair after refleca chemical
tion on a SAW resonator device (7,19). A schematic diagram for
sensor based on a SAW delay line is shown i n Fig. 4.
Other acoustic devices such
as a quartz microbalance or a piezoelectric
oscillator, based on the dependence
of the resonance frequency of a vibrating
quartz crystal on its mass, can also be used as biosensors. The quartz crystals
in a liquid, and if the
oscillate when immersed either partially or completely
solution properties such as density, viscosity, and conductivity are kept constant,
the change in mass of the quartz crystal can be detected (8,23). Imrnunosensors
based on the piezocrystal microbalance principles have already been commercially introduced (24).
Using conventional antibody-antigen interactions on thin substrates, detection limits of nanomolar down to picomolar concentrations have been claimed
with ideal test samples, although this is perhaps unlikely to be achieved with
normal food samples. Manufacture of the devices to produce cheap, disposable
351
Biosensors
Sample channel with
sensitizing layer for
specific bindingof
the analyte
lnterdigital
electrodes
- I' 1
Piezoelectric
substrate
SAW
delay
Fig. 4 Chemical sensor based on a surface acoustic wave (SAW) delay line.
units with uniform characteristics is also likely to pose problems, and provision
of multianalyte analysis on a single unit will be a considerable challenge.
C. Semiconductor
Field-effect transistor (FET) devices similar
to the ion-sensitive FET devices(ISFET) have been used for the detection of minute potential changes associated
with the formation of an antibody-antigen complex (25). Such direct immunosensors are called IMFETs. ISFET and other microelectronic gas or ion sensors
can also be used in indirect electrochemical immunoassays using enzyme labels
instead of the traditional ion-selective electrodes of gas probes (8,19,26). The
fast response,good signal-to-noise ratio,and small sizeof FET devices are particularly useful in flow-injection analysis techniques. Because of manual handling
in conventional ELISAs (chloramphenicol acetyl transferase, or CAT assays),
the variations in the binding time between the reactants need to be eliminated
by extending the timeso that slight variationswill not influence thefinal outcome.
In contrast, in flow-injection analysis, the only factor
that varies is the time during
Deshpande
352
which the pulse of the substrate is introduced. This makesit possible to keep the
variation between assays to less than 2%. Potentially, the FET devices can be
constructed as inexpensive disposable units, although their full potential has not
yet been explored by the food diagnostics industry.
A schematic diagram of a prototype IMFET devicein which the metal gate
is replaced by an antibody or antigen immobilized on a membrane is shown in
is controlled
Fig. 5. The conductivity of the n-channel region in the p-type silicon
by the strength of the electric field at the gate and is measured by the application
its proper
of a voltage between the source and drain electrodes. A prerequisite for
functioning, however, is to have the solution-membrane interface remain ideally
polarized and thus impermeable to the passageof the charge. Failure to meet this
criterion results in the poor sensitivity of the instrument.
Yet another semiconductor device, TELISA (or thermistor ELISA), has
great promise in the diagnostics industry. The heat-sensitive enzyme thermistor
in a small,wellmeasurestheheatevolved
in anenzyme-catalyzedreaction
of an enzymethermistor is shown
insulatedchamber.Aschematicdiagram
in Fig. 6. A small sample volume is injected
in a comparatively rapid buffer
stream. A short sample pulse results in a temperature peak that is proportional
a linear operating
to the concentration of the analyte (enzyme substrate) over
range of concentration. Normally, the thermograms are evaluated by peak height
Encapsulant
Membrane
insulator
"""""
n - Si source
p-Type
silicon
substrate
n Si drain
<
Antigen
Immobilized
antibody
Fig. 5 Immuno-Field-Effect Transistor (IMFET) sensor for direct potentiometric monitoring of the antibody-antigen complex.
353
Biosensors
Polyurethane
//
Heat Exchanger
\
Thermostatted
Insulation
Aluminum Block
Auxiliary or
Column
Reference
Column
Enzyme
Fig. 6 A schematic representation of an enzyme thermistor that usesflow injection analysis for analyte detection.
D. Electrochemical
Electrochemicalbiosensorssimilartoenzymeelectrodescan
also beusedin
developing rapid assays for food quality control. They have a unique blend of
sensitivity with simplicity that resultsin a family of low-cost, rapid, and portable
chemical sensors capable of operating in turbid solutions such as food analytes
(29).
Deshpande
354
e5mV
I
"
Reference
Electrode
Titanium Wire
Electrode
Immobilized anti-hCG
Fig. 7 A direct potentiometric immunosensor based on a titanium wire electrode.
Biosensors
355
7.
Potentiometric
Potentiometric membrane electrodes, such as pH, solid-state, polymer, and gassensing electrodes, have been used as internal sensing elements for biosensors.
A typical classification of these ion-selective electrodes
(ISE) is shown in Table 4.
Gas-sensing electrodes are by far the most popular choice because of the inherent
selectivity of gas-permeable membranes (32,33).
Potentiometric biosensors based on gas-sensing membrane electrode consist of a hydrophobic gas-permeable membrane anda pH electrode separatedby a
thin layer of internal electrolyte. The differences between the several gas-sensing
electrodes are mainly apparent with respect to their internal filling solutions and
their operating pH ranges. Gas-sensing electrodes are quite selective since their
membranes are permeable only to gases.Samplesolutionshavetobetotally
aqueous since the hydrophobicity of these membranes would be compromised
by organic solvents, thereby leading to membrane failure.
The usefulness of gas-sensing electrodes is often hampered by pH limitations imposed by samples or by the biocatalysts used in the construction of biosensors. Compromises often have to be made in order to satisfy both the pH
requirements of the electrodes and those of the biological components, with the
latter usually having stringent pH limitations due to their biological nature. The
pH requirements of the gas-sensing electrodes themselves arise from the fact that
the electrodes are responsive onlyto the gaseous formof the measured substrate.
Ion-selective electrodes, however, can tolerate a certain degree
of turbidity in
samples and are thus especially suited for food analysis.
Someexamples of contemporarypotentiometricmembraneelectrodes
are summarized in Table 5. The lifetimes of these biosensors vary from hours
Solid state
Liquid ion-exchange membrane
With coating over membranes of
ion-selective electrodes
ofmembrane
sensor
Table 5 SelectedExamplesofPotentiometricBiocatalyticMembraneElectrodes
Internal
sensing
Lifetime
Substrate
Ref.
Enzyme electrodes
Adenosine
L-Alanine
Glutamine
Guanine
Gluconate
Histidine
Salicylate
Tyrosine
Bacterial electrodes
L-Arginine
L-Aspartate
L-Cysteine
L-Glutamate
Glutamine
L-Histidine
NAD+
Nitrate
Nitrilotriacetic acid
Pyruvate
Serine
Sulfate
Sugars
Tyrosine
Uric acid
Plant tissue electrodes
Ascorbate
Cysteine
Dopamine
Glutamate
Phosphatelfluoride
Pyruvate
Urea
L-GlutaminehAsparagine
Tyrosine
Spermidine
Animal tissue electrodes
AMP
Guanine
Glucosamine 6-phosphate
kidneyPorcine
Glutamine
(d)
element
Biocatalyst
Adenosine deaminase
L-Alanine dehydrogenase
Glutaminase
Guanase
Gluconate kinase-6-phospho-~gluconate dehydrogenase
Histidine decarboxylase
Salicylate dehydroxylase
L-Tyrosine decarboxylase
Streptococcus fueciutn
Bacterirtm caduveris
Proteus morgnnii
Escherichia coli
Sarcina jlava
Pseudomonas spp.
Esclzerichia colilNADase
Azotobacter vinelundii
Pseudomonas spp.
Streptococcus faecium
Clostridium ncidiurici
Desulfovibrio desulfuricans
20
IO
1
42
4
30
12
0.42
IO
6
21
14
21 s
7
14
30
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
14
51
52
IO
53
54
3
8
55
50
56
Cucumber
Cucumber leaf
Banana pulp
Yellow squash
Potato tuberlGlucose oxidase
Corn kernel
Jack bean meal
Magnolia flower
5-6
57
28
58
59
7
28
7
94
14
60
Sugar beet
Pea seedling
8
20
65
66
Rabbit muscle
Rabbit liver
Porcine kidney
28
14
21
67
68
69
30
70
10
61
62
63
64
Biosensors
357
to weeks, depending upon the biocomponent used in the preparation of the electrode.
2. Amperometric
Amperometric biosensors are usually based on oxidoreductase enzymes, which
commonly use oxygen, NAD', or NADP' as the electron acceptor that recycles
as the electron
the enzyme after the substrate reaction. Enzymes using oxygen
acceptor are commonly called oxidases, while those usingNAD' or NADP' are
called dehydrogenases or reductases. Of these two groups, oxidase enzymes are
the most commonly used as biocomponents.
The first amperometric biosensors used the Clark oxygen electrode as the
physical transducer. The oxygen electrode is generally constructed using a twoelectrode system, e.g.,a platinum cathode and a silver anode. A gas-only permeable membrane (e.g., Teflon) covers the tip of the cathode. A biochemical layer
(most often an enzyme) is then placed on the oxygen electrode and secured with
a coarse membrane that allows the passageof analyte, thus forming a biosensor.
A negative potential is then applied to reduce oxygen at the cathode. The reduction current is directly proportional to the oxygen concentration. In the presence
of substrate, the enzymatic reaction decreases the oxygen concentration, and the
decrease in the oxygen reduction current is proportional to substrate concentration.
Theprincipaladvantage of anoxygenelectrode-basedbiosensor
is its
excellentselectivity. The selectivityoftheenzyme
is not compromised by
the oxygen electrodes, because only gases can pass through the membrane and
only substances that are reducible at the applied potential can interfere.
The limitations of this type of biosensor are that it is sensitive to ambient oxygen concentrationsandthegas-onlypermeablemembranecanbecomeblockedor
clogged, which often necessitates the use of a second coarse membrane (e.g., a
dialysis membrane). As the membrane layers become thicker and more complex,
the response and recovery times increase, and the biosensor may require more
frequent calibration (71). The maintenance of calibration for many biosensors is
dependent not only on enzymatic activity but also on the constancy of the diffusional pathways for reactants and products. The thicker and more complicated
the biochemical layer and its associated membranes, the more difficult
it is to
maintain constant diffusion of reactants and products, thus increasing the frequency of calibration.
Oxygen electrode-based biosensors have been used to determine a variety
of enzymatic substrates including glucose, monosaccharide, hypoxanthine,
lac(8).
tate, amino acids, sulfite, salicylate, oxalate, and pyruvate
Hydrogen peroxide-and dehydrogenase-based biosensors also operate on
similar principles.The former functionby oxidizing hydrogen peroxide,a product
358
Deshpande
of the enzymatic reaction, while the latter utilize the enzymatic oxidation of
NADH or NADPH.
The mainproblemwiththedehydrogenase-basedbiosensors
is thatthe
NADt/NADH and NADP+/NADPH redox couples are not always completely
reversible, and when the reduced formis reoxidized, some product can form. this
oftenirreversiblyadsorbsontheelectrodesurface,causingelectrodefouling.
After repeated use, the oxidized cofactor decreases
in concentration if the electrochemical recycling is not 100% efficient or if it is not confined to the electrode
surface. Thus, it may become a limiting factor in the enzymatic reaction. In conjunction with this problem, if the undesired product fouls the electrode surface,
the current response of the electrode decreases with time because the active electrode area is decreasing. Careful attention to the choice of electrode material and
electrolysis conditions can minimize these problems.
Alternatively, a mediator with better electrochemical properties can be used
to recycle the NAD'/NADH redox couple. This approach was used
in the construction of ethanolandlacticacidsensors,
in whichflavinmononucleotide
(FMN) was the mediator, and its reduced form, FMNH?, was oxidized at a platinum electrode, producing a current directly proportional to substrate concentration (72).
Amperometric biosensors that use oxidase enzymes normally rely on oxygen as an electron acceptorto recycle the enzyme after conversion of the substrate
to the product. The upper linear rangeof these biosensors is limited at high substrate concentrations because oxygen becomes a limiting factor due to its limited
solubility. As the substrate concentration increases, more oxygen is used
in the
enzymatic reaction; when the concentration of the oxygen becomes too low, it
becomes a limiting factor. Consequently, each increasein substrate concentration
results in less of an increase in the overall reaction rate, and the biosensor response begins to level off, eventually becoming independentof substrate concentration and limited by the oxygen concentration. Also, hydrogen peroxide proin sufficient concentration, is known to
duced from the oxygen, when present
deactivate many enzymes (7 1).
Electron mediators can be used
to alleviate both problems by replacing
oxygen as the electron acceptor. This approach usually extends the linear range
and often lowers the working potential, thereby reducing noise, background curto interferences.Byeliminatinghydrogenperoxide,the
rent,andsignalsdue
operating lifetime of the biosensors is often extended. The commonly used electronmediatorsincludetetrahiafulvalene,hexacyanoferrate,N-methylphenazinium, and ferrocene and its derivatives (7). Ferrocenes are popular because they
are hydrophobic, exhibit good electrochemistry, and can be structurally altered
to change their redox potential. Hydrophobicity is important because it prevents
the mediator from leaching from the electrode surface when used
in aqueous
systems.
Biosensors
359
Table 6 ApplicationsofBiosensorsintheFoodIndustry
Compound
measured
Applications
Amino acids
Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glutathione, histidine, leucine, lysine,
methionine, N-acetylmethionine, phenylalanine, sarcosine,
serine, tyrosine, tryptophan, valine
Amines,amides,hetero-Aminopyrine,aniline,aromaticamines,acetylcholine,chocyclic
compounds
line,
phosphatidylcholine,
creatine,
guanidine,
guanosine,
penicillin, spermine, creatinine, uric acid, urea, xanthine,
hypoxanthine
Carbohydrates
Amygdalin,
galactose,
glucose,
glucose-6-phosphate,
lactose,
maltose, sucrose, starch
Carboxylic
acids
Acetic
acid,
formic
acid,
gluconic
acid,
isocitric
acid,
ascorbic acid, lactic acid, malic acid, oxalic acid, pyruvic
acid, succinic acid, nitriloacetic acid
Gases
NHI, Hz, CH,, SO?, NO
NAD(P)H,
ATP,
Cofactors
AMP,
Hz02
Inorganic
ions
Fluoride.
nitrite,
nitrate,
phosphate,
sulfate,
sulfite,
Hg",
Zn"
Complex
variables
Antibiotics,
assimilable
carbohydrates,
assimilable
substances, biological oxygen demand, freshness of meat, mutagens, vitamins
Alcohols,
phenols
Acetaldehyde,
bilirubin,
catechol,
cholesterol,
cholesterol
ester, ethanol, glycerol, glycerol esters, methanol, phenol
VII.
DNAPROBETECHNOLOGY
360
Deshpande
or more of these label systems. Therefore, the detection systemsused in the field
of immunoassay are also applicable to DNA probing.
Nucleotide-probing techniques rely primarily on the ability of the labeled
DNA and RNA probes to detect antigen by binding to the complementary arrangements of target nucleotides. Since such binding, under appropriate assay
conditions, can be extremely strong and specific, DNA/RNA probe assays can be
extremely specific and sensitive. These characteristics are mandatory for devising
sensitive and specific diagnostic assays. Furthermore, these probes can be amplified by polymerasechainreaction(PCR)usingappropriateenzymesystems,
thereby providing the starting material in great quantity for testing.The PCR is a
three-step process that duplicates a DNA fragment bound by two oligonucleotide
primers.Eachprimer is complementary to one strand of thedouble-stranded
DNA template used. The first step of the process is initiated by denaturing or
separating the double-stranded DNA target into its single-stranded components
by heating the sample to 90-95C in the presence of a large excess of the two
primers. In the second step, the temperature of the reaction is lowered to about
10C below the melting temperature of the primers, and the primers are allowed
to anneal or hybridize to their complementary sequence on the DNA template
molecule. The third step of the reaction is carried out by raising the temperature
of the reaction to 70-73"C, the optimal temperature for extension of the primers
by a DNA polymerase. One cycle of denaturation, annealing, and primer extension results in a doubling of the DNA target sequence. By repeating the cycles
as many as 25-30 times, the DNA sequence flanking the primers can be amplified
as much as a millionfold. The PCR can also be used to assay RNA in a sample
if the RNA template is first transcribed into DNA by reverse transcriptase.
Blackburn et al. (73) described an electrochemiluminescence (ECL) for the
detection of PCR
reaction
products
using
ruthenium(I1)
tris(bipyridy1)
(Ru(bpy)32+)to label the probe. ECL assays detect the hybridization of labeled
of PCR products is demonstrated
probes to nucleic acid sequences. Quantification
in Fig. 8. I n this format, the PCR is first used to amplify the specific genes by
use of two primers, one of which is biotinylated. The double-stranded DNA is
then captured on streptavidin-coated microparticles and washed with an alkaline
solution to denature and separate the strandsof DNA. Incubation of the particlebound DNA with Ru(bpy)32'-labeled probes is followed by washing the sample
and quantifying the particle-bound label.
Other recent amplification systems include the ligase chain reaction (LCR),
Q-p replicase,Syva'sPA-singleprimeramplification,andself-sustainedsequence replication (3SRJ (8).
Although the current nucleotide probe-based techniques are slow and laborious in comparison to technologies such as immunoassays, several commercial
products are already being marketed. DNA probe technology will find its general
use in medicine, clinical sciences, and food technology. In the food technology
361
Biosensors
PCR Reaction
Biotin
mmmOiigo
Polymerase
extension
Bindina to Beads
BS
/"".
WBb
Fig. 8 Diagram of the procedure for the detection of polymerase chain reaction (PCR)
products in the DNAprobe assay. B = Biotin, S = streptavidin-coated microparticles;
Ru(bpy),".
Deshpande
362
IX.
BIOLUMINESCENCE ASSAYS
Luminescence techniques based on the detection of light generated by enzymemediated reactions represent someof the fastest assays currently availablefor the
of the few rapid technologiesto
detection of microbial contaminants and are some
be currently utilized extensively in the food industry. Techniques based on this
phenomenon can be divided into two broad categories: ATP bioluminescence
and bacterial bioluminescence.
Biosensors
363
Luciferase
LUCIFERIN.AMP
PPI
facturer
Deshpande
364
Table 7 CommerciallyAvailableATPBioluminescenceHygiene
Monitoring Kits
name Instrument/Test
Bio-Orbit"
GEM@
Hy-Lite'
InspectoPlSystem SureTM
Lightning@
Lumac@
Luminator"/PocketSwabTM
Uni-Lite@'/Uni-Lite@Xcel
Profile"
X.
EMERGINGTECHNOLOGIES
Biosensors
365
A.
ElectronicNose
The world of electronic nose technology mimicking the human nose and
its utility
in the flavor industry as both a research and quality assurance tool literally exploded in the United States in late 1994. Traditionally, much of the flavor testing
in the food and beverage industries as well as flavor and fragrance manufacturers
has relied on sensory evaluation by trained expert panels, such as U.S. Food and
Drug Administration (FDA) inspectors, who assess seafood freshness, coffee and
tea tasters, or brewmasters and wine stewards. Flavor and fragrance companies
rely heavily on sensory evaluation throughout the development process to
final
production. The results areusually highly subjective, prone to error, and difficult
to relate to other analyses. Current research seems to indicate that no two humans
really perceive the same aroma quite the same, nor can they communicate a common descriptor easily due to an array of cultural, presuppositive, and associative
memory effects (81).
20-300 daltons)
Flavor molecules are generally small (molecular weight
and polar and can be detected by humans at levels below one part per billion.
To date, more than 6000 flavor-active compounds have beenidentified, and more
are being discovered each passing day. Recognizable flavors or odors arise from
the specific contribution of complex mixtures of many odorous molecules, each
of different concentration. Attempting to detect complex odors at these levels by
conventional analytical techniques is very expensive and not always possible. It
is therefore not surprising that traditional (organoleptic) methods of odor assessment have survived for so long. Analytical techniques can objectively discriminate odors, but the sample must be separated into
its individual components using
gas chromatography/mass spectroscopy ( G U M S ) techniques. While these techniques can provide a chemical tally of volatiles in a food, flavor, or fragrance,
aroma, judge its character, or
none can tell if the identified component has an
predict synergistic effects with other compounds.
In contrast, human olfaction
can discriminate aromas without separating mixtures into individual compounds.
In the human brain, a kind of pattern recognition may also take place to decode
signals sent from receptor cells located in the human olfaction system (82).
In recent years, significant interest in the use of sensor arrays to discriminate between odors has arisen. If an array of nonspecific sensors could be compiled to rival the human olfactory system, then the sample need not be separated
(83,84). Analysis would prove
and could be monitored analytically as a whole
rapid, nondestructive, and continuous. The term electronic nose (or artificial
366
Deshpande
nose) has been coined to describe such an array of chemical sensors, where
each sensor has only partial specificity to a wide range of odorant molecules,
coupled with a suitable pattern recognition system software.
In order to discriminate aromas by mimicking the human olfaction system,
attempts to construct the so-called artificial nose by utilizing various gas sensors have been made since the early
1980s (85.86). Metal oxide semiconductor
sensors (85,87-89), surface acoustic waves (SAW) sensors (90), and quartz resoits own
nator sensors (91) seem potential methods. Although every sensor has
advantages and limitations, there are
not such sensors that can show perfect selectivity to specific compounds or a group of compounds. Pattern recognition of
into every analytical
the responses from a multisensor array was incorporated
procedure due to the nonselectivity of gas sensors. Early efforts were largely
focused on detecting specific gases or hazardous compounds on the basisof their
response patterns (88-90). From the viewpoint of aroma analysis, discriminating
is essential. As the first step for
a particular gas mixture from other mixtures
developing a simple aroma-monitoring system, pattern recognition analysis for
responses to food aromas from a gas sensor array was attempted using semiconductor gas sensors (92.93). Such an array may consist of as many as six or more
semiconductor gas sensors. These are preferred over other types of sensors because of their durability, high sensitivity for most reducing compounds. and insensitivity to water vapor.
The most popular sensors used to develop electronic noses suitable for use
within the food industry are described below.
Biosensors
367
3. Conducting Polymers
The unique electrical properties of conducting polymeric materials have been
exploited in the preparation of electronic noses. They have the advantage that a
wide variety of suitable materials exists. Two main classes are poly(pyrro1e)s
and poly(ani1ine)s. The materials are easyto process, so preparation of reproducible gas sensors is possible. One of the first applications of conducting polymers
as odor sensors was described by Persaud and Pelosi (86).
Several properties of the polymers can be used for measurement, including
changes in the work function of the polymers, as well as Inass changes that can
be monitored when polymers are depositedonto quartz resonator type substrates.
The most popular parameter to monitor, however,is the conductivity of the polymers, which is known to change rapidly and reversibly in the presence of odors.
The adsorbed odor molecules are believed to cause a swelling of the polymers
and to interfere with charge transfer within the polymer and interchain hopping
mechanisms (95). These sensors can be used at ambient temperatures and have
quite good sensitivities, typically 0.1- 100 ppm.
Electronic noses based on conducting polymer arrays are available commercially. Examples include the e-Noseo// 4000 Aroma Analysis System (NeotronicsScientific Inc., FloweryBranch, GA) andthe AromaScannero//from
Aromascan, Inc. (Hollis, NH).
The electronic nose technology does provide objective and reproducible
aroma discrimination on a wide variety of sample types with a sensitivitycomparable to human nose. Analysis times are on the order of minutes, yet the results
compare very favorably with GUMS, and sensory panel analyses that can take
much longer. There continues to be ongoing research into new sensor technologies, materials, and fabrication methods. At least one company offers multiple
hybrid systems. Toko has reported a taste sensor that has successfully discriminatedthebasic
taste qualities-sourness,saltiness,bitterness,sweetness,and
umami-in a number of food products and materials(8 I ) . The integrationof taste
sensors with electronic nose technology could truly revolutionize flavor analysis.
all
To date, all attempts to develop universal electronic nose suitable for
applications have failed. Problems associated with sampling. calibration, sensor
drift, and control of humidity and temperature are the 1na.jor causes for concern.
Improvements in associatedtechnologiessuchasnewodor-sensingmaterials,
Deshpande
368
development of hybrid noses, smarter pattern recognition techniques, and miniaturization are essential if the market potential of such an instruments is to be
realized (84,95). Ultimately, the development of application-specific noses that
are tailor-made for a given problem may prove more successful than attempts to
develop a single system suitable for universal applications.
B.
Molecular Imprinting
The technology of molecular imprinting could alleviate several problems associated with the biorecognition element of the biosensors. It provides highly stable
synthetic polymers that possess selective molecular recognition properties because of recognition sites within the polymer matrix that are complementary to
the analyte in the shape and positioning of the functional groups. Some of these
polymers have high selectivities andaffinity constants, comparable with naturally
occurring recognition systems such as monoclonal antibodies or receptors. This
makes them especially suitable as constituents in chemical (biomimetic) sensors.
Molecular recognition between a molecular receptor (host) and a substrate
(guest) in a matrix containing structurally related molecules requires discrimination and binding. This can happen only if the binding sites of the host and guest
molecules complement each otherin size, shape, and chemical functionality.
Biological systems, such as enzyme-substrate, antibody-antigen, and hormone-receptor systems, demonstrate molecular recognition properties that have developed
by natural selection.
One of the most intriguing areas for host-guest chemistry
is the development of biomimetic recognition systems. As described earlier, a wide range
of
analytical procedures depend on reliable and sensitive biological recognition elements such as antibodies, receptors, and enzymes. Because such biomolecules
can suffer from stability problems, synthetic counterparts are desirable. One such
approach to biomimetic recognition is the fabrication of molecularly imprinted
polymers (MIPS) (96).
Molecular imprinting is a powerful method for preparing synthetic recognitionsiteswithpredeterminedselectivityforvarioussubstances.
It canbeap(97)andthepreorganized
proached in twoways:theself-assemblyapproach
approach (98). These two approaches, which differ with
respect to interaction
mechanism in prepolymerization, follow common molecular recognition terminology.
The self-assembly nlolecular imprinting approach involves host-guest complexes produced from weak intermolecular interactions (e.g., ionic or hydrophobic interactions, hydrogen bonding, and metal coordinations) between the analyte
and the monomer precursors (97,99). These complexes are spontaneously established i n the liquid phase and are then sterically fixed by polymerization with a
high degree of crosslinking. After removal of the print molecules from the re-
Biosensors
369
sulting macroporous matrix, vacant recognition sites that are specific to the print
molecule are established.The shapeof the sites, maintained by the polymer backbone and the arrangement of the functional groupsin the recognition sites, results
in affinity for the analyte.
In the preorganized molecular imprinting approach, strong, reversible, covalent arrangements (e.g., boronate esters, imines, and ketals) are formed between
the monomers and the print molecules before polymerization. print
The molecule,
therefore, needs to be derivatized with the monomers prior to actual imprinting.
After cleaving the covalent bonds that hold the print molecules to the macropoto the analyte remainin the polymer
rous matrix, recognition sites complementary
(98).
MIPs have been successfully prepared with affinities for proteins, amino
acid derivatives, sugars and their derivatives, vitamins, nucleotide bases,
pesticides, and a wide varietyof pharmaceuticals (98). The binding of some of these
of some natural monoclonal
polymers has been comparable with the binding
antibodies ( 100,lO1).
The primary advantageof MIPs is that imprints can be made
of compounds
against which it is difficult to raise high-affinity antibodies. Other advantages
include their long-term stability and resistance to chemically harsh environments
( 102).
MIPs have unique properties that make them especially suitable for sensor
of medical,
technology. They exhibit good specificity for various compounds
environmental, and industrial interest, andthey have excellent operational stability. Their recognition properties are unaffected by acid, base, heat, or organic
in
phase treatment (102), making them highly suitable as recognition elements
chemical sensors. Some examples are shown in Table 8. To date, the most convincing demonstration of the usefulness of a real biomimetic sensor based on
molecular imprints is an optical fiber-like device in which a fluorescent amino
acid derivative (dansyl-L-phenylalanine)binds to the polymer particles, resulting
Table 8 ExamplesofBiolnilneticSensorsBasedonMolecularlyImprintedPolymers
(MIPs)
Range
Analyte
Morphine
Vitamin K,
Phenylalanine anilide
Dansyl-L-phenylalanine
Atrazine
Conductometry
Sialic acid
(pg/mL) Ref.
0- 10
0-4
Qualitative
33-3300I05
0-30
Transducer
Amperometry
Ellipsometry
Capacitance
Potentiometry
fluorescence
Fiberoptic
0-0.5
0-3
fluorescence
Optical
102
103
I04
106
I07
I08
Deshpande
370
XI.
FUTURE TRENDS
Biosensors
371
as cheaply as with the other typesof biosensors described. Among the biosensor
technologies described here, the mass-detection piezoelectric sensorsin the form
of acoustic wave guide devices, or one of the four or five optical devices described, are the most likely candidates as general purpose bioanalytical sensors
to produce and possess a potential for use over a
of the future. They are easy
wide rangeof analyte concentrations. They also approach the low detection limits
required for immunosensing, and the reproducibility of results is similar to that
achieved by conventional analytical methods. These devices rely on relatively
well-understood physical principles, and the associated instrumentation
is quite
in the optical sensor is, however,
easy to construct. The analyte detection element
much cheaper to construct and is more flexible in the uses to which it can be put
than the piezoelectric devices.
The fluorescent methods used with the optical sensors, although intrinsically more sensitive than the other optical methods, suffer from potentially greater
interference effects arising from the
test specimen matrix. It is for this reason
that the surface plasmon resonance (SPR) or integrated optical sensors are likely
to be commercialized as the next generation of general purpose analytical tools.
These devices have not yet met all the criteria listed for the ideal biosensor,
but they have met most of these criteria,
and future developments are likely to
enable all of them to be met within the next few years.
Although biosensors hold considerable promise and potential for on-line
still in its
quality and safety monitoring in the food industry, the technology is
of biosensors have been developed commercially.
infancy. Only a limited number
The problems associated with reliable mass production and commercialization
of this technology are enormous. They include long-term stability
of enzymes,
antibodies, and bioreceptors; sterilization of sensors; nonspecific adsorption of
other species; immobilization and mediation of enzyme-based sensors; reduction
of interferences from other substancesin the food; miniaturization of sensors and
sensor arrays; and variability in large-scale manufacturing. Progress on all these
fronts is continually being made in this rapidly growing area of research.
of fields involved in the developmentof biosenBecause of the wide variety
sors, successful research and developmentof biosensors will require a multidisciplinary approach. Biologists, chemists, physicists, and electrical and food and
bioprocess engineers will have to join together to solve the technical problems
facing biosensors. Nevertheless, it will not be too long before this technology
will become the mainstay of quality and safety monitoring in the food industry.
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10
New Techniques for Food Quality
Data Analysis and Control
Jinglu Tan
University of Missouri, Columbia, Missouri
1.
INTRODUCTION
Food quality data are gathered for various purposes and subjected to different
analyses. Instrumental food quality data are often analyzed and used for two
important objectives. First, to establish the usefulness
of an instrumental measurement as a food quality characteristic,it usually needs to be compared with human
sensory responses, which are the primary standard for food quality measurement.
Instrumental measurements are thus used as direct or indirect indicators of sensory quality attributes. Second, instrumental measurements are frequently used
for quality control purposes because instrumental techniques can be noninvasive,
rapid, convenient, and implemented on-line.
of variance and deterStatistical techniques are commonly used for analysis
mination of functional relationships. The routinely used techniques include correlationanalysis,analysis of variance(ANOVA),regressionanalysis,principal
component analysis (PCA), andpartial least squares (PLS) analysis. The conventional statistical methods are well known and can be found
in many textbooks.
In this chapter, we describe some recent developments
in food quality data analysis and food quality control.
First, we present a fuzzy set-based paradigm for
sensory data interpretation, which is followed by a neural network approach to
establishingrelationshipsbetweeninstrumentalmeasurementsandsensory
responses. Then we discuss the use of neural networks as recognizers of patterns
in quality data for the purpose of statistical process control (SPC). Finally, we
in real-time statisdemonstrate the use of instrumental food quality measurements
tical process control.
379
Tan
380
New Techniques
381
Tan
382
A.
By and large, sensory analysis is about modeling human senses and using human
senses as sensors for measurements. Human senses are sophisticated and powerful, and yet they are inaccurate
and lack repeatability. Exact quantification of
sensory responses is difficult if not impossible because they are naturally fuzzy.
As a result, sensory scales and attributes are also fuzzy. The following are fuzzy
set interpretations of some commonly used sensory scales, sensory attributes, and
sensory responses.
383
New Techniques
0. Interval and Ratio Scales. Among the interval and ratio scales in use
today, variations exist. Some do but others do not permit simultaneous partial
classification of a sample into more than one level or, simply termed, betweenlevel classification. For interval and ratio scales thatdo not permit between-level
classification, a suitable representation would also be a batteryof singleton fuzzy
Fig. 2 A battery of singleton fuzzy sets, C , to C,, describing nominal and ordinal scales,
and interval and ratio scales that do not permit between-level classification.
384
Tan
Fig. 3 A battery of fuzzy sets, C, to C,, with triangular membership functions depicting
interval and ratio scales that permit between-level Classification.
sets (Fig. 2 ) . For example, the nine-point hedonic scale is considered an interval
scale, and a sample must be classified into one
of the nine levels. Indeed, such
a scale is not fundamentally different from an ordinal scale except that the intervals are assumed equal.
Some widely used interval or ratio scales allow between-level classification. The US. Department of Agriculture (USDA) scale for grading marbling
abundance in beef steaks is a good example. It has nine levels, from devoid
to moderately abundant. A sample may be given a grade between two adjacent
levels with the closeness to the higher level indicated by a degree. For example,
a 30-degree slight grade means that the sample stands at the 70-30% split of
the interval between traces and slight. Such a scale can be exactly represented by a battery of fuzzy sets defined by the membership diagram in Fig. 3.
which is routinely seen in the fuzzy set and fuzzy logic literature. The fuzzy sets
or linguistic values, C,, C2, etc., are defined by a set of triangular membership
functions as shown by the solid lines in Fig. 3. When the intensity of an attribute
in the universe, x, its membership grade
under evaluation moves from left to right
decreases in one set and increases in another in a linear fashion. For instance, a
sample with an attribute intensity x, partially belongs to both C,and C2 (Fig. 3 ) .
Its membership grades in the two sets are pI(x,) and p?(xl), respectively, with
the two summing to 1. Evidently the membership grades are simply a different
interpretation of the degrees in the USDA marbling scale, and the fuzzy sets
rather accurately depict the marbling abundance levels.
The intervals between
the vertices of two adjacent membership functions may not be equal in general,
but in the case of an interval scale they are often assumed equal as discussed
before.
The widely used graphical line scale for intensity evaluation
is simply a
An
special case of the interval scale that permits between-level classification.
example line scale is shown in Fig. 4a. Two intensity extremes, such as exof the line. A
tremely low and extremely high, are marked near the ends
385
New Techniques
Extremely
Low
I
Extremely
High
I
Extremely
High
Fig. 4 (a) A graphical line scale. (b) Two fuzzy sets representing the line scale.
Tan
386
Table 1 SummaryofFuzzySetRepresentationsofCommonlyUsedSensoryScales
Fuzzy
Scale
Nominal scales
Ordinal scales
Interval and ratio scales
that do not permit
between-level classifications
Hedonic scales
Interval and ratio scales
that permit betweenlevel classifications
Line scales
As above
As above
Pi,jtX) =
if C, is chosenfor x by judge j
0 otherwise
(1)
New Techniques
387
CLi+l.J(X)
= 1
(2)
4.
Since human sensory responses are intrinsically inaccurate and imprecise, variations occur from judge to judge and from one measurement to the next by the
same judge. These variations constitute a measurement noise, which can be reduced by calibration (judge training) but cannot be eliminated.As a result, multiin sensory
ple judges are used to minimize the effect of this noise, especially
studies where data accuracy
is essential. When multiple judges are involved, there
exists the question of how the data from multiple judges should be represented;
in other words, how can we establish an overall response for a sample?
The conventional way of finding an overall response is to treat the individual judge responses as numerical scores and take the average
of them. This suffers
from the difficulty in providing the underlying equal-interval and linearity assumptions as discussed before. Sincethe responses are values of fuzzy variables,
a logical way would be to take the fuzzy average of them. However, the fuzzy
operations based on the extension principle (7) and consequently the fuzzy averages (8) require explicit knowledgeof the membership functions.In sensory analysis, the fuzzy setsin the scales are not explicitly defined in terms of some instrumentallymeasurablevariables;thus,theaveragingoperationbasedonthe
extension principle is not applicable.
While there may be many possible waysto represent and utilize multijudge
responses, one method would be the fuzzy histogram or membership vector described as follows. Though a fuzzy average over the fuzzy values
is not obtainable
without explicit knowledge of the membership functions, the membership grades
given by the judges can be averaged within each fuzzy set. Suppose that there
388
Tan
0.7 1
c, c, c,
c, c, c,
Fuzzy set or category Ci
c,
c,
c,
a
are n fuzzy sets (categories) in the scale used and there are m judges. For
sample with attribute x, judge( jj = 1,2, . . . m) decides that its membership grade
in fuzzy set i (i = I , 2, . . . n) is p,,,(x) as describedin the previous subsection. Note
that p),,(x)= 0 if fuzzy set C, is not chosen by judge j. The average membership
grade of the sample in fuzzy set C, voted by all the judges is
TP1.,(X)
pix) =
,=I
(i = 1 , 2 . . . n)
(3)
pi(x) represents an averageor consensus voteby all judges on the degree to which
the sample belongs to category C,; in other words, it is an expected membership
grade of x in fuzzy set C,. Note that the averaging operation is performed within
a fuzzy set and does not require knowledge of the membership functions and the
n fuzzy sets in the scale. Eq.
intervals between the fuzzy sets. Since there are
(3) implies n membership grades for x, which can be written into the following
fuzzy membership vector
P(X) = [PI(X) P?(X) . . CI,I(X)IT
'
(4)
New Techniques
389
are membership grades in the fuzzy sets of a scale, we may refer to Fig. 5 as a
fuzzy histogram of response. Because no assumptions are involved, the fuzzy
of response provides an unadulterated
membership vector or the fuzzy histogram
depiction of the overall response by a panel of judges.
It should be noted that a single judge is also used in practice because of
cost considerations as in the case of government-administered grading. A single
judge is just a special case of a judge panel with m = 1. The fuzzy membership
vector and fuzzy histogram of response defined are still applicable.
5. Defuzzification
The fuzzy membership vector or fuzzy histogram of response gives an unaltered
representation of judge responses for sensory analysis, and the fact
that a sample
may be partial membersof multiple categories depicts the fuzzy nature of sensory
responses. In commercial applications of sensory evaluation, however,it is sometimes necessary to assign a sample to a single, specific category. Then we need
to convert the multijudge responses into a unified consensus, or in other words,
to convert the fuzzy membership vector into a scalar of unity corresponding to
a single crisp set. The conversion
is similartodefuzzification in fuzzylogic
control. Several defuzzification methods have been proposed in the fuzzy logic
literature, including the maximum method, the mean-of-maximum method, and
the center-of-gravity method (9, IO).
The concepts of the defuzzification methods may be borrowed to convert
membership vectors into crisp sets. By the maximum method, the fuzzy set with
the maximum membership grade is chosen as the defuzzified crisp value (or category). Lincklaen Westenberg et al. (6) employed this method to determine the
in Fig. 5
final classification. For example, the fuzzy membership vector plotted
will be defuzzified by the maximum method to givea crisp classification of category C,. When there is a tie in the maximum membership grade, any one set
involved in the tie may be chosen as the crisp value. The mean-of-maximum and
the center-of-gravity methods use the center of peaks and the center of gravity,
respectively, of a combined membership function. This requires explicit knowledge of the membership functions. As the membership functions are generally
unknown in sensory analysis, these two methods are rarely applicable.
The maximum method is a logical choice for defuzzification of multijudge
sensory responses in many cases. The fuzzy set or category with the maximum
membership grade represents the majority opinion of the judges and thus seems
to be a natural and logical choice for the crisp value or consensus classification.
In contrast, the conventional defuzzificationby arithmetic averaging of assigned
numerical values is not as natural because an unverifiable equal-interval assumption must be made, as discussed before, and also because the resulting crisp value
does not generally possess the majority-opinion property.
Tan
390
Instrumental Measurements
Sensory responses are reactions of senses to stimuli. Stimuli can be measured
and characterized by instrumental measurements.
It is of great interest to researchers and product developers to establish relationships between sensory responses and instrumental measurements of stimuli. Such relationships can lead
to a fundamental understanding of human senses and help formulate products
that people like. Moreover, they establish the usefulness of instrumental measurements as indicators of food quality.
As discussed in the previous section, it is unreasonable to represent sensory
responses to a product sample as a precise number.
The fuzzy membership vector,
p, or the fuzzy histogram of response describes the judges' responses without
artificial modification. One obvious characteristic of p is that it is multivalued
in general. At the same time, characterization of a stimulus or a set of stimuli
the stimuli for human responses
usually requires a multitude of data. For example,
of marbling abundance in a beef steak may include a numberof marbling characof the
teristicssuchassize,uniformity,percentarea,andspatialdistribution
marbling flecks. As a result, relationships between instrumental measurements
and sensory responses are routinely multi-input and multi-output (MIMO) type.
Moreover, the relationships areusually nonlinear because of the nonlinear nature
of human senses.
To establish nonlinear relationships for MIMO data, the conventional statistical methods are extremely awkward.Artificial neural networks, fortunately, provideaneffectivetoolforthispurpose.
An artificial neuralnetworknormally
consists of a layer of input nodes, a layerof output nodes, and oneor more layers
o f hidden nodes in between. Between every two adjacent layers, the nodes are
interconnected with neurons, each of which represents some linear or nonlinear
relationship with two adjustable parameters: a weight and a
bias. Overall, each
output node is related to the input nodes through a complex nonlinear relationship. Many algorithms and techniques are available to determine the parameters
for an optimal fit of a functional relationship between the inputs and outputs.
to many available text(Readers unfamiliar with neural networks are referred
books, including Ref. 1 I.) In mathematical notation, an artificial neural network
represents the following nonlinear relationship, f
f(X)
(5)
with
x = [x, X? . . . XJT
[ Y I Y? . . . Y"1'
where X and Y are the input and output vectors, respectively, and k and
the numbers of inputs and outputs, respectively.
n are
New Techniques
391
C.ExampleApplications
We demonstrate the use of the proposed fuzzy set and neural network paradigm
withtwo example applications in beef quality evaluation. The current USDA
beef quality grading system is heavily based on visual appraisals of muscle color
and marbling abundance over a steak. It is therefore of interest to study human
of various
sensory response of muscle color and marbling abundance as functions
instrumentally quantifiable characteristicsof color and marbling.In these applications, color and marbling were characterized by using color image processing
(12).
1.
Samples
2. SensoryEvaluation
A IO-member panel of 7 males and 3 females was assembled to evaluate beef
steak muscle color and marbling abundance. All participants were screened for
formal training in fresh beef quality evaluation. The panelists were reacquainted
official
with the attributesof fresh beef qualityin training sessions with the aid of
photographical standards of color and marbling. Representative samples were
evaluated during the training sessions and group discussions helduntil the panelists gave relatively uniform scores to the training samples. The laboratory conditions for sensory evaluation were maintained in accordance with the guidelines
established by the American Meat Science Association ( 1 3).
Muscle color of each steak was scored according to the Beef Steak Color
Guide from the National Cattlemens Beef Association (formerly National Live
Stock and Meat Board, Denver, CO) in an eight-point scale:
[Very dark red. Dark red, Moderately dark red, Slightly dark red, Cherry
red, Moderately light cherry red, Very light cherry red, Bleached
red]
Marbling abundance was rated with the aid of USDA marbling score standards
in a nine-point scale:
Tan
392
(Devoid, Practically devoid, Traces, Slight, Small, Modest,
Moderate, Slightly abundant, Moderately abundant)
0.7
- 4
USample 1
Sample 2
Sample 3
i- 0.6
0.0
c,
c2 c,c,c, c, c,
c,
c9
New Techniques
4.
393
where p and (J denote mean and standard deviation, respectively, and subscripts
R, G, and B denote the color functions. The means indicated the average color
properties of a longissimus dorsi muscle, and the standard deviations showed the
color variations over it.
b. Marbling Stitnulus Charucterisfics. The size and density of marbling
flecks were considered most relevant
to sensory responses of marbling abundance. Each marbling fleck was individually identified and its size (area) calcuin 0.5mm2increments into 11 size
lated. The marblingfleckswereclassified
categories: A 5 0.5, 0.5 < A 5 1, 1 < A 5 1.5 . . . 5 < A 5 6.5, and A >
6.5; where A is fleck area in mm. The number of marbling flecks in each size
in a marbling
category was counted and divided
by the ribeye area, which resulted
for all the sizecategoriesformed a marbling
density. The marbling densities
density vector as
5. NeuralNetworkTraining
Feedforward neural networks were trained by using the back propagation algoMath
rithm to predict the sensory color and marbling responses. Matlab (The
Works, Natick, MA) was used for neural network training and testing. The samples were randomly separated into training and testing sets.
Two networks, one
for color and the other for marbling, were trained by feeding the training data
(6) and (7)]
in a sequential and recursive manner. The stimulus vectors [Eqs.
were the inputs, and the fuzzy membership vectors [Eq.
(4)1 were the outputs.
Different numbers of layers and neurons and different transfer functions were
Tan
394
tested to minimize the sum of squared errors. The resulting neural networks had
three layers with, respectively, 33, 20, and 8 neurons for color and 20, 25, and
9 neurons for marbling. A linear transfer function was used for the
first layer
and a sigmoid function used for the second and third layers. The networks were
quite complex, consistingof many layers and neurons. This complexity was considered to be a result of the complex nature of sensory responses to stimuli.
6. SensoryResponsePrediction
After the neural networks were trained, the
test data sets were used
to verify
them. The inputs in the test data were fed to the networks to generate predicted
outputs. The predicted fuzzy membership vectors were compared with
the real
sensory fuzzy membership vectors.
Figures 7 and 8 show comparisonsof predicted and sensory fuzzy membership vectors for a test sample. Results for other test samples were similar. The
predicted and sensory fuzzy membership grades had very similar patterns. For
a test data set of five samples, the mean squared errors in the predicted membership grades averaged 0.0059 for color prediction and0.0012 for marbling prediction. This shows that the neural networks could predict the sensory responses to
a good degree of accuracy for both muscle color and marbling.
It should be noted that the network outputs were fuzzy membership vectors
Sensory
E2223 Predicted
1
395
New Techniques
1 .o
h
i0
0.8
.f3.
0.6
xb
5
B
c,
0.4
E
0
M
0.2
e,
0.0
c,
c2
c,
c,
c5
C6
c,
c,
c,
of marbling
instead of simple crisp scores or grades. The predictions were for sensory reartificial simplification.This
sponses in virtuallytheiroriginalformswithout
allows analysis of sensory data with their natural fuzziness and complexity and
determination of instrumentalmeasurements as indicators of truesensoryresponses. When the maximum method was used to classify the
test samples into
specific categories, the predictions were
100%correct forboth color and marbling
of the test samples.
7. Comment
When a new approach is developed, one would naturally expect a quantitative
comparison with existing approaches in terms of prediction accuracy and so on.
Unfortunately, such a comparison cannot be easily made meaningful because the
or do not exist in
true values (true grading, true category, etc.) are unknown
sensory evaluations. This is usually the case when subjective judgments are involved. Nevertheless, differences existin the ways subjectiveor sensory information is analyzed and interpreted. Some are more logical and reasonable than others.Theparadigmandproceduresdescribedhaveapparentadvantagesover
conventional techniques in avoiding unverifiable assumptions and retaining the
a more realisintrinsic fuzzy characteristicsof sensory responses and thus provide
tic representation of reality.
Tan
396
111.
PATTERNRECOGNITION
Statistical process control (SPC) has been an important tool for quality control
in the processing industry. It has been regaining acceptance and popularity
in
recent years becauseof the increasing consumer demand for quality products and
the intensifying global economic competition.SPC is being employed as a major
quality control mechanism for more and more manufacturing processes.
It has
become an indispensable tool to the manufacturing industry for improving productivity and product quality (14).
An SPC system consists
of means for product quality data gathering, quality
data analysis to detect process abnormalities, and implementation of corrective
all product
actions when necessary(1 5). Quality variables are evaluated for either
units or product samples taken periodically. The quality variables are plotted as
functions of time on control charts such as Shewhart and cumulative sum (CUSUM) for production process monitoring (1 6). If a quality variable deviates excessively from normal values or exhibits abnormal patterns, one or more special
causes exist and appropriate corrective actions are determined and implemented.
Depending on the product and process, one or more steps
of the SPC procedure may require human involvement. These may include manual evaluation of
data characteristics and manual implementation of corrective actions. The need
for manual operations often leadsto a considerable time lag between data gathering and process correction, which can be costly. For a high-throughput process,
it is very desirable to have on-line problem detection and process control because
of lowany time delay in process adjustment can result in significant amount
grade products or wastes. This
is especially true for continuous processes that
cannot be stopped and restarted frequently. A good example
of such a process
is food extrusion, which currently relies
on constant attention of experienced
operators for control. To implement an on-line SPC system, a critical ability
is
automatic detection of abnormalities. The 30-criterion (three-standard-deviationcriterion) detects the existence of special disturbances
by performing limit checking. The abilityto identify special disturbances by recognizing unnatural patterns
in quality data is also very important in SPC applications.
Swift (1 7) proposed a dichotomous decision tree approach and used a series
of statistical hypothesis tests to identify six unnatural patterns in control charts
a highly inflated
(quality data plots). The approach was found useful but had
false rate because of the use of hypothesis tests. The capability of the pattern
recognition algorithm depends on the accuracy of the hypothesis tests.
Cheng ( 1 8) developed a process deviation reasoning system using a traditional syntactic pattern approach to recognize unnatural patterns. Specific protoin thesystem.Thesetemplatesareusertypepatterns or templatesareused
defined and could significantly affect the performance of the resulting algorithm.
In addition. the pattern-making processis tedious and time-consuming, especially
New Techniques
397
when the number of templates or patterns is large. This has impeded real-time
applications of the approach.
With the advent of computers, intelligent pattern recognition techniques
have been developing rapidly, making automated pattern recognition possible.
Hwarng ( 19) reported a neural network model to recognize six common patterns
in quality data. The model could successfully recognize computer-generated patterns. However, it requires transformation of real data into binary numbers as
input, leading to a large input layer size and making the neural network complicated and slow.In addition, the transformation inevitably changes some statistical
real
characteristics of quality data, which can make the algorithm less reliable for
applications.
In this section, a sliding-window schemeis described for developing neural
network pattern recognizers for quality data analysis. Synthesized and experimenof the approach.
tal data from food extrusion are used to show the usefulness
The recognizers are suitable for on-line applications.
A.
PatternsinFoodQualityData
398
Tan
Trend
Cycle
Stratification
Mixture
I
Sudden shift
t = w , w + 1, . . .
(8)
where t is the current time. Equation (8) simply indicates that each input vector
to the neural network is a window or segmentof a data sequence and the window
slides forward by one data point for each time increment. To train a neural net-
New Techniques
399
C.PatternDataGeneration
It is generally difficult to acquire
real quality data with distinct and repeated
patterns of importance for recognizer training. As
an alternative, a pattern generator may be used. A pattern generator may be expressed in a general form of a
process mean plus two disturbances:
Y(t) = I.I + d(t) +
at)
(9)
where y(t) is a quality measure at time t, p is the process mean for y(t) when
the process is in control, d(t) is a special disturbance, andE(t) is a random variable
following a normal distribution, i.e.,
E(t) = N(O, ro)
(10)
D. ExampleApplications
We use two examples reported in Tan and Sun (21) to demonstrate the development and application of neural network pattern recognizers. One example was
based on synthesized pattern data, and the other on experimental data.
1. Synthesized DataExample
N. Pattern Data. Equation (9) was used to generate data of six patterns
by usingthespecialdisturbancesdescribed
in Table 2. For robustness of the
trained neural network pattern recognizer, the pattern data were generated with
different combinations of magnitudes of the special disturbances and noise levels
shown in Table 3. Symbol yi,,(t) denotes a data sequence generated with special
is nonpedisturbance magnitude i and noise levelj. Since the sudden shift pattern
riodic, it was generated in such a way that each vector of size w would contain
one shift at a sequentially different point in the window.
Tan
400
Table 2 SpecialDisturbancesforPatternDataGeneration
rameters
on
disturbance
Notes
Special
Pattern
Trend
d(t) = k(t - ~ J o
k = slope of trend
terms
in of
o
4, = initial time
Cycled(t)
= kosin[2x(t - &,)/TI
k = amplitude of cycle intermsof
o
T = cycle period
Systematic
d(t) = k(- 1)'o
k = amplitude of pattern
terms
in
of o,
k>0
Stratification
d(t)
= d
d = offset
from
process
the
mean (center
of stratification)
Mixture
d(t, r) = k(- 1)"s
r = random
number,
0
<r< 1
k = magnitude of pattern in terms of o,
k>0
If r < p. w = 0, otherwise w = 1
P = prespecified probability value which
determines the shifting between distributions
Sudden
shift
d(t,
t,) = k(-l)'o
t, = time
when
sudden
shift
occurs
s = 0 if t 2 t,, and shift upward
s = 1 if t < t,, and shift downward
k = magnitude of shift in terms of o,
k = 0 i f t < t,, k > 0 if t 2 t,
Table 3 Magnitude of Special Disturbance and Noise Levels Used for Pattern Data
Generation
j
Magnitude
Pattern
2
level.
Noise
of disturbance,
k
~~
1.5
1.5
1.75
shift
0.1
2.5
Trend
Cycle
Systematic
2.5
Stratification
Mixture
Sudden
2.0
2.0
1 .5
I .5
0. I
0.3
0.5
0. I
0.1
0.0.3
I
0.6 0.4
0.3
0.3
0.3
0.2
0.5
0.5
0.5
0.5
New Techniques
401
b. RecognizerTraining.
The neuralnetworkrecognizerwasathreeAs a result
layer network with an input layer, a hidden layer, and an output layer.
of the data format, the input layer consisted of w nodes, and the output layer had
six nodes. Twenty-eight nodes wereused for the hidden layer. The transfer function of the hidden layer was a sigmoid function. The transfer function of output
layer was a linear function.
When 50 data vectors (windows of data) from a pattern were used to train
the recognizer and data from the same pattern at the same magnitude and noise
levels were used for testing, the results were excellent. When data from the same
pattern but at different magnitude and noise levels were used to
test the neural
network recognizer, the results were poor. For satisfactory robustness of the recognizer, the training data were not only from different patterns, but also from
different magnitude and noise levels. The first 300 training vectors consisted of
50 vectors from each of the six patterns at magnitude and noise levels of yz,z(t).
The next 100 training vectors were from the cycle and the mixture patterns
at
magnitude and noise levels of y,,,(t). Thelast 50 training vectors were from the
sudden shift pattern at magnitude and noise levels of y3,3(t).
The backward propagation algorithm was used to train the neural network.
Momentum and adaptive learning rate features were
used to facilitate the training
process. The Matlab Neural Network Toolbox was used for network training and
testing.
c. RecognizerPerformance.
The performance of thetrainedneuralnetwork pattern recognizer was evaluated with test data (data not used for training).
Because the recognizer might correctly or incorrectly identify a pattern or might
even fail to recognize a pattern as one of the six, three performance measures
were used.The target rate (TR) was the percentage of correctly identified patterns.
The error target rate (ETR) was the percentage of incorrectly identified patterns.
The false target rate (FTR) was the percentage of patterns unrecognized by the
recognizer(neithercorrectlynorincorrectlyidentified).Thefalsetargetrate
showed how frequently the recognizer failed to recognize a pattern.
Table 4 shows the target rate and error target rate for testing sequences of
different magnitude and noise levels (indicated by the subscripts of y; see Table
3). All target rates were more than 79% with a majority being perfect or nearly
or
perfect. All error target rates were less than 5.7%, with a majority being zero
nearly zero. Given the differences in the patterns and in the magnitude and noise
a properly
levels, the results are considered very good. This demonstrates that
trained neural network has robust ability to recognize patterns.
The false target rate depended on a threshold value used. Since the neural
network output could not always be a perfect binary number with one bit being
1 and the rest being 0, a threshold must be defined. When an output had a
bit
greater than the threshold, the output was considered
to indicate an identified
402
Tan
Table 4 PerformanceoftheNeuralNetworkRecognizer
for Patterns of Different Magnitude and Noise Levels
Data
sequence
Target
rate
(%)
Error target
rate
97-100
99- 100
95- 100
99- 100
0-0.17
0
100
100
IO0
88- 100
0
0
0
0-2. I7
79- 100
0-5.67
(%)
0-0.17
0-0.17
pattern corresponding to that bit. It is not surprising that the threshold value had
10 shows the false target rate
significant effects on the false target rate. Figure
as a function of threshold value for three magnituddnoise combinations. A high
to a high false target rate.
A low threshold value
threshold value would lead
would reduce the false target rate but increase the error target
rate; in other words,
with a lower threshold value, the recognizer would more easily claim data as
recognizable patterns but also more easily misclassify them. Selecting an appropriate threshold value is, therefore, important. From this work, a threshold value
of 0.6 appeared most reasonable.
Threshold value
Fig. 10 False target rate as a function of threshold value (a value above which a target
was considered identified),
New Techniques
403
2. ExperimentalDataExample
The methods and procedures described in the last section were usedin an experimental application in food extrusion. Although 15 patterns have been identified
and considered common in the literature, experimentally acquiring data of those
patterns from a specific process is not always practical. First, the responses of
quality variables depend on the process dynamics. It is usually difficult to determine by experimental trial and error what disturbances would result in a certain
pattern in quality data. On the other hand, patterns induced by disturbances frequently occurring in a process may somehow differ from the representative patterns described in the literature. Second, to obtain sufficient data of various pata huge and exhaustive array of experiments
terns for neural network training,
may be necessary, which can be practically unfeasible.
The food extrusion process is expensive to run and cannot be subjected to
extensive experimentation. To facilitate the experiment process and to minimize
the number of experiments necessary, process modeling was used to advantage.
A major source of process variation was identified and perturbed with a pseudorandom binary sequence (PRBS). A quality variable of interest was measured
and modeled as a function of the disturbance variable. The model was used to
determine the typeof disturbance that would rendera certain quality data pattern,
and further experiments were then conducted.The model was also used to generate pattern data for neural network training. Finally, the neural network recognizer was tested on the experimental data.
a. The Process. Afoodextruder is a high-temperature,short-timeprocess that can transform a variety of raw materials into intermediate and finished
In the
productssuch as ready-to-eatfoods, flat breads,andbreakfastcereals.
404
Tan
process, food materials are pressed through a barrel by a set of screws. They are
heated, pressurized, and subjected to shear. The materials often become highpressure, high-temperature, and gelatinized extrudate when they reach the die.
After exiting the die, the extrudate expands and is cut into a desirable size.
The product size is one of the most important quality attributes of many
extruded food products. Since itreflects the degree of expansion and bulk density
of expanded products andis relatively easy to measure, it is a routinely monitored
quality variable for quality control in food extrusion. For constant material feed
rate, cutter speed, and other processing conditions, the product size depends on
the degree of expansion, whichis affected by material properties. Since variations
rein material properties are inevitable, the product size varies. Based on past
search, a major source of process disturbance is feed moisture content. It significantly affects quality attributes such as product size.
h. EquipmentandExperiments.
An APV-BakerMPF-50125twin-screw
food extruder wasused. The extruder consistedof two setsof intermeshing screw
elements of different geometry fitted in an enclosed barrel. A die was mounted
at the end of the barrel. The screws were driven by a 28 kW DC motor, and the
screw speed could vary from 0 to 500 rpm. The feeder was a KTRON model
T35 twin-screw volumetric feeder.
An IVEK Digifeeder system was used
to inject
water into the barrel. The extruder had nine zones with independent barrel temperature controllers. The screw speed, feed rate, moisture addition rate, and cutter
speed were controlled by a host microcomputer.
To measure the extruded product size on-line, a computer vision system
1 color frame
was developed. The system consisted of a Data Translation DT-287
grabber, a DT-2878 advanced processor, two programming libraries (AURORA
and AIPL), a Sony DXC- 15 I CCD color video camera and a Sony PVM-I 342Q
color video monitor hosted by a microcomputer. Each digitized image frame had
512 X 480 pixels. The pixel value range was 0-255 (3 X 8 bit resolution). The
same exposure and focal distance were
used for all images. The selected exposure
was such that the image intensity histograms were roughly centered
at the middle
of the full-scale range (0-255), which gave the best resolution and clearest images. The focal distance was set so that a desired number of product samples
could fit in the image frame. An image processing algorithm was developed in
the C programminglanguagetosegmenttheproductobjectsfromthebackground, identify each sample individually, compute the size (side view area in
of several samples as
square millimeters) of each sample, and take the average
the measured product size.
A sampling mechanism was fabricated to bring a numberof samples from
the product streamto the camera view area
at each sampling instant.The sampling
a
mechanism and the vision computer acted as slaves to the host computer. On
signal from the host, the sampling mechanism would take samples, and the vision
New Techniques
201
405
401
601
801
1001
1201
Time (s)
computer would capture a sample image, compute the product size, and transmit
the measurements to the host computer. The sampling period used was 4 s.
The extrusion experiments were performed around an operating condition of screw speed of 300 rpm, feed rate of 45 kg/h, and total moisture content
of 19% (wet basis). Yellow corn meal was used to make a puffed product.
The barrel temperature profile was held constant as described in Chang and Tan
(22).
To produce the effect of natural moisture variations in feed materials, the
moisture addition rate was perturbed with various disturbances while the product
size was measured. First, a pseudo-random binary sequence (PRBS) was designed
and applied to the moisture addition rate to excite the process across its frequency
bandwidth. The PRBS signal is shown in Fig. 1 1 . The amplitude of the disturbance was such that the total extrudate moisture content varied between 17 and
21% (wet basis). The PRBS data were used to develop a model of product size
versus feed moisture content. From the model, other types of disturbances were
selected and applied to the moisture addition rate for more experiments around
the same average operating conditions of the process.
c. Process Modeling. The response of product size to moisture disturbance was modeled with the following ARMAX (auto-regressive movingaverage with auxiliary input) model:
(1
( 1 1)
where A(t) is product size (mm), M(t) is moisture content (%, wet basis), E(t)
is a white noise sequence, d is time delay (s), and a,, a?, m,, m!, and c I are constant
coefficients.
The model was developed using the PRBS experiment data. The model
structure and time delay were determined using a systematic search algorithm
406
Tan
27-0.904
-2.980
13.774
-0.081
-0.863
(22). The recursive least-squares algorithm in Matlab was used to determine the
coefficients. The time delay and coefficients are shown in Table 5.
d. Puttertz Recognizer Training and Testing. From the model (Eq. [ 1 I ] )
it was found that a sinusoidal wave, a square wave, and a step disturbance would,
respectively, induce a cycle, a mixture, and a sudden shift pattern in the product
size data. These three disturbances were then applied to moisture addition rate
in new experiments. The amplitude (or magnitude) of the disturbances was the
same as that used for the PRBS disturbance (17-21 %). The period of the sinusoidal and square wave disturbances was 360 s.
One set of experimental data is plotted in Fig. 12. The data approximately
exhibit the three patterns. There are significant noisesin the data, which resulted
from natural disturbances and were not part
of the patterns resulting from the
special disturbances. After a zero-phase-shift digital filter was used to remove
the noises, the filtered data and those predicted by the model are in good agreeof the special disturbances
ment. This shows that the model described the effects
very well.
Pattern data were generated with Eq. ( I I ) for the same three disturbance
inputs as those used in the experiments. The data were used to train the neural
network pattern recognizer by following the same procedures described in Sec.
1II.D. Data sets measured on-line with the computer vision system were used to
test the trained neural network. The target rate was more than 98% for the cycle
pattern, 95% for the mixture pattern, and 91% for the sudden shift pattern. The
false target rate was zero.
These results further verify the usefulnessof the procedure used to develop
the neural network pattern recognizer. In addition, a process model derived from
multifrequency perturbation is helpful to save experimental efforts.
IV.
REAL-TIMESTATISTICALPROCESSCONTROL
New Techniques
407
..
350
m i
200
181
240
361
541
72 1
901
1081
4
1
271
91
181
541
361
451
Time (s)
Fig. 12 Comparison of model-predicted patterns with measurements: (top) cycle, (middle) mixture, and (bottom) sudden shift. Dotted lines are measured, thicker solid lines are
filtered measurements, and thinner solid lines are predicted.
Tan
408
A.
ProcessandEquipment
The application process was twin-screw food extrusion. The process and equipment used were the same as those described in Sec. 1II.D. Yellow corn meal was
used to make a puffed product, and the quality variable of interest was product
to measure the product side view
size. The computer vision system was used
area in square millimeters, and length and width in millimeters.
B. ProcessVariations
During an extrusion run, feed material properties can vary considerably. For example, the moisture content of the corn meal used in this work could vary from
9 to 13% (wet basis)as a result of differences in batch, supply source, and storage
conditions. This variationcan significantly affect the product size and other quality attributes. To shorten the experimentalrun time, the effects of material moisture variation on product size was demonstrated
by introducing a disturbance into
the moisture addition rate. The disturbance was a sequence of step changes as
shown in Fig. 13, which caused the overall moisture content to vary from
17 to
21%.
The variations in product area are shown in Fig. 13. The dotted line in the
figure shows the area variations when a single sample was measured at every
sampling instant. The plot indicates that the variations consisted of two major
components: a fast or high-frequency variation on top of a slow trend. The fast
component of variationwasduetorandomornaturaldisturbances,andthe
slow component was due to
anassignableorspecialdisturbance,whichwas
moisture change in this case.
Natural disturbances are undeterminable, and thus random variations can-
409
New Techniques
400 r
..""
__
"_
50
Single-sampleoreo
Sinqle-somole
oreo
Subgroup-averagedare0
Sudgroup-averaged
Mosturecontent
Mosture
content
350
300
_.
250
200 I
"""
I"""
30
20
.""_I
1 50
50
1 00
150
200
250
Fig. 13 Product area variations resulting from natural and special disturbances (changes
in moisture content).
+ aR
LCL = X - a R
Tan
. ~ . ..
Length
Width
Moisture c o n t e n t
UCL=23.8
h
h.
Subgroup Number
Fig. 14 Productdimensionvariationsresultingfrommoisturecontentchanges
and LCL are upper and lower control limits, respectively).
(UCL
where X is the grand average of a measured quality variable over a long run
(average of subgroup means), a is a constant depending on the subgroup size,
and R is the average of within-subgroup ranges. Since R indicates the magnitude
by Eqs. (12) and
of random variations in a process, the control limits defined
(13) reflect the process capability to maintain uniformity in a quality variable.
The control limits were determined through experiments. For the product
area, X = 300 mm? and R = 80 mm'. For a subgroup size of 10, a = 0.308
( 16). Then,
UCL = 325 mm2 and LCL = 275 mm'
for the subgroup-averaged product area (solid line
in Fig. 13). Figure 13 is the
of
Shewhart control chart for the product size (area) without implementation
corrective actions. The size was out of the control limits because of the moisture
variation and the process was not in a state of statistical control.
The product length and width variations are shown in Fig. 14. The length
reduced with an increasein moisture content or vice versa. For the product length,
X = 22 mm, R = 6 mm, and a = 0.308 (subgroup size = lo), which give
UCL = 23.8 mm and LCL
= 20.2 mm
As shown in Fig. 14, the product length was also out of its control limits as a
result of the moisture variation.
The product width exhibited little variation relating to moisture change as
New Techniques
41 1
shown by Fig. 14. The size variations were almost exclusively reflected on the
product length. As a result, improved control of the product width was unnecessary for minimizing the effects of moisture variation on size uniformity of the
test product.
C. CorrectiveAction
To bring the process to a state
of statistical control, appropriate corrective actions
must be implemented. Sincethe process was continuous, quick actions are important. Upon detection of a state out of statistical control, corrective actions were
determined by using a simple feedback control scheme. The size measurement
by the vision system was used as the feedback signal to determine a proper cutter
speed to compensate for the effects of moisture variations.
The block diagram of the control system implementedis shown in Fig. 15.
In the figure, product size refers to either product area or length depending
on
which one of the two is of interest for control. The process block stands for a
functional relationship (transfer function) from cutter speed to product size. The
disturbance effect block represents the unknown relationship from moisture conof the controller was to minimize product size
tent to product size. The role
variation under the disturbance of moisture variations.
Since the process dynamics between cutter speed and product size
was
simple, the following PI (proportional and integral) controller was considered
appropriate for determining corrective actions:
u(t) = u(t - 1 )
+ K,[e(t)
e(t
-111 + K,e(t)
(14)
Cmtent
Effect
Cutter
Cmtroller
speed
Process
P r a h t %e
412
Tan
350
33
~~~3~s_""""""""_"""
E
E
E
E
v
0
31
300
LCL=275
250
- 25
. . . . . ,*.,. . . . . . . . . . . . . . . . . UCL=_7_338,
,.,.%
, I. \,......_,
*.\ ,l-s.l... ,,, ......_-___
:4, ,.' .
.- ,.,~
'<
.
...:*
\,
a:.;
;%.'e:..
,I
SI
-21
~.I\.
2oo"""""""-
150
"""""_
19z
212
LCL=20.2:
""_
-,
I""
:..lzz-,l
100
1 50
g,
- 17
Molsture content
50
19
"""
- __ Area
.....- .- L e n g t h
""
1 00
23
\"
.._.I.,
200
15
- 13
11
250
Subgroup Number
where u is the cutter speed control signal, e is the difference (error) between the
K, and K, are gain
desired product size (process mean) and the measured size,
constants, and t stands for the current sampling instant or subgroup number.
The proportional and integral gains, K, and K,, could be designed if a process model were known. As an alternative, they were determined through experimental tuning as widely practicedin industry by using the Ziegler-Nichols tuning
procedure (see, e.g., Ref. 24). The two controller gains were determined as K,=
0.01, K, = 0.03 for product area control andK, = 0.14 and K, = 0.42 for product
length control.
The vision-basedcontrolsystemwasimplementedon-lineandtested
against moisture disturbances. Figure 16 shows a control chart for both product
area and length when the process was subjected to the same moisture disturbance
shown in Figs. 13 and 14. The control chart illustrates the performance of the
system.
In comparison with the uncontrolled curves in Figs. 13 and 14, the system
improved the product size uniformity significantly. The controlled product area
and length were always within UCL and LCL, meaning that the process was in
a state of statistical control in spite of the moisture disturbance. The controlled
curves do not have anidentifiablepattern,indicatingthatthecontrolsystem
largely eliminated the variations caused by the special disturbance.
Figures 17 and 18 are, respectively, the product area and length histograms
with and without the vision-based process control. The histograms show that the
413
New Techniques
0.14
0.12
0.10
0.08
x
W
3
0-
EZd
Uncontrolled .
Controlled
.
0.06 -
LL
0.04 ,0.02
0.00
240 250 260 270 280 290 300 310 320 330 340 350 360 370 380
Area (mm*mm)
Fig. 17 Histograms of product area with and without the real-time SPC.
0.1 4
0.1 2
0.1 0
Uncontrolled
Controlled
6 0.08
3
0-
0.06
0.04
0.02
0.00
1 61 71 8
1 9 20 21
21
22 2 3 24 25 26 27 28 29
Length (mm)
SW.
Tan
414
V.
FUTURETRENDS
New Techniques
415
REFERENCES
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SS Stevens,ed.
Handbook of Experimental Psychology. New York: Wiley, 195 I .
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I.
Press,1993.
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Duin. Fuzzy set theory applied to product classification by a sensory panel. J. Sensory Stud 4155-72, 1989.
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Civ Eng Syst 2:201-208, 1985.
8. WM Dong, FS Wong. Fuzzy weighted averages and implementation of the extension
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IO. J Tan, Z Chang. Linearityand a tuning procedure for fuzzy logic controllers. Trans
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12. DE Gerrard, X Gao. J Tan. Determining beef marbling and color scores by image
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14. CRauwendaal.SPC-StatisticalProcessControl
in Extrusion. NewYork:Hanser
Publishers,1993.
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416
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18. CS Cheng. Group technology and expert systems concepts applied to statistical process control in small-batch manufacturing. PhD dissertation, Arizona State University, Tempe, AZ, 1989.
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Wiley,1989.
Index
[Bacteria]
Listeria spp.. 361
Salrnonellu, 129, 36 1
Yersinia enterocolitica, 361
Banana:
DLEintensity,103,106-109
gloss, 20
maturity, I 1 1
Bayesianclassifier,154,155, 158
Beans, color evaluation, 74
Beef
color and marbling, 391-393
fat content, 26
grading, 150
Beer-Lambert Law, 6, 8, 139
Bell pepper:
DLE intensity, 109, I 1 1
gloss, 20
picking, 150
orientation and shape, 1 10
Bioluminescence, 363-364, 370
Biosensors, 335, 337
acoustic, 350
amperometric, 357, 370
applications in food industry, 359
biocatalyst, 338-339
biocatalytic membrane, 356
biocomponent, 338-339, 345-346
biomimetic, 369-370
417
418
[ Biosensors]
biomolecules, 339
biorecognition, 344
electrochemical, 353
optical, 348, 370
potentiometric, 370
Blueberry, firmness, 270
BOD (biological oxygen demand),
337
Boltzmann constant, 203, 205
Bound water, 201
Butter:
composition, 28-29
creep, 306
Camera:
area-array, 55
BCCD, 57-58
calibration, 72
CCD, 55-57, 87, 90, 122, 124
CID, 56
CMOS, 55-57
digital, 58
frame transfer, 56
line-scan, 55, 58, 90
progressive scan, 58
TDI (time delay integrate), 55, 90
Cantaloupe:
firmness, 272
maturity,112
Carotene (carotenoid), 20, 109, 1 I O
Cam-Purcell pulse sequence, 179, 183
Carr-Purcell-Meiboom-Gill(CPMG),
179, 195- 199
Cavitation, 2 19
Cheese:
Cheddar, 307-3 I3
cut time, 227
fat globules, 84-86
meltability, 309
mozzarella, 304
process American, 307
shreds, 66
Cherry:
firmness, 272-273
ripeness, sugar, 19
index
CHESS (chemical shift selective)
sequence,190
Chlorophyll:
content, 99, 107-1 IO, 126, 257
lossldegradation,17, 19, 99,109
Chocolate:
blooming, 43, 56
melting profile, 226
Chromaticity:
coordinates, 4-5, 19
diagram, 5
CIE (Commission de Internationale de
I'Eclairage), 4-5, 19, 69
CLSM (confocal laser scanning microscopy), 83
Cod fillet:
firmness, 248
sealworms and bones, 223
Color:
calibration, 7 1
diagram, 5
index, 22
memory, 1
model. 5
Munsell, 5
rendering index (CRI), 70-71
temperature, 43
Colorimeter, Agtron, 19
Computer tomography (CT), 139- 147, 159
dual energy gamma, 159
Computer vision, 39-92, 1 IO, 130,
404-41 1
Corn:
color evaluation, 74
defects, 23. 24
extrudate characteristics,68
quality factors, 1I O
shape inspection, 66-67
Cracker, shape inspection, 67
Creep compliance, 291 -295, 301 -3 I O
retardation time, 294-308
Cucumber, chilling injury, 1 1 1
Cytometry, 129, 36 I , 362
Deborah number, 290
Debye-Stockes theory, 205
Index
419
420
Index
[Kiwifruit]
modulus, 266
MR image, 188
texture, 256
Knowledge base, 41, 62-63
Kubelka-Munk, 8, I O
Larmor equation, 166
frequency,168,171, 180
Laser air-puff firmness detector, 244,
259, 265
Laser Doppler vibrometer, 256-257,
275-276
Lemon:
color grade, I I O
DLE intensity, 107-1 10
Ligase chain reaction (LCR), 360
Lighting:
arrangements, 43
sources, 43
strobe, 43, 90
Linescan,139-141, 153
Loss tangent (tan 6). 300, 3 14-315,
321 -322
Lubein, 20
Lycopene,109
Machine vision, 39-92
Magness-Taylor (MT) puncture test,
244-246, 253. 263, 269, 271
Magnetic dipoles, 166
Mango, firmness, 256, 274
Maxwell model, 292, 294. 301, 306
Melon. firmness. 244. 248, 254, 270,
274-275
Microorganismlmicrobial:
index
421
[Microscopy]
electron, 83
fluorescence,123,129
3-D, 83
Microwave, 275
Milk:
coagulation, 84, 310. 31 1
gel, 310-312
composition, 26, 30, 224
Modulus:
bulk, 219
complex, 300
elastic, 219, 264
loss (viscous), 298
shear, 289
storage, 298, 320, 322
Young's, 232, 259, 289
Moisture content, measurement,
26
Molecular imprinting, 368-370
MRI (magnetic resonance imaging),
165,179-185, 2.57
Munsell color atlas. 5
Muscle food, fat thickness, 232
Muskmelon, DLE intensity, 113
Mycotoxins, I23
Oil:
adulteration, 28
composition,28-29,127,129
Olives, DLE intensity, 109
Onion:
diseases,137, 151
DLE intensity, 109
firmness, 273
gloss, 20
line scan image, 153
separation from clods, 252
On-line:
control, 63
firmness sorting, 278
inspection,142
moving scene analysis, 89
quality and safety monitoring, 371
statistical process control, 396
viscometer, 236
Optical density (OD), 7. 18, 20-26, 1 19
Orange:
DLE intensity, 102-1 10
firmness, 247, 252, 270, 273
gloss, 20
juice, 22
surface defects. 22
Nectarine:
DLE intensity, 109
firmness, 252
Neural network, 63, 74-75 92, 154,
157, 379-398
Neuro-fuzzy systems, 75
Newton's law, 289
Newtonian. 292-293, 297
NIR (near-infrared), I , 9, 1 1 , 18, 21 31, 54, 57, 86, 88. 115, 227, 244.
253, 280
image, 86-88
instruments, I2
sensors. 160
spectroscopy. 26, 257, 275
NMR (nuclear magnetic resonance),
165-21 1
imaging.165
spectroscopy,165,166,288
Papaya:
DLEintensity,103,108
maturity,112.113
Partial least squares (PLS), 379
Pattern recognition, 63, 64, 154, 232,
396-406
Pea, firmness, 275. 276
Peach:
DLE intensity,102, 109
firmness, 246, 249, 259. 270-275
ripeness, 19, 1 13
Peanut:
maturity, 20
moisture content, 26
Pear, firmness, 249, 270-273
Penetrometer, 245
Persimmon,DLEintensity.103.105,
109,113
Phase locked loop (PLL), 60
422
Phosphorescence, 7, 100
Photoluminescence, 1 13, 1 16
photoluminography,I30
Photonintensity,139
Photosynthesis, 100, 130
Pineapple, surface color, 22
Pistachio, color classification,75
Pixel, 41
square, 55, 59
jittering, 60
Plancks constant, 1 I , 167
Plum, DLE intensity, 109, 113
Poissons ratio, 251, 264, 266
Polymerase chain reaction, 360
Pomegranate, DLE intensity,109
Pork, muscle quality, 24-25
Potato:
color evaluation, 74
diseases, 23
firmness, 273
hollow heart, 23, 234
Principal component analysis (PCA),
22, 379
discriminant analysis, 22
Proportional and integral controller,
41 1
Prune:
ripeness, I9
sorting, I 14
Quanta, 1 I8
quantumnumber,166,168
Quenching,119,120
Quartz resonator sensor, 366
Radiofrequency (RF) pulse, 167- 174,
183,185,190
Reduced mass, 1 1-12
Reflection:
body, 9
coefficient, 223
diffuse, 7, 10, 17
regular, 7
specular, 7, IO, 43
total internal, 52, 348-349
Refractive index, 4, IO- 12,52-53.348
Index
Relaxation time, 17 I - 172, 184- 185,
189, 220, 289-290, 305
Retardation time, 294, 305
RGB (red-green-blue) system, 5, 42,
68-74, 393
Rice:
degree of milling, 24
gelatinization, 24
quality factors, I 10
RNA probe, 359-360
SAOS (small amplitude oscillatory
Index
423
[Ultrasonic]
pulse-echo method, 22 I , 232
pitch-and-catch method, 22, 222
through-transmission method, 22 I
velocity, 219,221,224,226,227,230
UV (ultraviolet), I , 54, 86, 100, I 15117,121,124
Viscoelasticity, 288-289, 301, 309. 324
Viscosity, 205. 243, 289, 307, 350
measurement, 3 12
complex, 3 18
Voxel,140,142
Water activity, 191,192
Waves:
acoustic, 291
evanescent, 348, 350
longitudinal, 2 18
mechanical, 2 17-2 18
shear, 2 18, 236
sound, 217
surface acoustic (SAW), 236, 350
Wheat:
fat content, I28
protein content, 28
smut content, 24
Xanthophyll, 20
X-ray:
absorption coefficient, 141,146
dualenergy,160
image, 86, 89, 139,141,150, 158
photons,144
source,139,146
Yogurt, gel stiffness, 3 I I