Inulins-Investigating The Influence of Inulin As A Fat

FOOSTR-15; No.
of Pages 13
food structure xxx (2014) xxxxxx
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Investigating the influence of inulin as a fat

substitute in comminuted products using rheology,
calorimetric and microscopy techniques
Derek F. Keenan a,*, Mark A.E. Auty b,2, Linda Doran b,2, Joseph P. Kerry c,3,
Ruth M. Hamill a,1
a
Teagasc, Food Research Centre Ashtown, Dublin 15, Ireland

Teagasc, Food Research Centre Moorepark, Fermoy, Co. Cork, Ireland
c
Food Packaging Group, School of Food and Nutritional Sciences, University College Cork, Co. Cork, Ireland
b
article info
abstract
Article history:
The present manuscript studied the effects of fat substitution with two commercial inulins
Received 16 January 2014
on the magnetic resonance, rheological, calorimetric and microscopic properties of break-
Received in revised form
fast sausages. Sausage formulations were evaluated using mixture design (D-optimal). A
17 April 2014
total of 17 experimental treatments were employed, with each representing a different
Accepted 16 June 2014
substitution level for fat. Sausage batters were formulated to contain lean pork shoulder,
Available online xxx
pork back fat/inulin, water, rusk and seasoning (44.3, 18.7, 27.5, 7 and 2.5% w/w, respective-
Keywords:
analyzed for each treatment group using nuclear magnetic resonance (NMR), rheology,
Sausage
differential scanning calorimetry (DSC), while their ultra-structural properties were ana-
ly). The resultant products water mobility, deformation and thermal behaviors were
Fat replacement
lyzed using light, confocal and scanning electron microscopy for selected extremes. Signifi-
Inulin
cant models were produced for water mobility with inulin inclusions in sausages increasing
Design of experiment (DOE)
the relative proton populations of bound water (T2b) values ( p < 0.0001) and decreasing free
Relaxation studies
Cryo-scanning electron microscopy
water (T22) population ( p < 0.0001). Inulin inclusions significantly altered the rheological
characteristics with increases in both the gel strength (G00 G000 ) and unit interaction strength
(An) ( p < 0.0001, respectively). Complementary temperature-dependent behavior was observed using rheology and DSC which showed increased elastic behavior (G0 ) circa 40 8C that
corresponded to the endothermic peaks for the onset of protein denaturation. Cryo-scanning electron and confocal laser microscopy techniques permitted visualization of the
aggregation of inulin micro-crystals and distribution of fat within the cooked sausage
matrix. Overall, the work presented has improved our understanding of the fundamental
properties of sausage products and will enable a more scientific-based approach to future
product development.
# 2014 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +353 1 8059500; fax: +353 1 8059550.

E-mail addresses: derek.keenan@teagasc.ie (D.F. Keenan), mark.auty@teagasc.ie (Mark A.E. Auty), joe.kerry@ucc.ie (J.P. Kerry).
1
Tel.: +353 1 8059500; fax: +353 1 8059550.

Tel.: +353 25 42222; fax: +353 25 42340.
3
Tel.: +353 21 4903000; fax: +353 21 4903000.
http://dx.doi.org/10.1016/j.foostr.2014.06.001
2213-3291/# 2014 Elsevier Ltd. All rights reserved.
2
Please cite this article in press as: Keenan, D. F., et al. Investigating the influence of inulin as a fat substitute in comminuted products using
rheology, calorimetric and microscopy techniques. Food Structure (2014), http://dx.doi.org/10.1016/j.foostr.2014.06.001
FOOSTR-15; No. of Pages 13
1.
Introduction
Meat gels can be thought of as an ordered three-dimensional

network formed by proteinprotein interactions in which fat
droplets, water, salts and other small components are trapped
(Peng & Nielsen, 1986). Sausage batters are structurally
complex meat products, comprising of mixtures of lean meat,
fat, fillers, seasoning and water with the dispersed phase
composed of fat globules and continuous phase made up of
protein/water (Morin, Temelli, & McMullen, 2004). This makes
them similar to both emulsions and suspensions from a
structural perspective (Honikel, 1983; Swasdee, Terrell, Dutson, & Lewis, 1982). Actin and myosin proteins must be
properly suspended and solubilized, denatured and heat
aggregated to form a gel structure with acceptable water
and fat binding and optimum texture properties (Ziegler &
Acton, 1984), with salt being commonly applied as a
solubilizing agent. Fat is stabilized within the solubilized
protein gel network in a sausage and contributes succulence
and texture (Feiner, 2006, chap. 12). Therefore, the mechanical
dispersal of the fat, which can depend on its hardness and
melting point, and the chemical composition of the lipid phase
also contribute to the subsequent physical properties of the
batters (Lee, Carroll, & Abdollahi, 1981; Lee, Hampson, &
Abdollahi, 1981; Whiting, 1987). Fat also helps prevent
shrinkage of the protein during cooking by acting as a filler
(Feiner, 2006, chap. 12). Overall, it has an important role in this
system due to its considerable influence on the binding
properties of the proteins (Morin et al., 2004).
Lowering fat levels in comminuted meat products to reduce
dietary intake of fat risks altering the structural properties of
the meat batter. During heating, the coagulating network of
proteins surrounds the melting fat particles, preventing them
from coalescing due to the mechanical fixation within the
meshes of the coagulated network. The larger these meshes
are, the less coalescence of fat that can occur and the better
the retention of fluids within the mixture (Hamm, 1975).
Structural deterioration occurs if the batter is not adequately
prepared leading to an unsightly and unsatisfactory product.
In such cases, uncoated fat surfaces, due to excessive
comminution, allow fat particles to come together and render
from the mixture during heating. This results in fat pockets or
greasing out of the emulsion leading to poor structural
integrity (Pearson & Gillett, 1996, chap. 9).
Diverse strategies to enhance the structural properties of
reduced fat products have been applied with variable results
(Choi et al., 2009; Hsu & Sun, 2006; Totosaus & Perez-Chabela,
2009; Youseff & Barbut, 2009). The long chain oligosaccharide,
inulin has been reported to exhibit synergistic effects with
most gelling agents, creating stronger gels than the sum of the
individual components (Kim, Faqih, & Wang, 2001). The
relative solubility of inulin in water allows it to form a
three-dimensional gel network of crystalline submicron
particles (100 nm diameter). These particles aggregate, trapping water and forming larger crystals (15 mm) in aqueous
solutions >25% inulin (short chains) or >13% inulin (long chain
with heating or shearing of the solutions). In this regard,
examining the potential of inulin to form a sufficiently stable
and acceptable gel, while partially compensating for the
removal of fat in a sausage matrix is of interest. Furthermore, a

more comprehensive understanding of the role of fat
substitutes in the structuring of low-fat products in both gel
forming and gel stability terms would be desirable.
To date, studies elucidating fundamental physico-chemistry
in meat systems have mainly utilized model systems, e.g.
combinations of oligosaccharides, purified myofibrils or myofibrillar proteins (Chou & Lin, 2010) and soy fractions (Tseng, Xiong,
& Boatright, 2008). While these studies are enlightening, they
cannot take full cognizance of the more complex matrix
properties of the full-scale product. Studies that do appear
(Keenan, Resconi, Kerry, & Hamill, 2014; Mendoza, Garcia, Casas,
& Selgas, 2001) concerning full-scale products are largely
concerned with technological characteristics of quality and do
not consider more fundamental physico-chemistry governing
these quality markers. However, more specialized techniques are
available for the analyses of the underlying physico-chemistry of
meat products. For example, the mobility of water has been
studied extensively using nuclear magnetic resonance (NMR)
relaxometry in meat (Andersen, Andersen, & Bertram, 2007;
Bertram & Andersen, 2004; Bertram et al., 2001; Shaarani, Nott, &
Hall, 2006) and could be a useful tool in determining the effects of
inulin on water in comminuted meat products, where water
plays a key role in the formation of the overall gel. Differential
scanning calorimetry can be used to analyze the thermal
behavior characteristics of sausages, particularly fat-containing
controls and those containing substituted-inulin. This may offer
some insight into the similarity of inulin gels which have been
reported to exhibit phenomena reminiscent of the melting
behavior of fat crystal networks due to the wide molecular
weight distribution of the inulin polyfructose (Bot, Floter,
Lammers, & Pelan, 2003; de Bruijne & Bot, 1999). The similarities
between the structuring properties of inulin when it crystallizes
and how fat structures meat products could be better elucidated
by rheological analysis (Bot, Erle, Vreeker, & Agterof, 2004).
Therefore, the principal objective of the present study was
to perform a detailed characterization of the impact of
substituting inulin for fat in sausages in rheological, calorimetric, NMR spectroscopic and microscopic terms using a
mixture design approach.
2.
Materials and methods
2.1.
Sausage preparation
The basic sausage formulation is given in Table 1. Sausage

formulations containing the three variable components
(X1 = pork backfat, X2 = Orafti1 GR, and X3 = Orafti1 HP (Orafti
Table 1 Sausage formulation.

Ingredient
Pork shoulder (95% lean)
Pork back fat
Inulin (Orafti1 HP/GR)
Water
Rusk
Seasoning
Composition (%)
44.3
018.7
018.7
27.5
7.0
2.5

products represented two commercial forms of inulin)) were

developed using Design Expert software (v. 7.6.1, Stat-Ease
Inc., Minneapolis, MN, USA). Pork shoulder (95% lean) and pork
back fat (Granby Meats, Dublin, Ireland) were minced (model
PT-82/22 Mainca Barcelona, Spain) twice (5 mm plate size) and
bowl chopped with powdered inulin, ice water, seasoning and
rusk for 2 min. Sausage batter was piped into a cellulose casing
and blast frozen (air speed 3.75 m/s) and stored (20 8C) for all
subsequent analyses as outlined in a previous study (Keenan
et al., 2014). Sausages (five per treatment, vacuum-packed)
were cooked using water bath immersion (85 8C) of five vacuumpacked sausages per treatment was until they had achieved a
core temperature of 73 8C. Core temperature profiles were
recorded during the process using an Ellab E-Val TM TM9608
data module (Ellab [UK] Ltd., Norfolk, England) connected to a
laptop. A standard Ellab SSA-12080-G700-TS temperature probe
was inserted through an Ellab GKM-13009-C020 packing gland
(20 mm) into the largest sample in the vacuum bag.
2.2.
Time domain nuclear magnetic resonance (TD-NMR)
Nuclear magnetic resonance (NMR) relaxation measurements

were carried out as previously described (McDonnell et al.,
2013), on a Maran Ultra instrument (Oxford Instruments,
Abington, Oxfordshire, UK) with a resonance frequency for
protons of 23.2 MHz. Transverse relaxation (T2) times were
measured using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with the resultant relaxation decays analyzed by triexponential unsupervised fitting in the RI Win-DXP software
(V. 1.2.3 Oxford Instruments, Abington, Oxfordshire, UK).
2.3.
2008) with slight modifications. Before testing, samples were

rested (5 min) to achieve a constant test temperature (5 8C) and
relaxation of residual stresses. Thermal gelation was induced by
heating samples from 5 8C to 85 8C at 1 8C min1 (Brunton, Lyng,
Zhang, & Jacquier, 2006). Temperature was controlled by the
aforementioned Pelltier plate and temperature hood. Samples
were sheared at a fixed frequency of 1.0 Hz with a strain of 0.02%.
Sample perimeter was coated with a thin layer of petroleum jelly
to prevent dehydration during testing. Changes in the storage
modulus (G0 ) were monitored throughout the gelling process.
2.4.
Differential scanning calorimetry (DSC)
Thermal transition properties were measured using a TA

Instruments DSC (Model No. DSC 2010, TA Instruments Inc.,
New Castle, DE, USA) equipped with nitrogen cooling. Indium
(melting point 156.6 8C) and baseline (empty pan) calibrations
were carried out prior to testing. Homogenized (Robot Coupe
Blixer 41 mono, Bourgogne, France) sausages samples
(1520 mg) were weighed into aluminum pans and hermetically sealed. Samples were equilibrated at 10 8C and then
heated from 10 to 90 8C at a heating rate of 10 8C min1
against a reference (empty) pan (McArdle, Kerry, Mullen, Allen,
& Hamill, 2011). Onset temperature, protein denaturation (To),
the peak temperature (Tp) and the denaturation enthalpy (DH)
were recorded. Samples were analyzed in triplicate.
Rheology
Rheological measurements were performed on a Physica MCR

301 rheometer (Anton Paar GmbH, Graz, Austria) fitted with
parallel plate (50 mm; smooth) geometry running Rheoplus
software package (version 3.21, Anton Paar GmbH, Germany).
Sausage batters were pressed from their casings and placed onto
the center of the base plate. The upper plate was moved into
position, i.e. the distance between the two plates (gap) was set to
1 mm. Excess material was trimmed from the plate edges and
samples were allowed to rest for 5 min to achieve a constant test
temperature (25 8C regulated by the rheometers Pelltier plate
and temperature hood), and for relaxation of residual stresses.
Viscoelastic properties were assessed by performing a preliminary amplitude sweep to identify the linear viscoelastic (LVE)
region of the samples and the strain (0.1%) that should be used
for the resultant frequency sweep. A frequency sweep from 0.1
to 10 Hz was performed and the results for storage modulus (G0 ),
loss modulus (G00 ), and complex modulus (G*) were recorded.
These data were modeled using the following power law
equations as suggested by Friedrich and Heymann (1998):
0
G0 G00 vn
(1)
00
G00 G000 vn
G An vn
(2)
(3)
Thermal gelation properties were assessed as previously

described (Cofrades, Serrano, Ayo, Carballo, & Jimenez-Colmenero,
Fig. 1 Representative distribution of T2 relaxation times

for breakfast sausages: (a) comparison between raw (
)
and cooked () in control sausages (run 10) and (b)
comparison between cooked control () sausages and
selected fat-substituted counterparts [- - - 100%
67% substitution HP:GR 1:1
substitution HP (run 10)]; [
(run 15)].
Table 2 Regression coefficients for significant quality and structural parameters of sausages.
Dependent variables
DHp1 Y1
DHp2 Y2
G00 Y3
G00 G000 Y4
A n Y5
n 0 Y6
G0 (30) Y7
G0 (72) Y8
T2b (P) Y9
T2b (T) Y10
T21 (P) Y11
T21 (T) Y12
T22 (P) Y13
T22 (T) Y14
*, **, ***
2.5.
Independent variables
X1
X2
X3
1.05
1.36
8.73
8.47
8.80
0.18
45.00
47.10
15.40
17.52
69.48
40.30
15.51
171.68
0.22
2.97
10.66
10.40
10.66
0.37
222.28
114.40
27.71
14.09
65.01
26.87
7.08
140.96
0.17
3.77
11.67
11.42
11.74
0.32
303.96
128.10
30.03
12.10
64.75
21.94
5.12
136.22
X1*X2
X1*X3
X2*X3
1.96
2.38
1.89
6.86
11.39
30.63**
5.45
14.77
27.13**
R2
p model
p lack of fit
0.85
0.90
0.89
0.89
0.84
0.40
0.75
0.35
0.90
0.61
0.70
0.96
0.96
0.85
0.0001
0.0001
0.0001
0.0001
0.0001
0.0286
0.0001
0.0468
0.0001
0.0013
0.0100
0.0001
0.0001
0.0001
0.25
0.78
0.84
0.84
0.86
0.76
0.87
0.60
0.79
0.98
0.56
0.99
0.56
0.08
X1*X2*X3
Significant at p < 0.05, p < 0.01 and p < 0.001 respectively.
Microscopy
Sausage samples (1 cm3) were flash frozen in liquid nitrogen

and stored at 80 8C. Sections were cut (20 mm) using a Leica
CM1950 cryostat (Leica Biosystems, Nussloch, Germany) after
equilibration to specimen chamber temperature (25 8C).
Light microscopy sections were stained with fast green and
iodine (ratio 10:1) stains and examined using a Leica DMLB
light microscope (Leica Microsystems AG, Wetzlar, Germany).
Confocal scanning laser microscopy (CSLM) was used in
conjunction with differential staining to visualize the distribution of the fat component within the sausages. Sections
were stained with Fast Green (FCF) and Nile Red stains and
examined under a Leica SP5 confocal microscope (Leica
Microsystems GmBH, Mannheim, Germany).
For Cryogenic Scanning Electron Microscopy (Cryo-SEM),
cooked samples were frozen in liquid nitrogen slush (210 8C)
and transferred to an Alto 2500 cryo preparation chamber
(Gatan Ltd., Oxfordshire, UK) at 185 8C. Samples were
fractured used a cooled knife and then warmed to 95 8C
Fig. 2 Contour plots of T2 relaxation data of (a) relative proton population for bound water (T2b %); (b) time constant for
bound water population (ms); (c) relative proton population for intra-cellular water (T21 %); (d) time constant for intracellular water population (ms); (e) relative proton population for extra-cellular water (T22 %); (f) time constant for extracellular water population (ms); for fat substituted sausages (A, pork fat; B, OraftiW GR; C, OraftiW HP; where A + B + C = 18.7%
of total mixture).

for 5 min to remove surface ice. Fracture surfaces were sputter

coated with platinum for 120 s at 130 8C and the sample
transferred to the cold stage in the SEM instrument. Images
were acquired at 125 8C and 2 kV accelerating voltage in a
Carl Zeiss Supra 40VP field emission scanning electron
microscope (Carl Zeiss Ltd., Hertfortshire, UK).
2.6.
Analysis of data
Mixture design experiments were designed and analyzed

using Design Expert (v. 7.6.1, Stat-Ease Inc., Minneapolis, MN,
USA) as previously described (Keenan et al., 2014). All
parameters of NMR spectrometry, rheometry and calorimetry
were assessed and modeled using linear, quadratic, or
Scheffes special cubic models. Models were subjected to
analysis of variance (ANOVA) to determine the significance
( p < 0.05), determination coefficient (R2) and lack of fit.
3.
in the present study and the quality attributes of cook loss and
total expressible fluid (R2 = 0.65 and 0.70, respectively). It
also supported the hypothesis that dietary fiber addition
improves water binding capacity (Rodrguez, Jimenez, Fernandez-Bolanos, Guillen, & Heredia, 2006). Previous studies
involving the water mobility of meat using TD-NMR have
shown no significant change in the T2b population (Bertram
et al., 2001; Mller et al., 2011). However, the data in the present
study appears to indicate evidence of additional water binding
due to the presence of inulin. This effect could be attributed to
inulins water solubility properties, i.e. its rich source of
hydroxyl groups that allow it to interact with water through
hydrogen bonding, resulting in gel formation by bridging the
inulin molecules together, allowing it to crystallise and
stabilize (Barclay, Ginic-Markovic, Cooper, & Petrovsky,
2010). It has a water binding capacity of about 2:1 (Silva,
1996) and the resultant ability to form stable gels at water
Results and discussion
Mean values for NMR, rheological and calorimetric responses for

all treatments are presented in the supplementary Tables 15.
3.1.
Water mobility by time domain-nuclear magnetic
resonance (TD-NMR) relaxometry
Fig. 1a shows a typical representation of raw and cooked T2
relaxation times obtained for control (full-fat) sausages. T2
relaxation measurements revealed three populations of water
present in all samples, namely; T2b, T21 and T22. A notable
decrease in all three water populations was observed in
cooked samples compared to their raw counterparts as
anticipated (through the water lost in cook-out). Fig. 1b shows
significant changes between different formulations of cooked
samples, i.e. control (full fat), a sample containing 33.3% of
each variable ingredient (fat/inulin type) and complete/total
fat substitution using inulin. T2b was the first peak identified
and occurred between 10 and 18 ms. T2b values have been
ascribed to water closely associated with macromolecules or
protons located on macromolecular structures plasticized by
water, i.e. bound/macromolecular water (Bertram et al.,
2001). Table 2 shows that the relative proton populations of T2b
values were fitted to a quadratic model which was found to be
significant ( p < 0.0001) with a good fit to the experimental data
(R2 = 0.90). The model showed that linear terms were the most
significant ( p < 0.0001) with T2b values decreased in fat
containing sausages compared to fat substituted sausages
(Fig. 2a). Furthermore, a significant interactive synergistic
effect ( p < 0.0024) was observed between the B and C
components, i.e. Orafti inulin GR and HP commercial forms.
With increasing T2b values, there was a concomitant decrease
in relaxation time. A linear model was fitted ( p < 0.0013) to the
T2b time data with a reasonable fit to the experimental data
(R2 = 0.61) (Fig. 2b). A decrease in relaxation time indicated a
reduced mobility of water (Mller et al., 2011). These data are in
agreement with the findings of a previous study (Keenan et al.,
2014) carried out by our group which focused on the physicochemical properties and eating quality of low-fat sausages and
showed negative Pearson correlation between T2b proton data
Fig. 3 Mechanical spectra of frequency dependant

rheological parameters of (a) storage; (b) loss; and (c)
complex moduli in control (~) sausages and selected fatsubstituted counterparts [& 100% substitution HP (run
10)]; [ 67% substitution HP:GR 1:1 (run 15)]; [+ 50%
substitution HP:GR 1:1 (run 16)]; [* 33% substitution HP:GR
1:1 (run 5)].
concentrations of 1350% due to these limited surfactant

properties (Kim et al., 2001).
T21 was the second peak identified and occurred between
2341 ms. T21 values represent water trapped by the dense
myofibrillar network, i.e. intra-myofibrillar water (Bertram
et al., 2001). The T21 population represented the majority of the
water present in the sausage matrix for all samples and this
overall trend is consistent with the findings of other authors
(Mller, Gunvig, & Bertram, 2010). T21 values were fitted to a
quadratic model which was found to be significant ( p < 0.01)
with a good fit to the experimental data (R2 = 0.70). The model
showed that linear terms were significant ( p < 0.0140) and that
fat formulations resulted in higher T21 values than their inulin
substituted counterparts (Fig. 2c). Additional interactions for
the B and C components (GR and HP) were observed
( p < 0.0076) giving a strongly antagonistic effect on the
sausage batter. Despite this trend, the relaxation times
followed a similar pattern to the T2b data, i.e. a decrease in
time (suggesting water immobilization) with increasing inulin
inclusion (Fig. 2d). T21 (time) values were fitted to a linear
model which was found to be significant ( p < 0.0001) with a
good fit to the experimental data (R2 = 0.96). The reason for
these contradictory observations is most likely due to an
assumption that the entire signal detected by TD-NMR is
attributable to water. In fact, the method also observes
protons on fat molecules and that the variation observed in
the present study is most likely a result of the changing fat
content between sausage formulations.
The final peak identified was T22 which occurred between
131 and 169 ms. T22 values represent free water within the
matrix, i.e. extra-myofibrillar water (Bertram et al., 2001). T22
values were fitted to a linear model which was found to be
significant ( p < 0.0001) with a good fit to the experimental data

(R2 = 0.95). T22 values decreased with increasing fat substitution (Fig. 2e). A linear model was fitted ( p < 0.0001) to the T22
relaxation time data which resulted in a good fit to the
experimental data (R2 = 0.85). With decreasing T22 values in
inulin-enriched formulations, there was also a decrease in
relaxation time indicating reduced mobility of water (Fig. 2f).
Other authors have reported a reduction in the T22 population
by substituting fat with potato starch in a force meat product
(Baranowska, Dolata, Piotrowska, & Manczak, 2004). The data
in the present study positively correlate (Pearson) with data
previously reported by our research group (Keenan et al., 2014)
for cook loss and TEF (R2 = 0.75 and 0.87, respectively). It
suggests that the interaction between the water molecules
and the polymer are the primary mechanism governing the
differences in the water state of the final formulation
(Baranowska, Dolata, Piotrowska, & Manczak, 2004).
3.2.
Rheology
3.2.1.
Frequency dependent behavior
Frequency sweeps for all sausage formulations were carried

out (25 8C) to determine the angular frequency dependence of
storage, loss and complex moduli (G0 , G00 and G*) within the
limits of the linear viscoelastic (LVE) range (Fig. 3ac). Overall
the line geometry of samples was similar with the main
differences occurring between the absolute values of the
respective moduli. The storage modulus can be seen to
dominate the loss modulus (G0 > G00 ) for all samples throughout the frequency range (Fig. 3a and b). Furthermore, a
frequency dependence was noted for all moduli, with
increasing G0 , G00 and G* values with increasing frequency,
Fig. 4 Contour plots of fitted rheology parameters of (a) initial storage modulus (G00 Pa); (b) initial loss modulus (G000 Pa); (c)
strength of rheological unit (An Pa); (d) structural stability (n0 unit less); (e) and (f) storage moduli (G0 ) at 30 and 72 8C,
respectively, for fat substituted sausages (A, pork fat; B, OraftiW GR; C, OraftiW HP; where A + B + C = 18.7% of total mixture).

implying a relatively weak structure, which is indicative of a

gel-like material consisting of a loosely ordered structure with
a viscoelastic consistency (Delgado-Pando, Cofrades, RuizCapillas, Triki, & Jimenez-Colmenero, 2012). Power law-fitted
parameters were derived from G0 , G00 and G* moduli as
previously described (Campo & Tovar, 2008). G00 and G000
(Eqs. (1) and (2)) are the initial storage and loss moduli,
respectively, and are a measure of the resistance of a test
material to elastic (storage) and viscous (loss) deformation
(Zhou & Mulvaney, 1998) at an angular frequency of
0.5 rad s1. The parameters were fitted with a linear model,
which was transformed using a natural log recommended by
the BoxCox method. The initial elastic behavior of sausages,
G00 , increased in sausages which contained more inulin,
particularly in full substitutions with Orafti HP, compared to
intermediate formulations and fat-only controls (Fig. 4a). The
predicted model for G00 was found to be significant ( p < 0.0001),
showing a good fit with experimental data (R2 = 0.90) and is in
agreement with the experimental findings from the mechanical spectra (Fig. 3ac). Higher storage modulus in sausages
prepared with Orafti HP can most likely be attributed to the
higher degree of polymerization of this inulin which is in
agreement with previous studies involving the use of this
ingredient in other food stuffs (Juszczak et al., 2012; Peressini &
Sensidoni, 2009). These authors postulated that both inulin
inulin interactions and increased water binding by the inulin
promoted an increase in the storage modulus.
The model of Friedrich and Heymann (1998) used a threedimensional structure in order to characterize the gel
properties. Gel firmness was assessed by studying the two
magnitude parameters, i.e. G00 G000 (related to viscoelastic gel
strength) and An (strength of the rheological unit interaction)
(Campo & Tovar, 2008). Transformed (natural log) G00 G000 data
resulted in significant model fitting ( p < 0.0001) with a good fit
to the experimental data (R2 = 0.89) and showed that the linear
components, i.e. A fat; B Orafti GR and C Orafti HP, were
the most significant terms (Fig. 4b). The same transformation
and subsequent fitting procedure was applied to An values
(Fig. 4c) which yielded a similar outcome ( p < 0.0001; R2 = 0.84).
These data indicate that fat-substituted samples, particularly
those containing HP inclusion, resulted in more rigid and
firmer gels than fat containing controls. These data positively
correlated with hardness values from TPA (R2 = 0.59 and 0.53,
respectively). However, despite the more rigid gels, there was
no apparent deterioration in the quality of the gel in the
present study, with sausages containing high quantities of
inulin displaying the highest water binding properties (lower
cook loss and higher T2b bound water) compared to fat
controls. This is due to inulins ability to stabilize the water in
the emulsion/gel structure (Bot et al., 2004). This implies
partial inulin substitutions may be more beneficial in fat
reduction strategies to promote better water binding, as
improved water binding properties have been purported to be
associated with greater structural gel strength (Delgado-Pando
et al., 2012). Phase angle (d) was consistent with the other
rheological parameters, i.e. indicating the viscoelastic behavior of sausage batters (458 > d > 08), and can be considered a
measure of the energy loss versus energy stored. No
differences were observed between sample formulations
indicating that no substantial structural changes occurred
during fat substitution. The structural stability and protein

network conformation were assessed by studying the exponent n0 derived from power law fitting of G0 , G00 and G*. n0 data
was fitted with a linear model and was found to be significant
( p < 0.0286) but only showed a fit of R2 = 0.40 with the
experimental data. n0 values were lowest (circa. 0.17) in
sausages containing higher amounts of fat and highest in
samples containing GR inclusions (Fig. 4d). Higher n0 values
indicate greater instability of the matrix when subjected to
changes frequency (Delgado-Pando et al., 2012; Zhou &
Mulvaney, 1998). Therefore, samples containing fat behaved
more like ideal elastics gel as the values were closer to zero
Fig. 5 Mechanical spectra of temperature dependant

rheological parameters of (a) storage; (b) loss; moduli and
(c) phase angle in control (~) sausages and selected fatsubstituted counterparts [& 100% substitution HP (run
10)]; [ 67% substitution HP:GR 1:1 (run 15)]; [+ 50%
substitution HP:GR 1:1 (run 16)]; [* 33% substitution HP:GR
1:1 (run 5)].
and exhibited a lower frequency dependence. The reason for a

weakening of the protein conformation is unclear as inulin
appeared to add structural stability to the sausage matrix (as
evidenced by the other rheological parameters). NMR data
showed more bound water which could be attributed to the
inulin molecules. Typically, sausage emulsions are formed by
the salt-soluble myofibrillar proteins emulsifying the fat and
immobilization of water (Feiner, 2006, chap. 12). Less available
water may have affected the emulsion structure, with less
protein being solubilized and hence reduced emulsification of
the fat component and consequently, increased its frequency
dependence.
3.2.2.
Temperature-dependent behavior
Temperature sweeps showed that the thermal behavior of

samples in terms of their storage, loss moduli and phase angle
(G0 , G00 and d) as a function of temperature (585 8C) (Fig. 5ac).
Differences in thermal rheological properties were observed
between the different formulations due to their differing
compositions (presence or absence of fat/inulins) as expected
and are supported by the previous texture and rheological
experiments. As was the case for frequency sweeps, storage
modulus (elastic behavior) exceeded the loss modulus
(viscous behavior) over the temperature range, indicating
gel network (elastic) formation containing a substantial
emulsion structure (viscous). These observations were in line

with previous studies on the rheological thermal gelling
properties of low fat pork liver pates formulated with other
polysaccharides (Delgado-Pando et al., 2012). Fig. 5a and b
shows a slight decrease in both G0 and G00 moduli for samples
containing higher fat content, such as the full fat control (and
to a lesser extent the intermediate formulations) at 3035 8C. If
we consider the absolute values of G0 at 30 8C in the mixture
design, it shows that data was significantly ( p < 0.0001) fitted
to a linear model with a good fit to the experimental data
(R2 = 0.75) and showed that the linear components were the
most significant terms. The modeled surface shows that G0
values were lower in higher fat containing formulations than
their inulin containing counterparts (Fig. 4e). This observation
is consistent with the melting of pork back fat (JimenezColmenero et al., 2012) and is in agreement with the DSC data
in the present study. This was followed by a dramatic increase
in G0 and G00 values for all samples between 40 and 80 8C during
which the main rheological changes occurred (Fig. 5a and b).
This can be attributed to conformational changes in the meat
proteins that occur in this temperature range leading to the
characteristic stiff elastic matrix of meat gels The absolute
values of G0 at 72 8C (finishing temperature of the sausages in
the present study) were successfully fitted ( p < 0.0468) to a
linear model (R2 = 0.35). Similarly, samples containing inulin
Fig. 6 (a) Representative thermal behavior of control (~) sausages and selected fat-substituted counterparts [& 100%
substitution HP (run 10)]; [ 67% substitution HP:GR 1:1 (run 15)]; [+ 50% substitution HP:GR 1:1 (run 16)]; [* 33%
substitution HP:GR 1:1 (run 5)]; and contour plots of thermal behavior: endothermic peaks (EP) (b) 1; and (c) 2; in
fat-substituted sausages by differential scanning calorimetry (DSC) (A, pork fat; B, Orafti1 GR; C, Orafti1 HP; where
A + B + C = 18.7% of total mixture).

had higher storage moduli than those containing fat (Fig. 4f).
The presence of inulin increased the storage and loss moduli
due to the formation of inulins own three-dimensional
network which differs to that of pork back fat. These data
corresponds to the thermal properties observed in the second
endothermic peak of DSC data (Fig. 6a). However, the effect
was not consistent for increasing levels of inulin, for example,
run 15 (containing 1/3 of each fat/inulin component) had
substantially higher G0 and G00 values than those containing
full substitution with inulin. Statistical analysis did not
support any possible interactive effect between the two inulin
types to account for this phenomenon. It could be attributed to
the heterogeneous nature of the sausage batter or due to the
mechanisms governing the formation of the inulin gel,
i.e. nucleation and crystallization, which are difficult to control

and rely on the mutual arrangement of the crystals that can
lead to gels with different rheological properties (Glibowski,
2010; Stasiak & Dolatowski, 2008). Furthermore, increases in G0
and G00 values were less pronounced in control formulations
compared to their inulin containing counterparts.
Further investigation of the absolute values of G0 , G00 and d
and specific temperature points in the mixture design
revealed some interesting observations. Significant differences were observed between different formulations for G0
values from 5 to 72 8C. However, this was only the case from 5
to 50 8C for G00 values. This could indicate that conditions that
conferred elasticity rather than viscosity were favored after
50 8C. Phase angle data showed no significant differences
Fig. 7 Light micrographs of cryostat sections of (a) control (full fat) sausages at 4T; (b) 10T; and (c) 20T magnifications and
(d) fully fat-substituted sausages at 4T; (e) 10T; and (f) 20T magnifications.
10
between formulations between 5 and 30 8C, implying a

similar underlying structure in all formulations. This
changed with increasing heat from 40 to 80 8C, with inulinformulated samples showing increases in viscoelastic properties (data not shown). These changes imply substantial
structural changes occurring during the application of heat
(as expected) and this changed significantly for different
levels of fat substitution (which is in agreement in with DSC,
rheological and textural parameters shown in this study). A
decrease in G0 and G00 values was observed between 80 and
85 8C for sausage samples containing inulin, which were
more notable in samples containing the inulin form Orafti
HP. This may be attributable to the greater degree of
polymerization (DP) of Orafti HP (>23) compared to Orafti
GR (>10). Inulin with high DP is thought to be thermally
unstable and temperatures above 80 8C have been reported to
inhibit the formation of gels (Bot et al., 2004; Glibowski &
Wasko, 2008).
3.3.
Differential scanning calorimetry (DSC)
Fig. 6a shows typical DSC heat curves for fat and inulinenriched sausages, with two endothermic peaks obtained for
all samples. The first endothermic peak had an onset
temperature between 24 and 31 8C and corresponds to the
melting point of fat. This finding is in agreement with data
presented by other authors (Morin et al., 2004). A linear model
was fitted to the reaction enthalpy (data, which was
transformed using an inverse square-root power law recommended by the BoxCox method). The model was significant
( p < 0.0001) with a good fit to the experimental data (R2 = 0.85).
The reaction enthalpy (DH) values for endothermic peak 1
(energy needed to melt the fat) decreased as fat was removed
from the formulation as expected (Fig. 6b). As fat was
substituted, DH values for endothermic peak 1 decreased,
while there was a concomitant rise in reaction enthalpy for
endothermic peak 2. The second endothermic peak had an
onset temperature of 47 and 66 8C and this corresponds to the
remaining components of the sausage matrix, e.g. protein and
carbohydrate. For example, the thermal properties of meat
proteins have been reported for myosin, collagen and actin in
the regions of 59, 66, and 82 8C, respectively (Brunton et al.,
2006), while wheat starch (present as rusk in our formulation)
gelatinization has been reported to occur between 58.8 and
62.6 8C (Alamri, Mohamed, & Hussain, 2013; Zaidul et al., 2008,
respectively)). A quadratic model was fitted to DH values for
endothermic peak 2. The model was significant ( p < 0.0001)
with a good fit to the experimental data (R2 = 0.90) and showed
that the linear components, i.e. A fat; B Orafti GR and C
Orafti HP, were the most significant terms. Increases in
enthalpy energy were higher in samples containing Orafti HP
and Orafti GR (Fig. 6c). The increase in energy required to
denature the proteins could be explained by a stabilizing effect
on the protein through the excluded volume principle and
differential interaction effects (Timasheff, 1998). This protein
stabilization is often attributed to simple sugars, such as
glucose, sucrose, raffinose and trehalose (Arakawa & Timasheff, 1982; Back, Oakenfull, & Smith, 1979; Schellman, 2003).
The polysaccharides provide multiple hydroxyl groups that
can replace hydrogen bond interactions as water is removed
Fig. 8 Confocal scanning laser microscopy (CSLM) of

cryostat sections of (a) control; (b) 67% substituted; and (c)
full fat-substituted sausages.

from a system (Hinrichs, Prinsen, & Frijlink, 2001). This helps

to maintain the proteins native confirmation and prevents
denaturation (Barclay et al., 2010). Other reported findings of
increased DH values soy protein dispersions containing both
sucrose and inulin (using water as a solvent) compared to
controls18 were postulated as a thermal stabilization effect
on native soy proteins by inulin. Other contributory factors
which are responsible for the increased thermal properties
observed in the second thermal peak may have been due to
the water binding effect of inulin. This could limit the
available water to other matrix components, e.g. rusk,
thereby limiting hydration of starch and profoundly
modifying the thermal properties of starch gelatinization
(Juszczak et al., 2012).
3.4.
Microscopy
Light microscopy conducted on iodine and fast green stained

cryostat sections of control (full fat) and fully fat-substituted
sausages at different magnifications (4, 10 and 20) are
presented in Fig. 7af. Both treatments consisted of a
continuous protein phase (green) containing fat globules
and adipose cells (unstained). Some larger muscle fragments
were also visible. Large aggregates of partially gelatinized
starch granules (stained purple in iodine) were dispersed
within the protein matrix. These are most likely rusk
particles. No obvious structural differences were observed
between the control and inulin-containing samples. The
presence of fat and its distribution within the sausage matrix
11
was subsequently visualized using confocal scanning laser

microscopy (CSLM) and differential staining with fast green
and Nile red (Fig. 8ac). Significant differences between the
sausage treatments were observed. Fig. 8a shows fat (stained
green) was abundantly present in full-fat control and was
coated by the protein/water continuous phase forming the
typical sausage emulsion-like structure. A large population
of adipose tissue is also visible in the bottom right of the
image. Fat substitution in different sausage formulations led
to a concomitant reduction of green stain, which can be
clearly seen in Fig. 8b and c representing 66% fat reduction
(run 15) and full fat substitution, respectively. The residual
green stain in the latter represents the native fat content of
the pork shoulder muscle. The presence of the inulin and its
distribution within the sausage matrix was visualized in a
fully fat-substituted formulation (Fig. 9c and d) using
cryogenic scanning electron microscopy (cryo-SEM) and
compared against control (Fig. 9a and b) formulation (full
fat). Gelatinized starch granules from the added rusk could
be clearly identified in the sausage matrix (labeled SG
Fig. 9a). Sausage treatment containing inulin had crystalline regions, often in the form of rounded spherulitic
structures. These structures were not present in the control
sample, which contained more conventional fat morphology (labeled F Fig. 9b), and it is strongly suggested that
they are inulin crystals (labeled I Fig. 9d). Previous studies
(Cooper & Carter, 1986; Cooper & Steele, 1991) have reported
that inulin particles normally crystallize from water as
ovoids of 110 mm diameter, much like the observations of
Fig. 9 Cryo-scanning electron microscopy (cryo-SEM) of (a and b) control; and (c and d) full fat-substituted sausages (where
GS, gelatinized starch; F, fat; and I, inulin).
12
the present study. Results suggest that the inulin had not
fully dissolved in the meat product, but remained in
localized concentrations.
4.
Conclusion
NMR, rheological, calorimetric and microscopy approaches

were applied as means to better elucidate the underlying
mechanisms governing the physico-chemical responses in
reduced-fat sausage formulations. NMR analysis gave a better
indication of the water properties within the sausages
compared to empirical methods of moisture assessment,
e.g. cook loss. Populations of bound, trapped and free water
were present in the sausage and increased water retention
was noted in samples with increasing inulin inclusion.
Rheological data showed an increase in the elastic (solid)
component of the sausage batters with increasing substitution
of fat with inulin which would have a direct effect on the
instrumental and sensory textural attributes. Heat ramps for
rheology and DSC analyses correlated thereby showing
complementary protein denaturation profiles and increasing
elastic behavior (gel hardening). Microscopy techniques
showed the differences in the distribution and morphology
of inulin enriched emulsions compared to their control (fat
containing) counterparts.
Industrial relevance
Most of the existing processes in meat product development
have been developed empirically by testing the effect of a
limited number of ingredients/inclusion levels/processing
conditions on product quality and yield. While this approach
in pragmatic, it does little to develop the understanding of the
processes occurring at the molecular level that govern the final
product. This study focussed on the implementation of a
systematic scientific design approach in the controlled and
efficient development of low fat sausages containing inulin by
consideration of the meat system as a matrix of interacting
components. The physical interactions between these components at the ultrastructural level were visualised using high
resolution electron and confocal laser microscopy
approaches. These data were integrated with information
on the molecular interactions within the meat matrix as
observed by nuclear magnetic resonance, rheological and
calorimetric techniques to give a better understanding of the
forces influencing the macro scale product. Overall, this work
permitted a more structured and predictive approach to
product formulation, facilitating the development of improved, healthier and more functional meat products for
the meat industry.
Acknowledgement
Research funding was provided under the National Development Plan, through the Food Institutional Research Measure
(08/RD/TAFRC/671), administered by the Department of
Agriculture, Food and the Marine, Ireland.
Appendix A. Supplementary data

Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.foostr.2014.06.001.
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Inulins-Investigating The Influence of Inulin As A Fat

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FOOSTR-15; No.

Available online at www.sciencedirect.com

Investigating the influence of inulin as a fat

Teagasc, Food Research Centre Ashtown, Dublin 15, Ireland

Received 16 January 2014

on the magnetic resonance, rheological, calorimetric and microscopic properties of break-

Received in revised form

total of 17 experimental treatments were employed, with each representing a different

Accepted 16 June 2014

Available online xxx

Design of experiment (DOE)

* Corresponding author. Tel.: +353 1 8059500; fax: +353 1 8059550.

Tel.: +353 1 8059500; fax: +353 1 8059550.

FOOSTR-15; No. of Pages 13

food structure xxx (2014) xxxxxx

Meat gels can be thought of as an ordered three-dimensional

removal of fat in a sausage matrix is of interest. Furthermore, a

Materials and methods

The basic sausage formulation is given in Table 1. Sausage

Table 1 Sausage formulation.

FOOSTR-15; No. of Pages 13

products represented two commercial forms of inulin)) were

Time domain nuclear magnetic resonance (TD-NMR)

Nuclear magnetic resonance (NMR) relaxation measurements

2008) with slight modifications. Before testing, samples were

Differential scanning calorimetry (DSC)

Thermal transition properties were measured using a TA

Rheological measurements were performed on a Physica MCR

Thermal gelation properties were assessed as previously

Fig. 1 Representative distribution of T2 relaxation times

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food structure xxx (2014) xxxxxx

Significant at p < 0.05, p < 0.01 and p < 0.001 respectively.

Sausage samples (1 cm3) were flash frozen in liquid nitrogen

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for 5 min to remove surface ice. Fracture surfaces were sputter

Mixture design experiments were designed and analyzed

Results and discussion

Mean values for NMR, rheological and calorimetric responses for

Fig. 3 Mechanical spectra of frequency dependant

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food structure xxx (2014) xxxxxx

concentrations of 1350% due to these limited surfactant

significant ( p < 0.0001) with a good fit to the experimental data

Frequency dependent behavior

Frequency sweeps for all sausage formulations were carried

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implying a relatively weak structure, which is indicative of a

during fat substitution. The structural stability and protein

Fig. 5 Mechanical spectra of temperature dependant

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food structure xxx (2014) xxxxxx

and exhibited a lower frequency dependence. The reason for a

Temperature sweeps showed that the thermal behavior of

emulsion structure (viscous). These observations were in line

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i.e. nucleation and crystallization, which are difficult to control

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food structure xxx (2014) xxxxxx

between formulations between 5 and 30 8C, implying a

Differential scanning calorimetry (DSC)

Fig. 8 Confocal scanning laser microscopy (CSLM) of

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