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Analysis of Soyabean Proteins in Meat Products: A

Review
b a a a
J. Belloque , M. C. García , M. Torre & M. L. Marina
a
Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, 28871
Alcalá de Henares, Madrid, Spain
b
Instituto de Fermentaciones Industriales, C. S. I. C., Juan de la Cierva 3, 28006 Madrid,
Spain.
Published online: 03 Jun 2010.

To cite this article: J. Belloque , M. C. García , M. Torre & M. L. Marina (2002) Analysis of Soyabean Proteins in Meat
Products: A Review, Critical Reviews in Food Science and Nutrition, 42:5, 507-532, DOI: 10.1080/20024091054238

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 Critical Reviews in Food Science and Nutrition, 42(5):507–532 (2002)

Analysis of Soyabean Proteins in Meat Products:

A Review
J. Belloque,1 M. C. García, M. Torre, and M. L. Marina*
Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, 28871 Alcalá de Henares,
Madrid, Spain; 1 Instituto de Fermentaciones Industriales, C. S. I. C., Juan de la Cierva 3, 28006 Madrid, Spain.
Downloaded by [University of Minnesota Libraries, Twin Cities] at 05:08 19 September 2013

Dr. Lourdes Amigo, Instituto de Fermentaciones, Industriales (CSIC), Juan de la Cierva, 3, 28006 Madrid, Spain

* To whom correspondence should be addressed.

ABSTRACT: The use of soyabean proteins as meat extenders has spread significantly due to the interesting
nutritional and functional properties that are present in soyabean proteins. Together with these, health and
economical reasons are the major causes for the addition of soyabean proteins to meat products. Nevertheless,
despite the good properties associated to soyabean proteins, there are many countries in which the addition of these
proteins is forbidden or in which the addition of soyabean proteins is allowed up to a certain extent. Thus, the need
of analytical methods enabling the detection of added soyabean proteins in meat products is obvious. Microscopic,
electrophoretic, immunologic, and chromatographic methods are the most widely used for this purpose. However,
the detection of soyabean proteins in meat products presents difficulties related to the composition (meat species,
meat quality, soyabean protein source, presence of other non-meat proteins, etc.) and the processing of the meat
products, and, although these analytical methods have tried to overcome all these difficulties, there is still not a
method enabling quantitative assessment of soyabean proteins in all kinds of meat products.

KEY WORDS: meat products, meat proteins, soyabean proteins, non-meat proteins, meat extenders, fat replacers,
quality control.

I. INTRODUCTION sion-type meat products, such as sausages, a large

In occidental countries, meat is considered
the top quality protein source, not only due to its
nutritional characteristics but also for its appreci-
ated taste. In order to take the greatest advantage
of animals, the food industry does not only use
the muscle meat but also other sections of the
animal with lower quality. This is for the manu-
facture of a wide range of marketable products,
such as sausages, hams, bologna, salami, etc.1
Nevertheless, these products used to present a
high level of fat. For instance, frankfurters and
bolognas may have as much as 30% fat, and fresh
pork sausages are allowed to contain up to 50%
fat. The presence of this high fat content adds
difficulties in the technological processes used
for the manufacture of this kind of meat product.
As an example, during the preparation of emul-
amount of fat is liberated. In order to prevent
coalescence of this fat during heating, an emulsi-
fying agent is needed. In meat this stabilization is
mainly due to meat proteins. Nevertheless, when
the lean meat content is low, meat proteins are
insufficient to stabilize the emulsion and, conse-
quently, difficulties in the manufacture of this
kind of meat products appear. This problem can
be solved by the addition of non-meat proteins,
such as milk or soyabean proteins.2
However, there are also other reasons that
justify the addition of non-meat proteins to meat
products. Indeed, health care professionals rec-
ommend consuming meat products with less fat
content in order to avoid coronary diseases. This
has generated the need for manufacturing meat
products with a lower fat content. Developing
lean products while assuring the necessary palat-

1040-8398/02/$.50
© 2002 by CRC Press LLC
507
 ability demanded by consumers is not as simple
as just removing fat. In fact, fat contributes to
flavor, mouthfeel, texture, juiciness, and storage
stability of meat. Thus, a way for reducing fat
content without altering physical properties of the
meat product is with the addition of fat replacers.
These ingredients, when added to formulated
foods, can remarkably diminish the caloric con-
tent, while preserving other characteristics such
as flavor, mouthfeel, viscosity, or other organo-
leptic properties.3,4 Fat replacers are either syn-
thetic compounds or compounds obtained from
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natural products such as lipid-, carbohydrate-, and
protein-based products, the latter including egg
proteins, caseinates, wheat gluten, whey proteins,
and certainly soyabean proteins.3-7
An additional reason for using vegetable pro-
teins as meat extenders is because they have a
lower price than muscle proteins and, conse-
quently, can reduce the cost of the meat product.
In fact, high meat prices have prompted the indus-
try to produce meat products with inexpensive
sources of proteins such as soyabean proteins.8,9
Furthermore, in many undeveloped countries ani-
mal proteins are very scarce, and food supplies
must be supplemented with vegetable proteins.10-12
Among protein additives used in meat com-
modities, soyabean proteins are the most widely
employed. Some advantages of using soyabean
proteins as additive are the following:2,13,14
1. Very little off-flavor.
2. Low cost.
3. High nutritional value (soyabean proteins
contain all the essential amino acids required
by humans and its digestibility is compa-
rable to that of meat, fish, milk or egg pro-
teins).
4. Interesting functional properties (soyabean
proteins can easily associate with water and
fat showing good hydration, gelling, and
emulsifying properties).13,14
5. Their presence in meat products improves
the appearance and organoleptic character-
istics of these products.
Soyabean proteins are available under differ-
ent forms, such as flour, grits, concentrates, iso-
lates, and textured.13,15 Every soyabean product
508
presents different functional properties, and the
election of a soyabean product for the manufac-
ture of a meat product depends on its functional-
ity and on the particular meat product. For in-
stance, soyabean protein isolates and concentrates
are used in the preparation of chopped meats due
to their water binding, fat emulsifying, and gel-
stabilizing properties.16-18 An advantage that
soyabean protein isolates shows over soyabean
protein concentrates is their low content in the
oligosaccharides raffinose and stachyose, which
are the main cause of flatulence.19 On the other
hand, in ground meats, such as patties, nuggets,
and meatballs, the preferred soyabean product is
the textured soyabean. Table 1 shows some uses
of soyabean products as additives in meat prod-
ucts. As it is observed, soyabean proteins are not
only added to chopped or ground meats, but also
to whole muscle meats such as ham. In this case
soyabean protein isolate and concentrate are in-
jected in the muscle piece improving its appear-
ance, firmness, and slicing characteristics.13,15
Regarding their organoleptic characteristics,
soyabean flour may present beany flavor and
undesirable physical mouthfeel, while soyabean
protein isolate and concentrate occasionally give
less desirable palability to soyabean-added meat
products.20 In this respect, it has been investigated
the potential of tofu, gelatinous curd prepared
from soyabean, as a meat extender, showing that
pork sausages with added tofu showed no differ-
ences in sensory attributes and presented even a
better nutritional value, that is, lower fat to pro-
tein ratio, than those containing other meat ex-
tenders derived from soyabean.20
II. NEED OF CONTROLLING THE
QUALITY OF MEAT PRODUCTS
The addition of non-meat proteins to meat
products may cause health problems. In fact, in-
dividuals that are allergic to the added non-meat
proteins can be affected greatly by the ingestion
of minute amounts of the allergen. Thereby, the
addition of foreign proteins to meat products is
subjected to legal limitations or even, in some
cases, is not allowed. Table 2 summarizes some
of the regulations in the United States for meat
 TABLE 1
Soyabean Products Used as Protein Additives in Meat13
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products with soyabean proteins added.13 Simi-
larly, most countries have established regulations
to control the addition of meat extenders. For
instance, according to French legislation, meat
products can contain up to 2% binding proteins,
while Spanish administration allows less than 1%
of soyabean proteins in cooked ham.21,22
Furthermore, there are some rules in the United
States for labeling meat products based on the
(dry soyabean ingredient:uncooked meat) ratio:13,23
1. When soyabean proteins are added at low
levels (lower than 1:13), the soyabean prod-
uct must be listed in the ingredient state-
ment.
2. At intermediate levels (1:10), the soyabean
product must be listed as a subtitle contigu-
ous to the product name as well as in the
ingredient statement.
3. At higher levels, the soyabean product must
be part of the descriptive name as well as
appearing in the ingredient statement.
All these legal limitations make necessary the
control of the level of additions and the
mislabelling of soyabean protein additives. The
traditional analytical procedure for estimating the
lean defatted meat content of a meat product,
based on the determination of the total nitrogen
content, is far from being useful, because non-
meat proteins are not distinguished from the ani-
mal proteins.24 In order to control these meat
products and prevent any potential fraud, an ana-
lytical procedure, which was able to quantita-
tively determine the amount of added proteins, is
needed.
In the 1970s and 1980s the detection of
soyabean proteins in meat products attracted much
509
 TABLE 2
Regulations in the United States for Meat Products Containing Soyabean Proteins13
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attention from food scientists. From these studies,
an official enzyme-linked immunosorbent assay
(ELISA) method for the detection of soyabean
proteins in meat products is available nowadays.25
This method, however, is not completely satisfac-
tory, and it requires knowing the source of the
soyabean added to obtain a real quantitative as-
sessment. In fact, although it is able to control
many commodities that would cause problems to
hypersensitive individuals and can even give an
estimation of the amount of soyabean proteins
present in a mixture, it does not solve the com-
plete quality control problem.25 Nevertheless, de-
spite the fact there is still no quantitative reliable
method, the accumulated knowledge through the
decades has to serve for new strategies that may
finally allow for the analytical quality control
requirements. Because of this, this article reviews
the methods that have been tried, their advan-
tages, and their deficiencies.
III. ANALYSIS OF ADDED SOYABEAN
PROTEINS IN MEAT PRODUCTS
The fact that processed meat products could
contain non-meat proteins has provoked interest-
ing but technically very difficult analytical prob-
lems for food analysis.24 In principle, the analyti-
cal methods employed for detecting soyabean
proteins in meat products have to fulfill two dif-
ferent objectives:
510
1. Detection of soyabean proteins added to meat
products that should not contain any amount
of soyabean protein as additive. This type of
method should have a good sensitivity and
specificity, but quantitative results are not
necessary.
2. Determination of soyabean proteins added
to meat products (whose addition is legal) at
concentrations above the maximum limit set
by the legislation. Because the objective in
this case is to discern between a legal addi-
tion and an excess, a specific and quantita-
tive method is needed, but sensitivity is not
required to be as good as in the first case
being enough a detection limit able to deter-
mine the maximum concentration allowed
by law.
Even though there are a reasonable number of
reliable methods for qualitative assessment, a
definite quantitative method is still needed for the
determination of soyabean proteins in meat prod-
ucts. However, this problem is not limited to de-
termining soyabean proteins in meat products,
but, in general, arises when quantifying foreign
protein in food commodities. These difficulties
can be summarized as follows:
1. Difficulties related to the composition of the
product:
Food products do not present a common
formulation. Indeed, meat products of the
 same kind can present a different composi-
tion and consequently this makes the analy-
sis more difficult. Differences in the compo-
sition of meat formulations can be due to:
• The meat species used, such as beef, pork,
chicken, and turkey. For instance, it is
common to find pork and beef mixed.
• The quality of the meat used. Within a
same species, meat can present different
qualities depending on the part of the
animal used. Lower quality meat is char-
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acterized for containing high proportion
of connective tissue, existing legal limi-
tations for the collagen content, charac-
teristic of this tissue. Other products, such
as sausages, contain blood and offals in
addition to muscle meat.
• The soyabean protein source used.
Physico-chemical characteristics and be-
havior of soyabean proteins toward analy-
sis depend both on the soyabean product
and on their genetic variant.
• The presence of other non-meat proteins.
Proteins such as those from wheat, sun-
flower, mustard, rapeseed, sesame or cot-
tonseed and, above all, milk proteins can
be used in place of or in addition to
soyabean proteins.
2. Difficulties related with the processing of
the product:
Technological treatments used in the manu-
facture of food can alter proteins. Modifica-
tions on proteins are mainly due to heating,
which causes physicochemical changes that
render aggregated and cross-linked proteins.
Furthermore, in the case of meat the influ-
ence of temperature is different as a function
of the kind of meat, proportion of collagen,
post-mortem rigidity, heating time, etc.,1,26
which makes an even more complex scenario
for the analysis of added soyabean proteins.
Analytical methods for the detection of
soyabean proteins in meat thus have to be inde-
pendent of all the factors described above.
Methods available for the determination of
the presence of soyabean proteins in meat prod-
ucts can be classified into two groups: (1) indirect
methods that detect the addition of soyabean in
meat products by the determination of substances
that come together with soyabean proteins, and
(2) those methods that are focused to the direct
analysis of soyabean proteins.
A. Analytical Methods Based on the
Determination of Substances
Accompanying Soyabean Proteins
Determination of compounds present in plant
cells and absent in meat has been studied as a
possibility for the determination of soyabean added
to meat products. However, due to the large amount
of soyabean preparations present in the market, all
presenting different characteristics, these methods
used to be limited to qualitative assessment.27
1. Microscopic Methods
With these methods the identification of
soyabean is based on the presence of characteristic
structural forms (e.g., calcium oxalate crystals ap-
pearing in the cotyledon cells of soyabean can be
seen in polarized light as polygonal green-colored
bodies) and/or on the color developed after the
staining of polysaccharides.28-32 For instance, car-
bohydrate staining with Toluidine Blue has been
employed for the detection of textured soyabean.32
The oldest method known to detect soyabean in
meat, dated on 1913, is actually an official qualita-
tive test to detect soyabean flour in meat and con-
sists of a microscopic analysis under polarized
light of the residue remaining after alkali treatment
of a meat sample.28 One of the main advantages of
this method is that, within a relatively short period
of time, all major constituents and their forms can
be identified and even a rough estimation of their
relative proportions can be obtained.29 In fact, this
method has been claimed to detect down to 1% of
soyabean in meat products.33
However, only if the soyabean additives are
present in the product at a detectable proportion,
examination by means of microscopy through the
detection of carbohydrates and oxalate crystals
provide a reliable and reproducible method, com-
511
 parable to electrophoretic and immunological tech-
niques.34,35 In fact, detection of additions of
soyabean protein concentrate and isolate, with a
lower amount of carbohydrates than soyabean flour,
to meat is more difficult.30,34 In this respect, Parisi
et al.36 claimed that the increased sensitivity
achieved when using periodic acid Schiff reagent
as carbohydrate staining allowed the detection of
added soyabean protein isolate in meat products.
2. Chemical Methods
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Certain compounds present in soyabean, such as
magnesium and fiber,37 hemicellulose,38 galactose plus
arabinose,39 alginate,40 phytate,41 amino acid canava-
nine,42 saponins,43 as well as 13C/12C isotopic ratios,44
have been analyzed with classic methods for detect-
ing soyabean in meat. In addition, other methods
based on the determination of sterols 45 and
isoflavonoids46,47 have been developed recently. All
these methods are less specific than microscopic ones
because other vegetable products different than
soyaben may contain the same substances.27
3. Biochemical Methods
Due to the increasing impact of deoxyribo-
nucleic acid (DNA)-based techniques on the de-
tection of minute amounts of DNA in foods that
has taken place during the last decade, a nested
polymerase chain reaction (PCR) technique has
been developed recently and tested in a variety of
soyabean, meat, and blended products.48 The pres-
ence of soyabean DNA was determined with two
pairs of specific oligonucleotides from the
soyabean lectin Le1 gene. The results were in
good agreement with those from ELISA, and the
test could detect DNA from textured soyabean in
meat products down to a level of 0.7%.48
B. Analytical Methods Based on the
Determination of Soyabean Proteins
In general, the procedures for the direct analy-
sis of soyabean proteins in meat products involve,
the following steps:
512
1. Removal of fat, which is in a considerable
amount in some meat products, such as sau-
sages. For that purpose, an extraction is per-
formed with an organic solvent such as ac-
etone, or with a combination of organic
solvents such as a mixture ethanol:chloroform.
The removal of fat is usually achieved by
several extraction steps that depend on the
type of sample and the degree of fat removal
required. Using soyabean protein isolate and
lean meat samples allows skipping this step.
2. Solubilization of proteinaceous material in a
solution containing denaturing agents (such
as urea or sodium dodecyl sulfate (SDS))
and thiol-reducing agents (such as
β-mercaptoethanol or dithiothreitol).
In some occasions, steps 1 and 2 are substituted
by direct extraction with a buffer solution, which
usually contains denaturing and reducing agents.
3. Protein separation and quantitative analysis
of the proteins.
1. Electrophoretic Methods
Electrophoretic methods are based on the char-
acteristic mobility showed by each protein in a gel
immersed in an electrical field and depends on the
nature of the protein and the experimental condi-
tions. In 1969, Olsman49 developed an electrophoretic
method that detected 0.5% soyabean proteins in
luncheon meats and liver paste that had been heated
to 115°C and 105°C, respectively. Since then, a
considerable number of electrophoretic procedures
have been tested for the analysis of soyabean pro-
teins in meat products shown in Table 3.50-80 Among
them, the classic SDS-PAGE with Comassie Blue
staining has been the most commonly employed.
The selection of bands within the electro-
pherogram is an important criteria to be consid-
ered. Figure 1 shows the electropherograms of
protein extracts from soyabean, meat, soyabean-
meat blend, and a sausage with added soyabean
proteins, showing the different pattern of bands
that allowed to distinguish soyabean from meat
 TABLE 3
Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products by
Electrophoretic Techniques
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513
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514
TABLE 3 (continued)
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FIGURE 1. Densitograms of the electrophoretic separations of extracts

of (a) textured soyabean, (b) ox muscle meat, (c) a mixture of 10%
soyabean proteins and 90% meat, and (d) a sausage containing soyabean
protein isolate. (From Parsons, A. L. and Lawrie, R. A., J. Fd. Technol.,
1972; 7: 455-462. With permission.)

proteins. Under a qualitative scope, it is necessary
to focus on bands that are characteristic of the
soyabean proteins and do not overlap with other
proteins from meat, offals, or other foreign pro-
teins. Hofmann and Penny52 identified three ma-
jor bands arising from soyabean-meat blends, two
from soyabean (53 kDa and 17.5 kDa) and one
from meat (40 kDa) that seemed to be actin.
Armstrong et al.70 studied the band pattern of
meat samples from different species (turkey,
chicken, lamb, and pig), different food commodi-
ties (ham and sausages), as well as different parts
of the animal (muscle, stomach, liver, heart, brains,
spleen, and tongue) and found a soyabean protein
band that did not interfere with proteins from any
of the above sources. Lee et al.63 studied mixtures
of meat proteins with proteins from soyabean and
from other sources such as cottonseed, peanut,
casein, milk whey, and egg white and found a
unique electrophoretic pattern for each of them,
which allowed for their identification.63 Olivera
Carrión and Valencia,79 who used the SDS-PAGE
Lee’s method,63 also checked a variety of proteins
of different sources, such as blood serum, globin,
egg, soyabean, caseinate, milk, whey, and gluten,
finding that simultaneous detection of soyabean,
globin, and egg proteins was possible without the
interferences from other common protein additives.
In this work, it was pointed out that the chosen
band (19.5 kDa) for soyabean was better than those
chosen by other authors that interfered with other
non-meat proteins.70,73 Less-crowded electrophero-
grams were achieved by Hamayounfar, who took
advantage of the higher sensitivity to heat of meat

515
 proteins relative to soyabean proteins. Thus,
samples were autoclaved at 117°C for 70 min
prior to analysis, removing most meat proteins.60,61
For quantitative studies, in addition to those
related to the presence of interferences, there are
other considerations to be taken into account. An
absolute measurement of the soyabean band(s) is
difficcult to perform due to variations of the back-
ground among gels and the irregularity of bands,
which produce erroneous results by the software
packages used for measurement of bands by den-
sitometry. To minimize this problem, ratios be-
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tween different bands can be used. Some authors
have proposed to determine the soyabean-protein
band/meat protein band ratio, for instance,
α-conglycinin/actin.52,76,78 However, this gives a
relationship between soyabean proteins and lean
meat that is not very useful, as the latter is sub-
jected to changes. Other authors have introduced
an internal standard in the sample, such as
hemocyanin, which reduces the background prob-
lem, while it does not depend on the meat con-
tent.70
With electrophoretic methods it has been pos-
sible to detect additions of soyabean proteins at
levels down to 0.5%.49,60,61,70,71 The quantitation
of soyabean proteins was accomplished by Lee et
al.62 in fresh and cooked samples by SDS-PAGE,
using Coomassie Blue as dye and measuring the
intensity of bands by densitometry. An extract-
ability of 96% of proteins and a quantitative lin-
ear range from 10 to 115 µg soyabean proteins
were achieved when the appropriate soyabean
bands were considered. Even though reliable
quantitation of soyabean proteins in meat prod-
ucts can be achieved in fresh products, high tem-
peratures cause such modifications to proteins
that the procedure fails when using heated meat
products. In fact, when meat-soyabean mixtures
have been heated at 121°C and further electro-
phoretically analyzed, none or diffuse bands have
been found, being able to make a qualitative as-
sessment at best.52,55,69,70,71 Therefore, solubiliza-
tion of the heated soyabean proteins has been one
of the major subjects for these studies. Guy et al.57
compared two methods for the solubilization of
samples, one based on Olsman’s work49 (Method I)
and the other on Parson’s work69 (Method II),
finding that the proteins were better extracted by
516
using Method II (Table 3). However, Method I
showed better quantitative results, and it could
quantify not only cooked products (sausages and
beef burgers) but also meat pie fillers and canned
meat loaf products heated at 115°C for 49 min.57
Furthermore, this method showed good reproduc-
ibility and little interference from collagen, blood
plasma, and eggs solids.58
Although SDS-PAGE has been the most com-
mon procedure used, urea-PAGE56 and poro-
PAGE,73 an SDS technique employing a gradient
porosity size along the gel has also shown to be
useful. By another way, isoelectric focusing, an
electrophoretic method in which proteins are sepa-
rated as a function of their isoelectric point, has
shown for soyabean-containing products complex
electropherogram patterns, which could be sim-
plified by a mild aqueous heat treatment to coagu-
late and remove most meat proteins.64-66 These
studies showed quantitative results for additions
larger than 5%, but failed at lower concentrations.
However, the authors claimed that the method
could be used as a better qualitative method than
other electrophoretic techniques due to its higher
resolving power.65,81 In 1990 Feigl80 detected down
to a 3% addition of soyabean meal to meat in
Brühwurst (a frankfurter-type sausage) by iso-
electric focusing using commercial gel plates.
An interesting approach was taken by Heinert
and Baumann, who took advantage of the heat
stability of glycoproteins to develop a different
detection system based on the detection of soyabean
glycoproteins.74 After the electrophoretic separa-
tion of these proteins, they were transfered to a
nitrocellulose membrane, and detected with a lec-
tin (Concavalin A) that binds carbohydrate moi-
eties on glycoproteins with a coupled peroxidase
detection system. This procedure, similar to
immunoblotting, enabled the detection of glyco-
protein bands in meats heated at 120°C for 60 min
with 1% added soyabean proteins. Moreover, gly-
coprotein bands did not interfere with either egg or
milk protein bands.74
2. Immunochemical Methods
Immunological techniques are very sensitive
and specific and have become very popular for
 the detection of small amounts of proteins.10,27
Indeed, many researchers have tried different
immunological techniques, such as immunodiffu-
sion, Western blotting, dot-blot, and ELISA, for
the analysis of soyabean proteins in products con-
taining these proteins,25,34,75,82-126 among those are
meat products. They are all based on the reactiv-
ity between the antigen (soyabean proteins that
are mainly glycinin and β-conglycinin) and the
antiserum, which recognize only particular re-
gions of the protein with a characteristic structure
(epitopes). Developing the appropriate antiserum
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is a major limiting step for the application of
immunological procedures. Indeed, denaturation
of proteins is responsible for many of the failures
of immunological methods. Thus, if the antise-
rum is rised against native soyabean proteins, it
could only recognize soyabean proteins in the
native state, decreasing its reactivity if the protein
presents an altered structure. For instance, anti-
bodies rised against native glycinin do not react
with its own subunits, and those prepared against
the subunits do not react with the native glycinin.82
β-Conglycinin, however, presents antigenic de-
terminants in both the native and the subunits.83
Besides, the antibody-antigen reactions in
heated soyabean proteins are very complex be-
cause it is not only the overall denaturation, but
the particular denaturation pathway of each pro-
tein that makes that the different proteins present
in the mixture respond differently. The antigenic-
ity of glycinin for antisera produced against na-
tive soyabean proteins decreases dramatically af-
ter heating to 70 to 90°C, because it presents a
reduced number of epitopes available to the anti-
bodies.86 An approach to solving this problem is
to produce anti-denatured-soyabean proteins anti-
sera instead of antisera against native soyabean
proteins. In fact, there are commercially available
antibodies that enable the detection of the sub-
units from glycinin when heating up to 117°C.87
These commercial antibodies allow the detection
of additions of 2% of soyabean proteins.87,88
Hammond et al.84 prepared an antiserum against
severely heated soyabean proteins to detect anti-
gens in processed products. Even though they
detected soyabean proteins in sterilized products
that did not respond to other anti-native-soyabean
proteins antisera, they still obtained negative re-
sults in products highly processed. Other authors
have found that antibodies rised against formal-
dehyde-treated soyabean could react against both
native and sterilized soyabean proteins, enabling
quantitative analysis.95,96 Ravestein97 used anti-
bodies against SDS-treated soyabean proteins and
also claimed that quantitation was accurate and
very low limits were possible to detect. More-
over, and due to their sensitivity to structural
changes, antigen-antibody reactions respond to
certain soyabean products but not to others.84,85 In
this regard, Berkowitz89 prepared an antiserum
designed to react with a broad spectrum of anti-
genic determinants, although textured products
still showed less reactivity than crude ones.
Taking advantage of the fact that most of the
aggregates that are formed as a consequence of
denaturation can be solubilized by the action of
denaturing agents, it is possible to obtain a better
response of the antigenic substrate if the sample is
subjected to a treatment that enables it to recon-
struct the target epitopes for the antiserum recog-
nition. Thanh and Shibasaki90 found that denatur-
ation of β-conglycinin in 6 M urea and further
removal of the denaturing agent, produced a re-
folded protein with regained immunoreactivity.
Koh91 used antibodies prepared from denatured/
refolded soyabean proteins and quantified
soyabean proteins in crude and cooked products.
The same principle was applied later by other
authors.92,93 Samples and calibration standards
were solubilized under denaturing conditions, then
the denaturing agents were removed and proteins
were allowed to refold in a “nearly-native” struc-
ture. However, these antibodies still showed di-
vergent reactivities on different soyabean pro-
cessed products, not allowing for a quantitative
assessment. In addition, Hitchcock et al.92 found
that the 7S fraction (mainly composed of
β-conglycinin) was more antigenic after renatur-
ation than the 11S (mainly composed by glycinin)
and the whey proteins. Berkowitz and Webert89
suggested that these differences may have been
due to changes occurring during heating that could
not be reversed by the action of denaturing agents.
They also suggested the possible role of forma-
tion of lysino-alanine (LAL) or other nondisulfide
covalent cross-links among proteins, as well as
deamidation of Asn into the acidic Asp as irre-
517
 versible changes. Some studies have included Cys
in the renaturation buffer in order to yield a better
rearrangement of disulfide bonds, and thus a bet-
ter refolding of the protein.94
Some of the most important immunological
methods used for the analysis of soyabean pro-
teins in meat products are summarized in Table 4.
Both qualitative and quantitative determinations
have been achieved. The efforts spent on this
subject led to the implementation in 1990 of a
semiquantitative official method for analysis of
soyabean proteins in fresh and heated meats by
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means of ELISA.28
a. Immunodiffusion Techniques
In the 1970s, before ELISA and immunoblotting
became the major choices for immunological detec-
tion of proteins, immunodiffusion techniques were
very popular. Single radial diffusion, Ouchterlony’s
double diffusion, and electroimmunodiffusion were
employed to detect soyabean proteins in meat prod-
ucts.10,66,77,84,87,88,91,119-122 In these techniques, the
sample, which contains the antigen, was allowed to
diffuse or was electrically driven in a solid phase
(gel) that contained the antiserum. Afterward, a pre-
cipitation reaction between antigen and antibody
within the gel formed a visible band. Ouchterlony’s
double diffusion was first used in meat for the analy-
sis of meat species as an official method.127 More-
over, recently Brauner-Glaesner and Kistow119 have
used this technique for the determination of non-
meat proteins in meat products after extracting these
proteins with a buffered or a saline solution. This
method was valid for non-heated products (raw meat
samples), but not for heated ones. Even though
ELISA and immunoblotting techniques have over-
come immunodiffusion techniques, the latter have
been proven to be very sensitive as the addition of
soyabean proteins as well as other non-meat pro-
teins to raw meat products has been detected to very
low amounts.119
b. Immunohistochemistry
A combination of microscopy with immuno-
logical detection, commonly used for histological
518
examination, was applied recently to determine
soyabean proteins, detecting additions of 0 to 5%
soyabean proteins in pork liver pate.21 This method
is based on measuring the areas occupied by la-
belled soyabean proteins in sections mounted on
slides. Immunofluorescence tests showed a good
sensitivity, being able to detect 0.1% soyabean
proteins in sausages.125 In addition, this method
avoided problems associated with protein dena-
turation.21
c. Immunoblotting Western Blot and
Dot-Blot
The Western blot combines the specificity
and sensitivity of immunological techniques with
the separation capabilities of electrophoresis.
Immunoblotting consists of performing an elec-
trophoresis, transfering the separated proteins to a
membrane (e.g., nitrocellulose), incubating the
membrane with the antiserum for specific bind-
ing, and detecting the bound antibody with a sec-
ondary antibody coupled to a detection system,
such as peroxidase or colloidal gold. The electro-
phoresis is usually performed under denaturing
conditions, solubilizing the proteins in a solution
containing urea or SDS and a reducing agent
(dithiothreitol or β-mercaptoethanol). Slab SDS-
PAGE is usually carried out in a pore gradient
mode, ranging from 5 to 8% to 15 to 22%
acrylamide,9,34,112-114 but some authors have also used
the disc version with a 15% acrylamide constant
concentration.74 The antisera utilized usually have
been rised against denatured-refolded soyabean pro-
teins, although gliadin antiserum has also been em-
ployed.113,114 Ravestein and Driedonks97 claimed that
the limit of detection by immunoblotting is lower
than by ELISA being able to detect down to 0.1%
additions. An interesting approach has been sug-
gested recently by Körs and Steinhart34 who used
a N-cetyl-N,N,N-trimethylammonium bromide
(CTAB) buffer, which is not as denaturing as
SDS, to extract the proteins. Immunological de-
tection was performed by using an antiserum spe-
cifically developed against renatured soyabean
proteins, which had been denatured previously
with urea and heat.34 With this method, it was
possible to detect 0.5% soyabean proteins in meat
 TABLE 4
Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products by
Immunological Techniques
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519
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520
TABLE 4 (continued)
 products, although at this level no clear bands
appeared when the sample was heated at 100°C or
higher. At higher concentrations of soyabean pro-
teins (2 to 10%) added to meat products, the
intensity of the bands did not depend on the heat-
ing temperature.34
The dot-blot technique is a simplified version
of the Western blot. It is easy to perform, fast, and
low cost, which is important for routine work. In
this case, the protein solution is placed directly on
a membrane omitting the previous electrophoretic
separation of the sample components that occurred
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in immunoblotting techniques. Janssen et al.112,114
studied the application of this technique in detect-
ing the presence of soyabean proteins and other
non-meat proteins in meat products. Using both
peroxidase and immunogold detection systems,
they detected soyabean proteins down to 0.1%.
However, as the dot-blot technique lacks the sepa-
ration capabilities of the Western blot technique,
the authors suggested that samples with positive
results by dot-blot should be double checked by
performing a Western blot.114
d. ELISA
The immunological technique for excellence
is ELISA due to its easy automation and avail-
ability. ELISA presents an advantage over those
methods that use a gel, because in the latter some
aggregates cannot enter the gel becoming unde-
tected, whereas in ELISA aggregates showing the
right epitopes react freely. ELISA has been useful
whereas in analyzing soyabean proteins in meat
products.9,28,75,89,92,93,97,117,118,121 Most of these meth-
ods have used an indirect ELISA method for
quantitation.28,75,92,93,97,111,115-118 For the detection
of soyabean proteins in heated meats, Hitchcock
et al.92 designed a strategy based on the denatur-
ation-renaturation of soyabean proteins in both
the sample to be tested and the antigenic prepara-
tion used for the antiserum production. This
method was developed further using commercial
antibodies.93 Two collaborative studies were then
carried out, which led finally to an ELISA method
that became official.75,111 In this method, the
sample was treated with organic solvents to re-
move fat and the residue containing the proteins
was solubilized in a denaturing solution. In order
to regain immunoreactivity, the denaturing agents
were diluted and the solution was incubated to
allow proteins to refold. This method was consid-
ered to be semiquantitative, but, as claimed by the
authors, could be quantitative (succeeded down
to 1.5%) if the nature of the soyabean additive
was known and if the specific soyabean was avail-
able for calibration.117 In this respect, other au-
thors claimed that their method could determine 1
to 10% soyabean proteins in raw and heated meats
in less than 1 day, which is a much shorter time
than the employed in the official method. 94 Fur-
thermore, the detection limit for the determina-
tion of soyabean proteins in sausage has been
reduced recently down to 0.1%.121
Using antibodies against SDS-denatured
soyabean proteins has enabled to quantitate down
to 0.5% of soyabean proteins in raw and sterile
meat products with no interferences of other non-
meat proteins regardless of the variety and type of
soyabean.97 ELISA has also been tested against a
peptide obtained by autoclaving and trypsin diges-
tion of soyabean proteins.118 Using antiglycinine
antiserum on heated products, this analysis allowed
detection down to 0.4% of added soyabean pro-
teins whatever the soyabean cultivar and without
interferences from other non-meat proteins.118
Moreover, it has been proposed a method that
avoids delipidation and protein isolation during
sample preparation, achieving good results.116 Re-
cently, a method was developed to detect trace
amounts of soyabean proteins in foods, detecting
amounts as small as 2 ppm.126
Comparing radial double diffusion, electro-
immunodiffusion, and histological examination
with ELISA, it has been possible to notice that
although any of the above gave good results for
raw meat samples, ELISA was the only that
enabled working with sterile samples, even
though this treatment decreased recovery in
20%.124
3. Chromatographic Methods
Chromatographic methods have also been
applied for the detection of soyabean proteins in
meat products. These methods consisted of deter-
521
 mining whole soyabean proteins, characteristic
soyabean peptides, or the amino acid composi-
tion. Table 5 groups these chromatographic meth-
ods.
a. Analysis of Soyabean Proteins by High-
Performance Liquid Chromatography
High-performance liquid chromatography
(HPLC) is a very popular technique for protein
separations. Despite this, very scarce literature
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has been found concerning the analysis of
soyabean proteins in meat products. The chro-
matographic modes used for this purpose were
reversed-phase and anion-exchange, while mo-
lecular exclusion and cation-exchange supports
did not enable the separation of soyabean proteins
from meat proteins.64
Parris and Gillespie128 separated soyabean
from beef proteins in a blend of beef and soyabean
protein isolates. They tested chemically modified
and unmodified proteins by both reversed-phase
and anion-exchange chromatography. Ion-ex-
change chromatography showed the vegetable
proteins well separated from the meat proteins
using a steep NaCl gradient. However, they did
not attempt quantitation because the great over-
lapping of soyabean protein peaks could lead to a
misinterpretation of the results if the different
soyabean proteins under the same peak presented
different recoveries. 4-Vinylpyridine derivatives
of the proteins allowed for the separation between
beef and soyabean proteins in a reversed-phase
column using a gradient water-trifluoroacetic acid
(TFA)-acetonitrile (ACN). Soyabean proteins were
detected at a 2% level. In both cases, the extrac-
tion was carried out directly with a buffer con-
taining tris(hydroxymethyl)aminomethane, urea,
and dithiothreitol, which solubilized 90% of the
soyabean proteins and 60% of meat proteins.
Ashoor and Stiles129 performed a more com-
plete study. They set up an HPLC method for
analysis of soyabean proteins in raw meats from
different species (beef, pork, chicken, and turkey)
and also analyzed mixtures with other proteins
commonly used as additives (whey proteins and
caseins). They extracted proteins with a solution
containing SDS and β-mercaptoethanol and used
522
a reversed-phase column and a TFA/water:TFA/
ACN gradient for the separation of caseins and
soyabean, whey, and meat proteins. As an ex-
ample, Figure 2 illustrates the separation of
soyabean and meat proteins from a solution of
soyabean protein isolate, beef protein isolate, and
a mixture of both. As shown, it is possible to
detect the addition of soyabean proteins by the
appearance of three peaks characteristics of
soyabean proteins. They also found characteristic
peaks for caseinate that enabled its quantitation
within the range 1 to 5% addition. Recovery of
soyabean proteins was 85.0 to 91.2%.129
In none of these attempts were heated samples
tested. However, as the buffers used for extraction
contained denaturing and reducing agents, it could
be expected that in these cases proteins could also
be recovered from processed meat samples.
An additional consideration when using HPLC
is the structure of the proteins to be separated. Let
us consider the separation on a reversed-phase
column. In the native state, a protein only exposes
a portion of its residues. In consequence, only
those hydrophobic residues at the surface would
be able to interact with the hydrophobic station-
ary phase. However, if the protein is denatured,
many more hydrophobic residues would show up,
leading to more retention. Here we find a similar
problem than in other techniques that find differ-
ences when dealing with non-heated or heated
products. To obtain reproducible results, the de-
gree of denaturation should be the same whatever
the product is employed. The addition of denatur-
ing agents to the mobile phase may solve the
problem, but may cause mechanical problems.
For instance, urea in high concentration is a good
denaturant but increases the viscosity of the sol-
vent and SDS may form bubbles. Therefore, it is
possible that the lack of literature referring to
soyabean and meat proteins separations by HPLC
is related to the above problems, which may lead
to short column life and poor resolution.
b. Analysis of Peptides by Ion-Exchange
Chromatography
In order to avoid some problems related to
working with whole proteins, it was proposed the
 TABLE 5
Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products by
Chromatographic Techniques
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523
 TABLE 5 (continued)
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analysis based on peptides, precisely on trypsin
hydrolysates of the proteins.130 First of all, samples
were preheated to bring all proteins to a “standard
denaturing condition”. Using a cationic-exchange
resin, a sodium citrate buffer as eluent, and a
ninhydrin detection system, a pentapeptide (SP1:
Ser-Gln-Gln-Ala-Arg) characteristic of soyabean
digestions can be separated. This idea was studied
further and the sample preparation modified.131,132
Agater et al.132 determined soyabean and meat
proteins in raw and processed meat. They identi-
fied a peptide from soyabean (SP2) that was
present in all hydrolysates obtained by trypsin
hydrolysis on different commercial soyabean prod-
ucts and that seemed to belong to the 11S fraction
(glycinin) of soyabean proteins. Furthermore, meat
proteins could be determined simultaneously by a
meat characteristic peptide (MP1). Figure 3 shows
the chromatograms corresponding to extracts ob-
524
tained by trypsin digestion of beef meat, SPI, and a
mixture of both being possible to observe that in the
mixture, the characteristic peaks of soyabean (SP2)
and meat (MP1) were well resolved. They detected
SP2 in samples heated up to 120°C for 3 h. Caseinate,
egg powder, and dried milk powder did not over-
lapped with SP2. However, although the method was
reproducible, quantitative results were not accurate.
These quantitative divergences, which were poorer in
soyabean protein isolate and concentrate than in tex-
tured soyabean, were assigned to an incomplete hy-
drolysis, because only 66% of the proteins underwent
hydrolysis in soyabean protein isolate. The minimum
amount of soyabean protein detected was 1% (w/w
total), and the method was considered valid for quali-
tative detection in strongly heated products.130 Never-
theless, no additional publications were found con-
cerning this method, probably due to the long
time required for obtaining results (5 to 6 days).
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FIGURE 2. RP-HPLC chromatograms corresponding to a solution of soyabean protein isolate,
a proteic extract of beef meat, and a mixture of both. (From Ashoor, S. H. and Stiles, P. G., J.
Chromatogr., 1987; 393: 321-328. With permission.)
c. Determination of Amino Acid
Composition
The best way to avoid problems arising from
working with whole proteins is to hydrolyze them
down to amino acids. The sample is hydrolyzed gen-
erally under acid conditions and high temperatures, and
the resulting amino acid mixture is analyzed by con-
ventional LC or HPLC. The resulting amino acid com-
position of blends is then mathematically analyzed by
computer-assisted chemometric analysis and can yield
quantitative analysis.133 Lindberg et al.134 applied this
technique to protein mixtures of meat, soyabean meal,
and rind. They determined rind proteins quantitatively,
but muscle protein determinations were not accurate
when high concentrations of soyabean meal was present
due to the similarity in amino acid composition be-
tween soyabean and meat proteins.134
Zarkadas et al.135 used hydrolysates of meat and
soyabean protein concentrate blends and found a
decrease of Lys and Met and an increase of Glu, Trp,
and Cys when increasing the amount of soyabean
protein added. However, the actual percentages of
added soyabean to meat products were too small to be
detected by these differences. Zhi-Ling et al.136 per-
formed a more complete study analyzing by HPLC,
the amino acid composition of blends composed of
muscle, collagen, shrimp, soyabean, wheat, and whey
proteins, and casein. These authors also pointed out
that similarities between soyabean and muscle meat
protein amino acid composition caused interferences
in the muscle meat content predictions.136
4. Other Methods
A fluorimetric detection method has also been
tried to determine soyabean in meat blends based
on the fluorescence spectra, related to soyabean
proteins, obtained at 440 nm (when exciting at
360 nm).31,137,138 The method involves simple ex-
traction, filtration, and measurement of the fluo-
rescence of the solution. However, in order to use
this method it is necessary to know the type of
product added and, in addition, it cannot be appli-
cable to heat-processed meat-soyabean blends
because Maillard browning causes fluorescence.39
Another interesting approach has been the
estimation of the level of soyabean proteins in
fresh meat protein-soyabean protein mixtures by
pyrolysis–high-resolution gas chromatography.
This analysis showed some unique peaks from
soyabean pyrolysates that could be detected at a
10% (w/w) addition.30 Nevertheless, it would be
interesting to investigate whether this method
works with heated meat samples.
C. Analytical Methods Based on the
Determination of Meat Proteins
The determination of meat proteins as an al-
ternative way to determine foreign proteins in
meats has also been proposed.109,139 The determi-
nation of methyl amino acids, particularly 3-me-
thyl-histidine, which is an integral component of
525
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FIGURE 3. Chromatographic profiles corresponding to extracts obtained by

trypsin digestion of (a) defatted beef, (b) soyabean protein isolate, and (c) a
mixture of both. (From Agater, I. B., Briant, K. J., Llewellyn, J. W., Sawyer,
R., Bailey, F. J., and Hitchcock, C. H. S., J. Sci. Food Agric., 1986; 37: 317-
331. With permission.)

526
 actin and myosin, has given good results for de-
termining the muscle meat content.39,140-142 Col-
lagen can also be determined by the content of
another modified amino acid, 4-hydroxyproline.
Thus, the foreign protein content could be calcu-
lated using the difference:
Foreign proteins = Total proteins - Muscle pro-
teins - Collagen proteins
Even though 3-methyl-histidine can be used
as an index for meat content in prime cuts, it fails
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if other cuts and offals are present because their
3-methyl-histidine content is lower. On the other
hand, due to the similarity in amino acid compo-
sition, even using chemometrics to analyze the
different components by amino acid analysis,
mixtures of soyabean and meat could not be dis-
tinguished.136 In addition, it has been pointed out
that the evaluation of the minor component (the
foreign protein) from the determination of the
major component (meat content) by difference is
very doubtful because of the high relative error.53
Khan and Cowen 143 developed two different
methods for measuring the amount of beef pro-
teins in meat-soyabean protein mixtures. One of
the methods was based on the determination of
phosphocreatine in meat. Phosphocreatine is re-
stricted to animal tissue and is present in fairly
constant amounts in lean beef. The other method
was based on the presence of myofibrillar pro-
teins, which are only present in meat tissues. These
two parameters were significantly correlated to
the muscle protein content of the mixture. Fur-
thermore, the determination of both parameters
neither requires sophisticated equipment nor elabo-
rate and cumbersome procedures.143
IV. FUTURE PERSPECTIVES
In general, the complexity of determining for-
eign proteins in protein containing matrices has
been largely exemplified in this review by the
determination of soyabean proteins in meat prod-
ucts. As well as these difficulties, the changes in
legislation, whose tendency is to allow more ingre-
dients and more complex products, and the large
variety of products, ingredients, additives, and
processing conditions available and allowed have
even made more difficult the analysis of these
products. During the last decade new approaches
have been tried to resolve this problem. Indeed,
methods based on techniques used previously for
the same purpose have been enhanced or newly
developed. Furthermore, new methods such as the
biochemical methods and some chemical and im-
munochemical methods that had never been ap-
plied before were also used recently. Nevertheless,
and despite the huge effort performed, the problem
is still not resolved, and new methods are needed
to control and prevent the misuse and abuse of
additives in meat products. Moreover, additional
difficulties with the analysis of soyabean proteins
in meat products may arise due to the use of new
processing techniques and the addition of geneti-
cally modified variants of soyabean. In order to
overcome these new difficulties and to face the
analysis of soyabean proteins in meat products,
techniques used previously, such as HPLC and
chemometrics, and techniques that have never been
applied before, such as capillary electrophoresis
and mass spectrometry, could provide clues for
their solution. In conclusion, there are still many
possibilities to be explored that hopefully may pro-
vide an answer to the determination of soyabean
proteins in meat products, and may also provide a
basis for the analysis of proteins in protein-based food
products that, in general, show similar problems.
ACKNOWLEDGMENTS
The authors thank the European Comission
and the Comisión Interministerial de Ciencia y
Tecnología (Spain) for project 2FD97-1300.
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