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Analysis of Soyabean Proteins in Meat Products: AReviewJ. Belloque b , M. C. García a , M. Torre a & M. L. Marina aa Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, 28871Alcalá de Henares, Madrid, Spainb 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 MeatProducts: 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.

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 interestingnutritional and functional properties that are present in soyabean proteins. Together with these, health andeconomical 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 theseproteins is forbidden or in which the addition of soyabean proteins is allowed up to a certain extent. Thus, the needof 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 meatproducts, and, although these analytical methods have tried to overcome all these difficulties, there is still not amethod 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

In occidental countries, meat is consideredthe top quality protein source, not only due to itsnutritional characteristics but also for its appreci-ated taste. In order to take the greatest advantageof animals, the food industry does not only usethe muscle meat but also other sections of theanimal 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 ahigh level of fat. For instance, frankfurters andbolognas may have as much as 30% fat, and freshpork sausages are allowed to contain up to 50%fat. The presence of this high fat content addsdifficulties in the technological processes usedfor the manufacture of this kind of meat product.As an example, during the preparation of emul-

sion-type meat products, such as sausages, a largeamount of fat is liberated. In order to preventcoalescence of this fat during heating, an emulsi-fying agent is needed. In meat this stabilization ismainly due to meat proteins. Nevertheless, whenthe lean meat content is low, meat proteins areinsufficient to stabilize the emulsion and, conse-quently, difficulties in the manufacture of thiskind of meat products appear. This problem canbe solved by the addition of non-meat proteins,such as milk or soyabean proteins.2

However, there are also other reasons thatjustify the addition of non-meat proteins to meatproducts. Indeed, health care professionals rec-ommend consuming meat products with less fatcontent in order to avoid coronary diseases. Thishas generated the need for manufacturing meatproducts with a lower fat content. Developinglean products while assuring the necessary palat-

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ability demanded by consumers is not as simpleas just removing fat. In fact, fat contributes toflavor, mouthfeel, texture, juiciness, and storagestability of meat. Thus, a way for reducing fatcontent without altering physical properties of themeat product is with the addition of fat replacers.These ingredients, when added to formulatedfoods, can remarkably diminish the caloric con-tent, while preserving other characteristics suchas flavor, mouthfeel, viscosity, or other organo-leptic properties.3,4 Fat replacers are either syn-thetic compounds or compounds obtained fromnatural products such as lipid-, carbohydrate-, andprotein-based products, the latter including eggproteins, 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 alower 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 inexpensivesources of proteins such as soyabean proteins.8,9

Furthermore, in many undeveloped countries ani-mal proteins are very scarce, and food suppliesmust be supplemented with vegetable proteins.10-12

Among protein additives used in meat com-modities, soyabean proteins are the most widelyemployed. Some advantages of using soyabeanproteins 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 requiredby humans and its digestibility is compa-rable to that of meat, fish, milk or egg pro-teins).

4. Interesting functional properties (soyabeanproteins can easily associate with water andfat showing good hydration, gelling, andemulsifying properties).13,14

5. Their presence in meat products improvesthe 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

presents different functional properties, and theelection 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 concentratesare used in the preparation of chopped meats dueto their water binding, fat emulsifying, and gel-stabilizing properties.16-18 An advantage thatsoyabean protein isolates shows over soyabeanprotein concentrates is their low content in theoligosaccharides raffinose and stachyose, whichare the main cause of flatulence.19 On the otherhand, in ground meats, such as patties, nuggets,and meatballs, the preferred soyabean product isthe textured soyabean. Table 1 shows some usesof soyabean products as additives in meat prod-ucts. As it is observed, soyabean proteins are notonly added to chopped or ground meats, but alsoto whole muscle meats such as ham. In this casesoyabean 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 andundesirable physical mouthfeel, while soyabeanprotein isolate and concentrate occasionally giveless desirable palability to soyabean-added meatproducts.20 In this respect, it has been investigatedthe potential of tofu, gelatinous curd preparedfrom soyabean, as a meat extender, showing thatpork sausages with added tofu showed no differ-ences in sensory attributes and presented even abetter 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 THEQUALITY OF MEAT PRODUCTS

The addition of non-meat proteins to meatproducts may cause health problems. In fact, in-dividuals that are allergic to the added non-meatproteins can be affected greatly by the ingestionof minute amounts of the allergen. Thereby, theaddition of foreign proteins to meat products issubjected to legal limitations or even, in somecases, is not allowed. Table 2 summarizes someof the regulations in the United States for meat

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products with soyabean proteins added.13 Simi-larly, most countries have established regulationsto control the addition of meat extenders. Forinstance, according to French legislation, meatproducts 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 UnitedStates for labeling meat products based on the(dry soyabean ingredient:uncooked meat) ratio:13,23

1. When soyabean proteins are added at lowlevels (lower than 1:13), the soyabean prod-uct must be listed in the ingredient state-ment.

2. At intermediate levels (1:10), the soyabeanproduct must be listed as a subtitle contigu-ous to the product name as well as in theingredient statement.

3. At higher levels, the soyabean product mustbe part of the descriptive name as well asappearing in the ingredient statement.

All these legal limitations make necessary thecontrol of the level of additions and themislabelling of soyabean protein additives. Thetraditional analytical procedure for estimating thelean defatted meat content of a meat product,based on the determination of the total nitrogencontent, is far from being useful, because non-meat proteins are not distinguished from the ani-mal proteins.24 In order to control these meatproducts and prevent any potential fraud, an ana-lytical procedure, which was able to quantita-tively determine the amount of added proteins, isneeded.

In the 1970s and 1980s the detection ofsoyabean proteins in meat products attracted much

TABLE 1Soyabean Products Used as Protein Additives in Meat 13

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attention from food scientists. From these studies,an official enzyme-linked immunosorbent assay(ELISA) method for the detection of soyabeanproteins in meat products is available nowadays.25

This method, however, is not completely satisfac-tory, and it requires knowing the source of thesoyabean added to obtain a real quantitative as-sessment. In fact, although it is able to controlmany commodities that would cause problems tohypersensitive individuals and can even give anestimation of the amount of soyabean proteinspresent in a mixture, it does not solve the com-plete quality control problem.25 Nevertheless, de-spite the fact there is still no quantitative reliablemethod, the accumulated knowledge through thedecades has to serve for new strategies that mayfinally allow for the analytical quality controlrequirements. Because of this, this article reviewsthe methods that have been tried, their advan-tages, and their deficiencies.

III. ANALYSIS OF ADDED SOYABEANPROTEINS IN MEAT PRODUCTS

The fact that processed meat products couldcontain 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 soyabeanproteins in meat products have to fulfill two dif-ferent objectives:

1. Detection of soyabean proteins added to meatproducts that should not contain any amountof soyabean protein as additive. This type ofmethod should have a good sensitivity andspecificity, but quantitative results are notnecessary.

2. Determination of soyabean proteins addedto meat products (whose addition is legal) atconcentrations above the maximum limit setby the legislation. Because the objective inthis case is to discern between a legal addi-tion and an excess, a specific and quantita-tive method is needed, but sensitivity is notrequired to be as good as in the first casebeing enough a detection limit able to deter-mine the maximum concentration allowedby law.

Even though there are a reasonable number ofreliable methods for qualitative assessment, adefinite quantitative method is still needed for thedetermination 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 foreignprotein in food commodities. These difficultiescan be summarized as follows:

1. Difficulties related to the composition of theproduct:Food products do not present a commonformulation. Indeed, meat products of the

TABLE 2Regulations in the United States for Meat Products Containing Soyabean Proteins 13

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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 iscommon to find pork and beef mixed.

• The quality of the meat used. Within asame species, meat can present differentqualities depending on the part of theanimal used. Lower quality meat is char-acterized for containing high proportionof connective tissue, existing legal limi-tations for the collagen content, charac-teristic of this tissue. Other products, suchas sausages, contain blood and offals inaddition 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 productand 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 canbe used in place of or in addition tosoyabean proteins.

2. Difficulties related with the processing ofthe 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 thatrender aggregated and cross-linked proteins.Furthermore, in the case of meat the influ-ence of temperature is different as a functionof the kind of meat, proportion of collagen,post-mortem rigidity, heating time, etc.,1,26

which makes an even more complex scenariofor the analysis of added soyabean proteins.

Analytical methods for the detection ofsoyabean proteins in meat thus have to be inde-pendent of all the factors described above.

Methods available for the determination ofthe presence of soyabean proteins in meat prod-

ucts can be classified into two groups: (1) indirectmethods that detect the addition of soyabean inmeat products by the determination of substancesthat come together with soyabean proteins, and(2) those methods that are focused to the directanalysis of soyabean proteins.

A. Analytical Methods Based on theDetermination of SubstancesAccompanying Soyabean Proteins

Determination of compounds present in plantcells and absent in meat has been studied as apossibility for the determination of soyabean addedto meat products. However, due to the large amountof soyabean preparations present in the market, allpresenting different characteristics, these methodsused to be limited to qualitative assessment.27

1. Microscopic Methods

With these methods the identification ofsoyabean is based on the presence of characteristicstructural forms (e.g., calcium oxalate crystals ap-pearing in the cotyledon cells of soyabean can beseen in polarized light as polygonal green-coloredbodies) and/or on the color developed after thestaining of polysaccharides.28-32 For instance, car-bohydrate staining with Toluidine Blue has beenemployed for the detection of textured soyabean.32

The oldest method known to detect soyabean inmeat, dated on 1913, is actually an official qualita-tive test to detect soyabean flour in meat and con-sists of a microscopic analysis under polarizedlight of the residue remaining after alkali treatmentof a meat sample.28 One of the main advantages ofthis method is that, within a relatively short periodof time, all major constituents and their forms canbe identified and even a rough estimation of theirrelative proportions can be obtained.29 In fact, thismethod has been claimed to detect down to 1% ofsoyabean in meat products.33

However, only if the soyabean additives arepresent in the product at a detectable proportion,examination by means of microscopy through thedetection of carbohydrates and oxalate crystalsprovide a reliable and reproducible method, com-

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parable to electrophoretic and immunological tech-niques.34,35 In fact, detection of additions ofsoyabean protein concentrate and isolate, with alower amount of carbohydrates than soyabean flour,to meat is more difficult.30,34 In this respect, Parisiet al.36 claimed that the increased sensitivityachieved when using periodic acid Schiff reagentas carbohydrate staining allowed the detection ofadded soyabean protein isolate in meat products.

2. Chemical Methods

Certain compounds present in soyabean, such asmagnesium and fiber,37 hemicellulose,38 galactose plusarabinose,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 methodsbased on the determination of sterols45 andisoflavonoids46,47 have been developed recently. Allthese methods are less specific than microscopic onesbecause other vegetable products different thansoyaben 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 thathas taken place during the last decade, a nestedpolymerase chain reaction (PCR) technique hasbeen developed recently and tested in a variety ofsoyabean, meat, and blended products.48 The pres-ence of soyabean DNA was determined with twopairs of specific oligonucleotides from thesoyabean lectin Le1 gene. The results were ingood agreement with those from ELISA, and thetest could detect DNA from textured soyabean inmeat products down to a level of 0.7%.48

B. Analytical Methods Based on theDetermination of Soyabean Proteins

In general, the procedures for the direct analy-sis of soyabean proteins in meat products involve,the following steps:

1. Removal of fat, which is in a considerableamount 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 organicsolvents such as a mixture ethanol:chloroform.The removal of fat is usually achieved byseveral extraction steps that depend on thetype of sample and the degree of fat removalrequired. Using soyabean protein isolate andlean meat samples allows skipping this step.

2. Solubilization of proteinaceous material in asolution containing denaturing agents (suchas urea or sodium dodecyl sulfate (SDS))and thiol-reducing agents (such asβ-mercaptoethanol or dithiothreitol).

In some occasions, steps 1 and 2 are substitutedby direct extraction with a buffer solution, whichusually contains denaturing and reducing agents.

3. Protein separation and quantitative analysisof the proteins.

1. Electrophoretic Methods

Electrophoretic methods are based on the char-acteristic mobility showed by each protein in a gelimmersed in an electrical field and depends on thenature of the protein and the experimental condi-tions. In 1969, Olsman49 developed an electrophoreticmethod that detected 0.5% soyabean proteins inluncheon meats and liver paste that had been heatedto 115°C and 105°C, respectively. Since then, aconsiderable number of electrophoretic procedureshave been tested for the analysis of soyabean pro-teins in meat products shown in Table 3.50-80 Amongthem, the classic SDS-PAGE with Comassie Bluestaining 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 ofprotein extracts from soyabean, meat, soyabean-meat blend, and a sausage with added soyabeanproteins, showing the different pattern of bandsthat allowed to distinguish soyabean from meat

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TABLE 3Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products byElectrophoretic Techniques

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TABLE 3 (continued)

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proteins. Under a qualitative scope, it is necessaryto focus on bands that are characteristic of thesoyabean proteins and do not overlap with otherproteins from meat, offals, or other foreign pro-teins. Hofmann and Penny52 identified three ma-jor bands arising from soyabean-meat blends, twofrom soyabean (53 kDa and 17.5 kDa) and onefrom meat (40 kDa) that seemed to be actin.Armstrong et al.70 studied the band pattern ofmeat samples from different species (turkey,chicken, lamb, and pig), different food commodi-ties (ham and sausages), as well as different partsof the animal (muscle, stomach, liver, heart, brains,spleen, and tongue) and found a soyabean proteinband that did not interfere with proteins from anyof the above sources. Lee et al.63 studied mixturesof meat proteins with proteins from soyabean and

from other sources such as cottonseed, peanut,casein, milk whey, and egg white and found aunique electrophoretic pattern for each of them,which allowed for their identification.63 OliveraCarrión and Valencia,79 who used the SDS-PAGELee’s method,63 also checked a variety of proteinsof 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 theinterferences from other common protein additives.In this work, it was pointed out that the chosenband (19.5 kDa) for soyabean was better than thosechosen by other authors that interfered with othernon-meat proteins.70,73 Less-crowded electrophero-grams were achieved by Hamayounfar, who tookadvantage of the higher sensitivity to heat of meat

FIGURE 1. Densitograms of the electrophoretic separations of extractsof (a) textured soyabean, (b) ox muscle meat, (c) a mixture of 10%soyabean proteins and 90% meat, and (d) a sausage containing soyabeanprotein isolate. (From Parsons, A. L. and Lawrie, R. A., J. Fd. Technol.,1972; 7: 455-462. With permission.)

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proteins relative to soyabean proteins. Thus,samples were autoclaved at 117°C for 70 minprior to analysis, removing most meat proteins.60,61

For quantitative studies, in addition to thoserelated to the presence of interferences, there areother considerations to be taken into account. Anabsolute measurement of the soyabean band(s) isdifficcult to perform due to variations of the back-ground among gels and the irregularity of bands,which produce erroneous results by the softwarepackages used for measurement of bands by den-sitometry. To minimize this problem, ratios be-tween different bands can be used. Some authorshave proposed to determine the soyabean-proteinband/meat protein band ratio, for instance,α-conglycinin/actin.52,76,78 However, this gives arelationship between soyabean proteins and leanmeat that is not very useful, as the latter is sub-jected to changes. Other authors have introducedan internal standard in the sample, such ashemocyanin, 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 atlevels down to 0.5%.49,60,61,70,71 The quantitationof soyabean proteins was accomplished by Lee etal.62 in fresh and cooked samples by SDS-PAGE,using Coomassie Blue as dye and measuring theintensity of bands by densitometry. An extract-ability of 96% of proteins and a quantitative lin-ear range from 10 to 115 µg soyabean proteinswere achieved when the appropriate soyabeanbands were considered. Even though reliablequantitation of soyabean proteins in meat prod-ucts can be achieved in fresh products, high tem-peratures cause such modifications to proteinsthat the procedure fails when using heated meatproducts. In fact, when meat-soyabean mixtureshave been heated at 121°C and further electro-phoretically analyzed, none or diffuse bands havebeen 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 oneof the major subjects for these studies. Guy et al.57

compared two methods for the solubilization ofsamples, 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

using Method II (Table 3). However, Method Ishowed better quantitative results, and it couldquantify not only cooked products (sausages andbeef burgers) but also meat pie fillers and cannedmeat loaf products heated at 115°C for 49 min.57

Furthermore, this method showed good reproduc-ibility and little interference from collagen, bloodplasma, 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 gradientporosity size along the gel has also shown to beuseful. By another way, isoelectric focusing, anelectrophoretic method in which proteins are sepa-rated as a function of their isoelectric point, hasshown for soyabean-containing products complexelectropherogram patterns, which could be sim-plified by a mild aqueous heat treatment to coagu-late and remove most meat proteins.64-66 Thesestudies showed quantitative results for additionslarger than 5%, but failed at lower concentrations.However, the authors claimed that the methodcould be used as a better qualitative method thanother electrophoretic techniques due to its higherresolving power.65,81 In 1990 Feigl80 detected downto a 3% addition of soyabean meal to meat inBrühwurst (a frankfurter-type sausage) by iso-electric focusing using commercial gel plates.

An interesting approach was taken by Heinertand Baumann, who took advantage of the heatstability of glycoproteins to develop a differentdetection system based on the detection of soyabeanglycoproteins.74 After the electrophoretic separa-tion of these proteins, they were transfered to anitrocellulose membrane, and detected with a lec-tin (Concavalin A) that binds carbohydrate moi-eties on glycoproteins with a coupled peroxidasedetection system. This procedure, similar toimmunoblotting, enabled the detection of glyco-protein bands in meats heated at 120°C for 60 minwith 1% added soyabean proteins. Moreover, gly-coprotein bands did not interfere with either egg ormilk protein bands.74

2. Immunochemical Methods

Immunological techniques are very sensitiveand specific and have become very popular for

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the detection of small amounts of proteins.10,27

Indeed, many researchers have tried differentimmunological techniques, such as immunodiffu-sion, Western blotting, dot-blot, and ELISA, forthe analysis of soyabean proteins in products con-taining these proteins,25,34,75,82-126 among those aremeat products. They are all based on the reactiv-ity between the antigen (soyabean proteins thatare mainly glycinin and β-conglycinin) and theantiserum, which recognize only particular re-gions of the protein with a characteristic structure(epitopes). Developing the appropriate antiserumis a major limiting step for the application ofimmunological procedures. Indeed, denaturationof proteins is responsible for many of the failuresof immunological methods. Thus, if the antise-rum is rised against native soyabean proteins, itcould only recognize soyabean proteins in thenative state, decreasing its reactivity if the proteinpresents an altered structure. For instance, anti-bodies rised against native glycinin do not reactwith its own subunits, and those prepared againstthe 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 inheated soyabean proteins are very complex be-cause it is not only the overall denaturation, butthe particular denaturation pathway of each pro-tein that makes that the different proteins presentin 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 areduced number of epitopes available to the anti-bodies.86 An approach to solving this problem isto produce anti-denatured-soyabean proteins anti-sera instead of antisera against native soyabeanproteins. In fact, there are commercially availableantibodies that enable the detection of the sub-units from glycinin when heating up to 117°C.87

These commercial antibodies allow the detectionof additions of 2% of soyabean proteins.87,88

Hammond et al.84 prepared an antiserum againstseverely heated soyabean proteins to detect anti-gens in processed products. Even though theydetected soyabean proteins in sterilized productsthat did not respond to other anti-native-soyabeanproteins antisera, they still obtained negative re-

sults in products highly processed. Other authorshave found that antibodies rised against formal-dehyde-treated soyabean could react against bothnative and sterilized soyabean proteins, enablingquantitative analysis.95,96 Ravestein97 used anti-bodies against SDS-treated soyabean proteins andalso claimed that quantitation was accurate andvery low limits were possible to detect. More-over, and due to their sensitivity to structuralchanges, antigen-antibody reactions respond tocertain soyabean products but not to others.84,85 Inthis regard, Berkowitz89 prepared an antiserumdesigned to react with a broad spectrum of anti-genic determinants, although textured productsstill showed less reactivity than crude ones.

Taking advantage of the fact that most of theaggregates that are formed as a consequence ofdenaturation can be solubilized by the action ofdenaturing agents, it is possible to obtain a betterresponse of the antigenic substrate if the sample issubjected 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 furtherremoval of the denaturing agent, produced a re-folded protein with regained immunoreactivity.Koh91 used antibodies prepared from denatured/refolded soyabean proteins and quantifiedsoyabean proteins in crude and cooked products.The same principle was applied later by otherauthors.92,93 Samples and calibration standardswere solubilized under denaturing conditions, thenthe denaturing agents were removed and proteinswere 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 quantitativeassessment. In addition, Hitchcock et al.92 foundthat the 7S fraction (mainly composed ofβ-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 beendue to changes occurring during heating that couldnot be reversed by the action of denaturing agents.They also suggested the possible role of forma-tion of lysino-alanine (LAL) or other nondisulfidecovalent cross-links among proteins, as well asdeamidation of Asn into the acidic Asp as irre-

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versible changes. Some studies have included Cysin the renaturation buffer in order to yield a betterrearrangement of disulfide bonds, and thus a bet-ter refolding of the protein.94

Some of the most important immunologicalmethods used for the analysis of soyabean pro-teins in meat products are summarized in Table 4.Both qualitative and quantitative determinationshave been achieved. The efforts spent on thissubject led to the implementation in 1990 of asemiquantitative official method for analysis ofsoyabean proteins in fresh and heated meats bymeans of ELISA.28

a. Immunodiffusion Techniques

In the 1970s, before ELISA and immunoblottingbecame the major choices for immunological detec-tion of proteins, immunodiffusion techniques werevery popular. Single radial diffusion, Ouchterlony’sdouble diffusion, and electroimmunodiffusion wereemployed to detect soyabean proteins in meat prod-ucts.10,66,77,84,87,88,91,119-122 In these techniques, thesample, which contains the antigen, was allowed todiffuse or was electrically driven in a solid phase(gel) that contained the antiserum. Afterward, a pre-cipitation reaction between antigen and antibodywithin the gel formed a visible band. Ouchterlony’sdouble 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 haveused this technique for the determination of non-meat proteins in meat products after extracting theseproteins with a buffered or a saline solution. Thismethod was valid for non-heated products (raw meatsamples), but not for heated ones. Even thoughELISA and immunoblotting techniques have over-come immunodiffusion techniques, the latter havebeen proven to be very sensitive as the addition ofsoyabean proteins as well as other non-meat pro-teins to raw meat products has been detected to verylow amounts.119

b. Immunohistochemistry

A combination of microscopy with immuno-logical detection, commonly used for histological

examination, was applied recently to determinesoyabean proteins, detecting additions of 0 to 5%soyabean proteins in pork liver pate.21 This methodis based on measuring the areas occupied by la-belled soyabean proteins in sections mounted onslides. Immunofluorescence tests showed a goodsensitivity, being able to detect 0.1% soyabeanproteins in sausages.125 In addition, this methodavoided problems associated with protein dena-turation.21

c. Immunoblotting Western Blot andDot-Blot

The Western blot combines the specificityand sensitivity of immunological techniques withthe separation capabilities of electrophoresis.Immunoblotting consists of performing an elec-trophoresis, transfering the separated proteins to amembrane (e.g., nitrocellulose), incubating themembrane 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 denaturingconditions, solubilizing the proteins in a solutioncontaining urea or SDS and a reducing agent(dithiothreitol or β-mercaptoethanol). Slab SDS-PAGE is usually carried out in a pore gradientmode, ranging from 5 to 8% to 15 to 22%acrylamide,9,34,112-114 but some authors have also usedthe disc version with a 15% acrylamide constantconcentration.74 The antisera utilized usually havebeen rised against denatured-refolded soyabean pro-teins, although gliadin antiserum has also been em-ployed.113,114 Ravestein and Driedonks97 claimed thatthe limit of detection by immunoblotting is lowerthan by ELISA being able to detect down to 0.1%additions. An interesting approach has been sug-gested recently by Körs and Steinhart34 who useda N-cetyl-N,N,N-trimethylammonium bromide(CTAB) buffer, which is not as denaturing asSDS, to extract the proteins. Immunological de-tection was performed by using an antiserum spe-cifically developed against renatured soyabeanproteins, which had been denatured previouslywith urea and heat.34 With this method, it waspossible to detect 0.5% soyabean proteins in meat

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TABLE 4Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products byImmunological Techniques

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TABLE 4 (continued)

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products, although at this level no clear bandsappeared when the sample was heated at 100°C orhigher. At higher concentrations of soyabean pro-teins (2 to 10%) added to meat products, theintensity of the bands did not depend on the heat-ing temperature.34

The dot-blot technique is a simplified versionof the Western blot. It is easy to perform, fast, andlow cost, which is important for routine work. Inthis case, the protein solution is placed directly ona membrane omitting the previous electrophoreticseparation of the sample components that occurredin immunoblotting techniques. Janssen et al.112,114

studied the application of this technique in detect-ing the presence of soyabean proteins and othernon-meat proteins in meat products. Using bothperoxidase 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 positiveresults by dot-blot should be double checked byperforming a Western blot.114

d. ELISA

The immunological technique for excellenceis ELISA due to its easy automation and avail-ability. ELISA presents an advantage over thosemethods that use a gel, because in the latter someaggregates cannot enter the gel becoming unde-tected, whereas in ELISA aggregates showing theright epitopes react freely. ELISA has been usefulwhereas in analyzing soyabean proteins in meatproducts.9,28,75,89,92,93,97,117,118,121 Most of these meth-ods have used an indirect ELISA method forquantitation.28,75,92,93,97,111,115-118 For the detectionof soyabean proteins in heated meats, Hitchcocket al.92 designed a strategy based on the denatur-ation-renaturation of soyabean proteins in boththe sample to be tested and the antigenic prepara-tion used for the antiserum production. Thismethod was developed further using commercialantibodies.93 Two collaborative studies were thencarried out, which led finally to an ELISA methodthat became official.75,111 In this method, thesample was treated with organic solvents to re-move fat and the residue containing the proteins

was solubilized in a denaturing solution. In orderto regain immunoreactivity, the denaturing agentswere diluted and the solution was incubated toallow proteins to refold. This method was consid-ered to be semiquantitative, but, as claimed by theauthors, could be quantitative (succeeded downto 1.5%) if the nature of the soyabean additivewas known and if the specific soyabean was avail-able for calibration.117 In this respect, other au-thors claimed that their method could determine 1to 10% soyabean proteins in raw and heated meatsin less than 1 day, which is a much shorter timethan the employed in the official method. 94 Fur-thermore, the detection limit for the determina-tion of soyabean proteins in sausage has beenreduced recently down to 0.1%.121

Using antibodies against SDS-denaturedsoyabean proteins has enabled to quantitate downto 0.5% of soyabean proteins in raw and sterilemeat products with no interferences of other non-meat proteins regardless of the variety and type ofsoyabean.97 ELISA has also been tested against apeptide obtained by autoclaving and trypsin diges-tion of soyabean proteins.118 Using antiglycinineantiserum on heated products, this analysis alloweddetection down to 0.4% of added soyabean pro-teins whatever the soyabean cultivar and withoutinterferences from other non-meat proteins.118

Moreover, it has been proposed a method thatavoids delipidation and protein isolation duringsample preparation, achieving good results.116 Re-cently, a method was developed to detect traceamounts of soyabean proteins in foods, detectingamounts as small as 2 ppm.126

Comparing radial double diffusion, electro-immunodiffusion, and histological examinationwith ELISA, it has been possible to notice thatalthough any of the above gave good results forraw meat samples, ELISA was the only thatenabled working with sterile samples, eventhough this treatment decreased recovery in20%.124

3. Chromatographic Methods

Chromatographic methods have also beenapplied for the detection of soyabean proteins inmeat products. These methods consisted of deter-

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mining whole soyabean proteins, characteristicsoyabean 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 proteinseparations. Despite this, very scarce literaturehas been found concerning the analysis ofsoyabean proteins in meat products. The chro-matographic modes used for this purpose werereversed-phase and anion-exchange, while mo-lecular exclusion and cation-exchange supportsdid not enable the separation of soyabean proteinsfrom meat proteins.64

Parris and Gillespie128 separated soyabeanfrom beef proteins in a blend of beef and soyabeanprotein isolates. They tested chemically modifiedand unmodified proteins by both reversed-phaseand anion-exchange chromatography. Ion-ex-change chromatography showed the vegetableproteins well separated from the meat proteinsusing a steep NaCl gradient. However, they didnot attempt quantitation because the great over-lapping of soyabean protein peaks could lead to amisinterpretation of the results if the differentsoyabean proteins under the same peak presenteddifferent recoveries. 4-Vinylpyridine derivativesof the proteins allowed for the separation betweenbeef and soyabean proteins in a reversed-phasecolumn using a gradient water-trifluoroacetic acid(TFA)-acetonitrile (ACN). Soyabean proteins weredetected 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 thesoyabean proteins and 60% of meat proteins.

Ashoor and Stiles129 performed a more com-plete study. They set up an HPLC method foranalysis of soyabean proteins in raw meats fromdifferent species (beef, pork, chicken, and turkey)and also analyzed mixtures with other proteinscommonly used as additives (whey proteins andcaseins). They extracted proteins with a solutioncontaining SDS and β-mercaptoethanol and used

a reversed-phase column and a TFA/water:TFA/ACN gradient for the separation of caseins andsoyabean, whey, and meat proteins. As an ex-ample, Figure 2 illustrates the separation ofsoyabean and meat proteins from a solution ofsoyabean protein isolate, beef protein isolate, anda mixture of both. As shown, it is possible todetect the addition of soyabean proteins by theappearance of three peaks characteristics ofsoyabean proteins. They also found characteristicpeaks for caseinate that enabled its quantitationwithin the range 1 to 5% addition. Recovery ofsoyabean proteins was 85.0 to 91.2%.129

In none of these attempts were heated samplestested. However, as the buffers used for extractioncontained denaturing and reducing agents, it couldbe expected that in these cases proteins could alsobe recovered from processed meat samples.

An additional consideration when using HPLCis the structure of the proteins to be separated. Letus consider the separation on a reversed-phasecolumn. In the native state, a protein only exposesa portion of its residues. In consequence, onlythose hydrophobic residues at the surface wouldbe 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 similarproblem than in other techniques that find differ-ences when dealing with non-heated or heatedproducts. To obtain reproducible results, the de-gree of denaturation should be the same whateverthe product is employed. The addition of denatur-ing agents to the mobile phase may solve theproblem, but may cause mechanical problems.For instance, urea in high concentration is a gooddenaturant but increases the viscosity of the sol-vent and SDS may form bubbles. Therefore, it ispossible that the lack of literature referring tosoyabean and meat proteins separations by HPLCis related to the above problems, which may leadto short column life and poor resolution.

b. Analysis of Peptides by Ion-ExchangeChromatography

In order to avoid some problems related toworking with whole proteins, it was proposed the

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TABLE 5Experimental Conditions Used for the Analysis of Soyabean Proteins in Meat Products byChromatographic Techniques

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analysis based on peptides, precisely on trypsinhydrolysates of the proteins.130 First of all, sampleswere preheated to bring all proteins to a “standarddenaturing condition”. Using a cationic-exchangeresin, a sodium citrate buffer as eluent, and aninhydrin detection system, a pentapeptide (SP1:Ser-Gln-Gln-Ala-Arg) characteristic of soyabeandigestions can be separated. This idea was studiedfurther and the sample preparation modified.131,132

Agater et al.132 determined soyabean and meatproteins in raw and processed meat. They identi-fied a peptide from soyabean (SP2) that waspresent in all hydrolysates obtained by trypsinhydrolysis on different commercial soyabean prod-ucts and that seemed to belong to the 11S fraction(glycinin) of soyabean proteins. Furthermore, meatproteins could be determined simultaneously by ameat characteristic peptide (MP1). Figure 3 showsthe chromatograms corresponding to extracts ob-

TABLE 5 (continued)

tained by trypsin digestion of beef meat, SPI, and amixture of both being possible to observe that in themixture, the characteristic peaks of soyabean (SP2)and meat (MP1) were well resolved. They detectedSP2 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 wasreproducible, quantitative results were not accurate.These quantitative divergences, which were poorer insoyabean protein isolate and concentrate than in tex-tured soyabean, were assigned to an incomplete hy-drolysis, because only 66% of the proteins underwenthydrolysis in soyabean protein isolate. The minimumamount of soyabean protein detected was 1% (w/wtotal), 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 longtime required for obtaining results (5 to 6 days).

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c. Determination of Amino AcidComposition

The best way to avoid problems arising fromworking with whole proteins is to hydrolyze themdown to amino acids. The sample is hydrolyzed gen-erally under acid conditions and high temperatures, andthe resulting amino acid mixture is analyzed by con-ventional LC or HPLC. The resulting amino acid com-position of blends is then mathematically analyzed bycomputer-assisted chemometric analysis and can yieldquantitative analysis.133 Lindberg et al.134 applied thistechnique to protein mixtures of meat, soyabean meal,and rind. They determined rind proteins quantitatively,but muscle protein determinations were not accuratewhen high concentrations of soyabean meal was presentdue to the similarity in amino acid composition be-tween soyabean and meat proteins.134

Zarkadas et al.135 used hydrolysates of meat andsoyabean protein concentrate blends and found adecrease of Lys and Met and an increase of Glu, Trp,and Cys when increasing the amount of soyabeanprotein added. However, the actual percentages ofadded soyabean to meat products were too small to bedetected by these differences. Zhi-Ling et al.136 per-formed a more complete study analyzing by HPLC,the amino acid composition of blends composed ofmuscle, collagen, shrimp, soyabean, wheat, and wheyproteins, and casein. These authors also pointed outthat similarities between soyabean and muscle meatprotein amino acid composition caused interferencesin the muscle meat content predictions.136

4. Other Methods

A fluorimetric detection method has also beentried to determine soyabean in meat blends basedon the fluorescence spectra, related to soyabeanproteins, obtained at 440 nm (when exciting at360 nm).31,137,138 The method involves simple ex-traction, filtration, and measurement of the fluo-rescence of the solution. However, in order to usethis method it is necessary to know the type ofproduct added and, in addition, it cannot be appli-cable to heat-processed meat-soyabean blendsbecause Maillard browning causes fluorescence.39

Another interesting approach has been theestimation of the level of soyabean proteins infresh meat protein-soyabean protein mixtures bypyrolysis–high-resolution gas chromatography.This analysis showed some unique peaks fromsoyabean pyrolysates that could be detected at a10% (w/w) addition.30 Nevertheless, it would beinteresting to investigate whether this methodworks with heated meat samples.

C. Analytical Methods Based on theDetermination of Meat Proteins

The determination of meat proteins as an al-ternative way to determine foreign proteins inmeats has also been proposed.109,139 The determi-nation of methyl amino acids, particularly 3-me-thyl-histidine, which is an integral component of

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.)

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FIGURE 3. Chromatographic profiles corresponding to extracts obtained bytrypsin digestion of (a) defatted beef, (b) soyabean protein isolate, and (c) amixture 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.)

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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 ofanother 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 usedas an index for meat content in prime cuts, it failsif other cuts and offals are present because their3-methyl-histidine content is lower. On the otherhand, due to the similarity in amino acid compo-sition, even using chemometrics to analyze thedifferent components by amino acid analysis,mixtures of soyabean and meat could not be dis-tinguished.136 In addition, it has been pointed outthat the evaluation of the minor component (theforeign protein) from the determination of themajor component (meat content) by difference isvery doubtful because of the high relative error.53

Khan and Cowen 143 developed two differentmethods for measuring the amount of beef pro-teins in meat-soyabean protein mixtures. One ofthe methods was based on the determination ofphosphocreatine in meat. Phosphocreatine is re-stricted to animal tissue and is present in fairlyconstant amounts in lean beef. The other methodwas based on the presence of myofibrillar pro-teins, which are only present in meat tissues. Thesetwo parameters were significantly correlated tothe muscle protein content of the mixture. Fur-thermore, the determination of both parametersneither 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 hasbeen largely exemplified in this review by thedetermination of soyabean proteins in meat prod-ucts. As well as these difficulties, the changes inlegislation, whose tendency is to allow more ingre-dients and more complex products, and the largevariety of products, ingredients, additives, and

processing conditions available and allowed haveeven made more difficult the analysis of theseproducts. During the last decade new approacheshave been tried to resolve this problem. Indeed,methods based on techniques used previously forthe same purpose have been enhanced or newlydeveloped. Furthermore, new methods such as thebiochemical 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 problemis still not resolved, and new methods are neededto control and prevent the misuse and abuse ofadditives in meat products. Moreover, additionaldifficulties with the analysis of soyabean proteinsin meat products may arise due to the use of newprocessing techniques and the addition of geneti-cally modified variants of soyabean. In order toovercome these new difficulties and to face theanalysis of soyabean proteins in meat products,techniques used previously, such as HPLC andchemometrics, and techniques that have never beenapplied before, such as capillary electrophoresisand mass spectrometry, could provide clues fortheir solution. In conclusion, there are still manypossibilities to be explored that hopefully may pro-vide an answer to the determination of soyabeanproteins in meat products, and may also provide abasis for the analysis of proteins in protein-based foodproducts that, in general, show similar problems.

ACKNOWLEDGMENTS

The authors thank the European Comissionand the Comisión Interministerial de Ciencia yTecnología (Spain) for project 2FD97-1300.

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