Food Hydrocolloids Vol.3 no.6 pp.485-490, 1990

Modified immunoassay for the detection of soy milk in pasteurizedskimmed bovine milk

M.M.Hewedy and C1.Smith!

Department ofDairying, Faculty ofAgriculture, Fayoum, Egypt and 1Faculty ofResearch and Innovation, The North East Wales Institute, Deeside, Clwyd, UK

Abstract. Utilization of soy milk and soy protein as a food supplement or as a replacement for milkand milk products is increasing. This form of food modification presents several different problems:quality assurance cannot always be given, people with soy allergy may be unknowingly exposed andregulatory bodies have difficulty in detecting the cases of deliberate adulteration. Therefore, areliable means of detecting and quantifying soy protein in milk is needed. In the present study wehave modified an ELISA technique to overcome the problem of quantitation of soy protein in milk.The results are reproducible, show low cross-reactivity with milk protein and sensitivity is good(assay range 3.5-70 ug soy protein/em:'). The assay measures soy protein and the correlation withsoy milk added to bovine milk is linear.

Introduction

In recent years soy milk and soy proteins have received considerable attentionfrom food manufacturers as alternative sources of economical and nutritiveprotein. This is especially true in developing countries where shortages of animalprotein (milk and meat) exist and soy proteins may be particularly useful incombating malnutrition. Moreover, soy milk and dairy-like products containingsoy protein are now being marketed as an ideal alternative for both vegetariansand patients with bovine milk allergies (1). However, despite the goodnutritional and functional properties of soy proteins, the authorities in manycountries are reluctant to give legal clearance for the use of non-milk proteins assupplements in bovine milk and its products.

Regardless of this lack of legal acceptance, developments in food processingmethods have made it difficult to detect the presence of non-milk proteins indairy products. Trantik and 1aksic (2) found that cheese and yoghurt made fromcows' milk containing 10-20% soy milk did not differ organoleptically fromcontrols made from cows' milk alone. In Egypt, trials carried out to study theeffects of soy milk addition on the production of local cheeses (3-6) showed thatno modification of production parameters was required for soy levels of up to20%, at higher concentrations clotting times increased. Organoleptic panelsfound fresh Domiati cheese containing soy milk unacceptable, but the flavourimproved after ripening (5). The authors suggested that production of soy milkwith a bland flavour would overcome this.

Apart from the good nutritional and functional properties of soy proteins,they can be used in meat and dairy products simply to increase the protein levelsof poor-quality products to the legal minimum requirement. This can be donewithout it being necessary for the manufacturer to indicate that soy protein hasbeen added as a supplement because it is an accepted food additive.

©IRLPress 485

M.M.Hewedy and C.J.Smith

The detection of this type of adulteration presents a peculiar problem to thefood analyst who needs to identify specific characteristics of the adulterant whichcan be used to detect the presence of that adulterant. The analyst need not,however, determine the nature of the marker being used for that detection.Many techniques have been used to identify and quantify soy protein in meatproducts, including SDS-gel electrophoresis (7), a stereological procedure (8)and peptide analysis (9). More recently immunological methods for thedetection of soy protein in meat mixtures have been developed (10). Thedetection of soy milk raises a slightly different problem in that this is a liquidsystem and as such cannot include insoluble soy protein which can be added tosolid foods. The detection of soy milk in bovine milk using either SDS-gelelectrophoresis or fast protein liquid chromatography (FPLC) has been reportedpreviously (11). Unfortunately such techniques are time consuming, expensiveand need highly trained personnel. In contrast, immunoassays can be simplifiedto eliminate these disadvantages. Enzyme-linked immunosorbent assays(ELISAs) are being used increasingly in the food industry for a number ofpurposes; e.g. meat speciation (12), and detection of gliadin (13), casein (14)and fungal toxins (15,16). The development of an immunoassay system for thedetection of soy milk in bovine milk is therefore apposite.

Methods

Samples

Bovine milk, soy milk and mixtures of soy and bovine milk were prepared asdescribed in our previous paper (11). Briefly, pasteurized skimmed bovine milkand processed soy milk were mixed to give samples containing 0, 1,5, 10, 15,20and 100% (v/v) soy milk. The samples were then freeze-dried and stored at 4°Cfor subsequent analysis.

Reagents

Soy protein standards, soy protein controls and soy protein sensitized microwellmodules were kindly supplied by Cortecs Diagnostics Ltd, Newtech Square,Deeside, Clwyd.

Antibody was raised in rabbits as described previously (10). Cross-reactionswith casein were eliminated by affinity chromatography. Casein (40 mg,technical grade, Sigma Ltd) was denatured by heating, (100°C for 1 h) indenaturation buffer (10 crrr', 0.125 mol/drrr' Tris, pH 8.6, containing 10 M ureaand 14 mmol/drrr' dithiothreitol) and then immediately renatured by cooling to50°C and adding L-cystine-NaCI buffer (20 em", 7.5 mmol/dnr' L-cystine, 0.06mol/dm' NaCl, pH 9.0), allowed to cool to room temperature and diluted to100 cnr' with renaturation buffer. This material was covalently linked to CNBr­Sepharose 4B (Pharmacia) according to the manufacturer's procedure. Anti­serum was loaded in buffer (0.05 mol/dnr' Tris, 0.15 mol/drrr' NaCl, pH 8.6) andpurified anti-soya antibody eluted in the same buffer. The column wasregenerated after each preparation by washing with glycine buffer (0.05 rnol/drrr'glycine, 0.15 mol/drrr' NaCl, pH 2.3).

486

Immunoassay for the detection of soy milk

Assay

Extraction of soy protein

Samples (40 mg) of each of the freeze-dried milk samples were accuratelyweighed, transferred to 100 crrr' conical flasks and extracted with denaturationbuffer as described above. Samples were then renatured with cystine-NaClbuffer. Soy protein standards were treated in the same way.

Samples of extracts and standards (0.1 em") were mixed with assay buffer(0.02 mol/dnr' phosphate, 0.15 mol/drrr' NaCl, 0.1% BSA, 0.01% thimerosal,pH 7.2) and aliquots (0.05 ern") dispensed into the microtitre plate wells. Alltests were carried out in quadruplicate. Controls were set up which containedonly buffer. To all wells, except the substrate blank well, purified rabbit anti­soya protein (0.05 cm') was added. The plate was placed on an orbital shaker for10 min at room temperature, after which wells were aspirated and thoroughlywashed with wash buffer (0.02 mol/drrr' Tris, 0.5% Tween 80, pH 7.8). Pre­diluted goat anti-rabbit peroxidase conjugate (0.1 cnr', Sigma) was added to allwells, except the substrate blank well, and the incubation and washing stepsrepeated. At the end of this second washing step the enzyme substrate [0.015%H20 2 , 2,2' -azino-bis-(3-ethylbenzthiazoline) suIphonic acid, 15 mg/crrr' in 0.1mol/dnr' citrate buffer, pH 4.0) was added and a third incubation step carriedout. After 10 min, the absorbance at 405 nm was measured.

The detection and quantitation of soy protein in foods using immunoassaytechniques is dependent on the extraction of specific, water-soluble proteins.Antibodies can be raised to the different protein components of soy but theadvantage of immunoassay in being able to identify and quantitate a singlecomponent is potentially lost by the need to carry out multiple assays, one foreach protein type. The immunogen used here was prepared by a modification ofKoy's (17) procedure which produces a modified soy protein. Antibodies raisedto this 'renatured' soy protein are capable of reacting with soy protein which hasbeen renatured using the same procedure (10). Unfortunately this treatmentalso modifies other proteins. Casein does not react with the anti-soy antibodyprior to the extraction treatment, but following treatment there is significantcross-reactivity which presents an obvious difficulty for assay applicationsinvolving dairy products. This phenomenon, as yet unexplained, necessitates thepartial purification, by affinity chromatography, of the anti-soya antiserum toremove the cross-reacting components (Figure 1).

Using the partially purified antiserum it was possible to measure the levels ofsoy protein in prepared mixtures of soy milk and bovine milk. The results of acompetitive ELISA used to measure soy protein standards are shown (Figure 2).From this curve the concentration of soy protein in test samples assayed in thesame manner was determined (Table I). A comparison of the knownconcentration of soy milk in the test samples and the concentration determinedby the ELISA shows there is a significant difference. This difference is explainedby the fact that the mixtures were prepared from liquid milks, thus they are not100% protein. The ELISA measures only the soy protein concentration not thesoy milk concentration. A plot of the known versus the observed values (Figure

487

M.M.Hewedy and C.J .Smith

120

100~

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60...;S] 40

~

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10 100 1000

Soy Protein Concentration I1 g/m1

Fig. 1. Comparison of the cross-reactivity of anti-soy antibody pre- and post-affinity purification toremove anti-casein activity. Crude anti-serum inhibited by casein (.) and soy (0); purified anti­serum inhibited by casein (D) and soy (+). Maximum absorbance was 1.338.

1.2

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Concentration of Soy Protein Standards

ug/ml

Fig. 2. Standard inhibition curve for the soy assay. The points are the means of replicate results . theSD for each of the points was < 5% .

Table I. Values for soy protein concentration in soy milk/bovine milk mixtures determined from thestandard curve

Sample in bovine milk OD at 405 nm Estimated concentration of soy protein (%)

0% soy milk1% soy milk5% soy milk

10% soy milk15% soy milk20% soy milk

1.311.2080.7930.6030.4770.38

o0.1401.5233.2635.3237.853

AJI assays were run in duplicate and the results given are the mean from three assays. Soyconcentrations were determined from the standard curve (Figure 2) . The background (i.e. decreasein absorbance produced by bovine milk compared to the absorbance produced by buffer) has beensubtracted.

488

Immunoassay for the detection of soy milk

y =0.80302 + 2.5513x RA2 =0.992

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ACTUAL % SOY PROTEIN DETECTED

Fig. 3. Comparison of actual soy protein detected with percentage soy milk added. The curve and thebest straight-line fit for the results are shown. The latter was used to derive the equation andcalculate the slope.

3) shows there is a linear relationship between these values. The intercept on they-axis indicates that there is stilI some residual cross-reactivity with bovine milkproteins. The slope can be used to calculate the conversion factor from proteinto soy milk.

In conclusion, the results revealed that ELISA can be used to detect andquantify soy protein in soy milk/bovine milk mixtures. We have previouslyreported that it is possible to use either FPLC or polyacrylamide gelelectrophoresis to detect soy protein in milk, but that, although detection waspossible in mixtures containing 1% soy milk, quantitation of unknown mixtureswas difficult (11). The ELISA described readily detects soy protein in mixturesof 1% soy milk in bovine milk and is also capable of determining theconcentration of samples of unknown composition. This is a distinct advantagewhich provides a means of enforcing quality control regulations if required.

References

1. Snyder,H.E. and Kwon,T.W. (1987) Soybean Utilisation. Van Nostrand Reinhold, New York,Chap. 9.

2. Trantik,L. and Jaksic,B. (1982) Mljekarstovo, 32, 48-51.3. EI-Safty,M.S. and Mehanna,N. (1977) Egypt. J. Dairy ScL, 5, 55-59.4. Metwalli,N.H., Shalabi,S.l., Zahran,AS. and El Demerdash.O. (1982) J. Food Technol., 17,

71-77.5. Metwalli,N.H., Shalabi,S.I., Zahran,AS. and EI Demerdash.O, (1982b) J. Food Technol., 17,

297-305.6. Abo EI-Ella,W.M., Farahat,S.M. and Ghandour,M.A (1978) Milchwissenschaft, 33, 245-297.7. Guy,R.C.E., Jayaram,R. and Wilcox,C.J. (1973) J. Sci. Food Agric., 24,1551-1563.8. Flint,F.e. and Meech,M.V. (1978) Analyst, 103, 252.9. Bailey,F.]. (1976) J. Sci. Food Agric., 27, 827-830.

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M.M.Hewedy and C.J.Smith

to. Rittenburg,J.H., Adams,A., Palmer,J. and Allen,J.e. (1987) J. Assoc. attic. Anal. Soc., 70,582-587.

11. Hewedy,M.M. and Smith,e.J. (1989) Fd Hydrocoll., 3, 399-405.12. Ayob,M.K., Ragab,A.A., Allen,J.e., Farag,R.S. and Smith,e.J. (1989) J. Sci. Food Agric., 49,

103-116.13. Ayob,M.K., Rittenburg,J.H., Allen,J.e. and Smith,e.J. (1988) Fd Hydrocoll., 2, 39-49.14. Rittenburg,J.H., Ghaffar,A., Smith.Ci.l., Adams,A. and Allen,J.e. (1984) Roy. Soc. Chern.

Special Publ., 47, 319-320.15. Candlish,A.A.G., Stimson,W.H. and Smith,J.E. (1985) Lett. Appl. Microsc., 1,57-61.16. Morgan,M.R.A., McNerney,R. and Chan,H.W.-S. (1983) J. Assoc. Offic. Anal. Chem., 66,

1481-1484.17. Rittenburg,J.H., Adams,A., Palmer,J. and Allen,J.C. (1987) J. Assoc. Offic. Anal. Chem., 70,

582-587.

Received on September 20, 1989; accepted on November 22, 1989

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