Review: Soya protein products‐their processing,functionality, and application aspects

Abstract

Soya protein products are very interesting ingredients because of their ready availability, functionality, nutritional value, and price. This review covers the processing, functionality, and application of the various soya protein products, that is, full‐fat soya flours, defatted soya flours and grits, soya protein concentrates, soya protein isolates, textured soya products, and modified soya proteins. The choice of food manufacturers between these soya protein products depends on the functional properties, performance in the end products, and price. According to our experience soya protein concentrates offer a proven functionality at the right price, especially for applications in meat products. Soya protein products can help to meet the protein requirements of the modern food industry economically.     

Detection of soya proteins in heated meat products by "blotting" and "dot blot"

Soy proteins (isolates, concentrates and texturates) as well as meat products containing soya isolate were analysed by SDS-electrophoresis. The separated proteins were blotted on nitrocellulose and stained with a selective immunoperoxidase system with the following sequence: primary (anti-soya) serum, goat anti-rabbit IgG serum and peroxidase-antiperoxidase complex (rabbit allotype). By developing the blot with a peroxidase substrate the antigenic soya fractions were visualised while the meat proteins did not stain. All major (reduced) soya fractions alpha, alpha', beta conglycinin, the acid and basic subunits of glycinin as well as some minor fractions became visible with a commercially available anti-soya serum as primary antiserum. The pattern thus obtained provides a high evidence for the presence of soya protein in meat products. Detection level is about 0.02% of soya protein. During a 24-h incubation at room temp. (before heat processing) of a meat product containing soya product and raw liver a remarkable loss of antigenic material was observed.     

Detection of Adulteration of Meat and Meat Products with Buffalo Meat Employing Polymerase Chain Reaction Assay

The primer pair was designed based on mitochondrial d-loop gene for detection of adulteration of buffalo meat in admixed meat and meat products by polymerase chain reaction (PCR) assay. Amplification of 537-bp DNA fragments was observed from buffalo, without any cross-reaction with cattle, sheep, goat, pig, and chicken. The amplification was further confirmed by BamHI restriction enzymes. No adverse effect of processing was found on PCR amplification of buffalo meat DNA extracted from processed meat and meat products, even from meat emulsion autoclaved at 121 °C, 20 psi for 15–20 min. The detection limit for buffalo meat was found to be 1% in the admixed meat and meat products; however, very faint and inconsistent results were obtained in autoclaved meat emulsion at 1% level. The developed PCR assay was found to be specific for buffalo and could be a useful tool for detection of meat adulteration.     

Model studies on the detectability of genetically modified feeds in milk

Detecting the use of genetically modified feeds in milk has become important, because the voluntary labeling of milk and dairy products as "GMO free" or as "organically grown" prohibits the employment of genetically modified organisms (GMOs). The aim of this work was to investigate whether a DNA transfer from foodstuffs like soya and maize was analytically detectable in cow's milk after digestion and transportation via the bloodstream of dairy cows and, thus, whether milk could report for the employment of transgene feeds. Blood, milk, urine, and feces of dairy cows were examined, and foreign DNA was detected by polymerase chain reaction by specifically amplifying a 226-bp fragment of the maize invertase gene and a 118-bp fragment of the soya lectin gene. An intravenous application of purified plant DNA showed a fast elimination of marker DNA in blood or its reduction below the detection limit. With feeding experiments, it could be demonstrated that a specific DNA transfer from feeds into milk was not detectable. Therefore, foreign DNA in milk cannot serve as an indicator for the employment of transgene feeds unless milk is directly contaminated with feed components or airborne feed particles.     

The rheological properties of exudates from cured porcine muscle: effects of added non‐meat proteins

Meat exudates were collected from massaged cured porcine M semimembranosus using a model massaging unit. Exudates were used to observe changes in gelation properties due to the incorporation of commercially available non‐meat proteins. These included: soya isolate (90%), sodium (Na) caseinate (85%) and high gelling whey protein concentrates (WPCs, A‐35%, B‐75% and C‐β‐lactoglobulin (55%), as well as a regular 76% protein, WPC D. Compositional analysis ( n=6) showed that incorporation of non‐meat proteins significantly (P<0.05) increased the protein concentration of test exudates in all cases compared to controls. The viscoelastic properties of control and test meat exudate samples (n=6) were analysed using control stress rheology in oscillatory mode. All exudates were heated from 20 to 80°C at 1°C min⁻¹, and subsequently cooled after 30 min back down to 20°C at 1°C min ⁻¹. Addition of WPCs at a 1, 2 and 3% residual powder level and soya isolate at a 1% residual level, resulted in increased storage modulus G′ (Pa) values compared with controls. A 1% residual level of Na caseinate was detrimental to meat exudate gelation, resulting in lower final G′ (Pa) values than those observed for the control. © 1999 Society of Chemical Industry     

Detection of bovine DNA in raw and heat-processed foodstuffs, commercial foods and specific risk materials by a novel specific polymerase chain reaction method

The identification of beef in animal foods is a major concern not only for the prevention of commercial fraud, but also to avoid safety risks deriving from the presence of prohibited bovine material that might be harmful to both human and animal health. Here we report a novel set of bovine-specific primers, CYTbos1 (forward) and CYTbos2 (reverse), which allow the specific amplification of a 115 base pair fragment of the bovine cytochrome b gene (cytb) between nt 844 (mitochondrial site 15,590) and nt 958 (mitochondrial site 15,704), no cross-reaction being observed with DNA from another 12 frequent commercial meat species. The polymerase chain reaction product obtained is cleaved specifically by endonucleases ScaI and TspE1 to achieve further confirmation evidence. The sensitivity of the proposed method was 0.025%. The CYTbos primers successfully detected bovine DNA in meat samples processed for 20 min at 133 °C/300 kPa or for 2 h at 121 °C. CYTbos primers also detected bovine DNA in heat-processed commercial meat products exhibiting a complex nature, as well as in bovine specific risk materials. The proposed polymerase chain reaction method, aimed at detecting a small and specific fragment of the bovine mitochondrial DNA, may be especially useful for the direct identification of bovine DNA in foodstuffs subjected to severe heating under overpressure conditions.     

A simple, sensitive enzymatic method for quantitation of soya proteins in soya-meat blends

A method was developed for the detection and quantitation of soya proteins in soya-meat blends. The procedure involves acid hydrolysis of soya, meat or soya-meat blends and determination of free galactose plus arabinose using galactose dehydrogenase. Acetone powders of the various systems were also examined. Average galactose levels in soya flours, soya concentrates and soya isolates were 674,600 and 66 μM galactose equivalents per gram of product, respectively. Beef and pork contained less than 1 μM per gram of raw product. The galactose plus arabinose values for soya-meat blends were linearly dependent on the amount of soya added. The method is sensitive and capable of detecting less than 0·2% soya flours or concentrates and less than 2% soya isolates in meat products.     

Nutritional evaluation of meat–soya bean protein product blends

The nutritive value of protein of blends consisting of meat and soya bean protein products, which replaced either 20 or 40 % of protein of meat, were determined in rat feeding experiments. Protein efficiency ratio (PER) and net protein utilisation (NPU) were significantly lower (P<0.05) in blends containing either 20% protein from soya isolate or 40% protein from soya concentrate or soya flakes than blends containing sirloin only. However, there were no significant differences in PER and NPU when model meat (consisting of 70 and 30% protein from sirloin and connective tissue, respectively) was replaced by up to 20% protein from soya isolate or up to 40% protein from soya concentrate.     

The determination of soya and meat protein in raw and processed meat products by specific peptide analysis. An evaluation

The detection and measurement of characteristic peptides formed on enzymatic hydrolysis of soya protein products, meats and offals is described. Samples were heated at 120°C for 3h prior to digestion with trypsin overnight, and the resultant peptide mixtures passed through an Amicon ultrafiltration membrane. After concentration the ultrafiltrates were analysed by ion exchange chromatography on Aminex A5 resin. Peptides were detected by post-column reaction with ninhydrin. Characteristic peaks designated SP 2 and MP 1 were seen in chromatograms of digests of soya protein isolate and beef respectively, and these peaks were well resolved in beef and soya protein isolate mixtures. The SP 2 peak was shown to contain peptides derived from soya 11 S globulin. The soya protein and beef contents of a series of mixtures of freeze-dried, defatted beef and soya protein isolate were determined by measurement of the SP 2 and MP 1 peaks respectively. Soya protein content could be determined within 2% of the true value over the range 30–70% soya protein isolate and beef content could be determined within 5% of the true value in the range 20–100% beef. Analysis of five soya protein isolates, four soya protein concentrates, six soya flours and 13 textured soya products indicated considerable interproduct variation in the yield of SP 2. The MP 1 peak was seen in a range of meats, both cooked and raw. It was also present in digests of offals which contained smooth or striated muscle but not in ‘non-muscle’ offals. The protein origin of the MP 1 peak was not established but the yield appeared lower in meat products which had been heated during manufacture than in those which had received no such treatment. Analysis of a series of laboratory prepared canned and heated pork and soya protein isolate mixtures enabled the pork content to be determined to within 8% of the true value, 2% soya protein isolate could be detected but not quantified accurately.     

Analysis of commercial soya additives in meat products

The quantitative aspects of the analysis of commercial soya additives in meat products have been investigated using a polyacrylamide gel electrophoresis technique. Extraction of the proteins from the food products was carried out either in 8 M -urea and 1%, 2-mercaptoethanol at 18 to 20 °C for 16 h (Method 1), or 10 M -urea and 4%, 2-mercaptoethanol at 100 °C for 30 min (Method 2). The proteins in the extracts were separated by electrophoresis on gels containing 6% polyacrylamide and either 6 M -urea (Method 1) or 8 M -urea (Method 2) using a 0.06 M -Tris-glycine buffer, pH 8.6. Densitograms of the stained protein bands were used for the quantitative estimation of the soya proteins. Both methods were found to give quantitative results for the analysis of soya protein additives in beefburgers and sausages. Method 1 was also found to give quantitative results for meat pie fillings and canned meat loaf products, which had been autoclaved at 115 °C for 40 min.     

Discrimination of two alleles of the melanocortin receptor 1 gene to discern European wild boar (<Emphasis Type="Italic">Sus scrofa scrofa</Emphasis>) and domestic pig (<Emphasis Type="Italic">Sus scrofa domestica</Emphasis>) in meat products by real-time PCR

A duplex real-time polymerase chain reaction (PCR) technique for the discrimination of two subspecies of sus scrofa in meat products has been developed. Primers and probes target at a sequence in the melanocortin receptor 1 (MCR1) gene being associated in the expression of wild-type coat color. Both PCRs amplify a 56-bp product of DNA from wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) likewise. One of the TaqMan probes specifically anneals to the wild boar sequence and the other one to the domestic pig sequence, their base sequence differing only in a single nucleotide (single nucleotide polymorphism, SNP). This qualitative-method allows the detection of genomic DNA from wild boar and domestic pig as low as 25 pg in a 25-μl reaction volume. No cross reactivities were found with either genomic DNA from various other meat species, or with other ingredients of meat products (e.g. spices). The PCR efficiency is >95% for both targets. Although both PCRs are impaired by the presence of bovine and porcine DNA (wild boar detection is impaired by domestic pig DNA and vice versa), the method is applicable for the detection of low levels of wild boar and domestic pig meat simultaneously in commercial meat products. The limit of detection (LOD) in meat samples is 2% for wild boar and 5% for domestic pig.     

Quantitative immunoassay for soya protein in raw and sterilized meat products

An enzyme-linked immunosorbent assay (ELISA) is described for the quantitative analysis of soya protein in meat products. The assay is applicable to raw and sterilized products and is not dependent on soya variety and type of soya ingredient (concentrate, isolate, etc.). Therefore, the assay can be applied without knowledge of product composition, heat pretreatment and other processing conditions. The ELISA is specific for soya; no interference of other product components has been found. The detection limit of the ELISA is 0.5% soya protein. Qualitative analysis by immunoblotting is possible at much lower concentrations. The antibodies used are specific for sodium dodecyl sulphate (SDS) denatured soya protein subunits. The products are extracted with SDS and 2-mercaptoethanol, which guarantees optimum recovery of the protein components. Product extracts can be analysed directly by ELISA without removal of the SDS by using nitrocellulose as a solid phase.     

Application of DNA Technique for Identifying the Species of Different Processed Products of Swordfish Meat

ABSTRACT: Polymerase chain reaction technology and sequence analysis were used to identify the species in fresh, frozen, cooked, sterilized, and dressed dried fried meat of swordfish Xiphias gladius . The specific primers L-HS I, II, III, and IV, in conjunction with H-CSBDH, produced 357-, 238-, 137-, and 87-bp fragments, respectively, in the control region of swordfish mitochondrial DNA, but not for other billfish. These fragments were useful for detecting the species used in processed products claiming to be X. gladius . The primers L-HS IV and H-CSBDH produced 87-bp mtDNA fragments to identify the species of dressed dried fried swordfish meat products. Using L-HS IV and H-CSBDH primers'gene fragment to judge, it was found that only 45.8% (11/24) commercial samples of dressed dried fried products were made from swordfish.     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Determination of soya proteins in food using an enzyme-linked immunosorbent assay procedure

Classical immunoanalytical techniques are based on native antigens and their precipitation with specific antibodies: they are not very appropriate for the investigation of processed denatured mixtures. This report describes an immunoassay based on the more recent but well-established enzyme-linked immunosorbent assay (ELISA) procedure, which in this case is specific for soya proteins that have been solubilised in urea and then ?renatured’? by removing or diluting the denaturant. Levels of soya protein 100 g^?1 total protein (nominally 100%) were determined with 34 commercial soya products, using a standard reference antigen. Normal expected levels were observed with flours (average 107%) and isolates (108%), while results with concentrates (82%) and texturates (79%) were somewhat lower. Some specialised products gave little or no response, presumably because they are hydrolysed. Meat, milk, egg, wheat and field bean proteins displayed negligible interference. These preliminary results suggest that the ELISA procedure will provide a convenient general method for the qualitative characterisation and quantitative estimation of individual proteins in food products even after severe processing; it seems more attractive than many other methods reported. Immunoassay is capable of large numbers of inexpensive but sophisticated determinations of specific food components. The food analyst should regard ELISA (for the determination in situ of specific antigens in mixtures) as a powerful technique complementary to classical chromatographic and electrophoretic methods which depend on separation before determination.     

Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products

Verification of pork adulteration in commercial meat products is increasingly important for the authentication of Halal labels in processed foods. Here, we documented a PCR–restriction fragment length polymorphism (RFLP) assay with high precision and reproducibility for the tracing of porcine DNA in commercial meat products. The assay combined the species-specific primers to selectively amplify a short fragment (109 bp) of porcine cytochrome b gene from a heterogeneous background of genomic DNAs followed by RFLP analysis to authenticate real amplicon. The analysis of PCR products and restriction digests was automated in a chip-based capillary electrophoresis incorporated in Agilent 2100 bioanalyzer. The swine specificity of the assay was checked with 11 different meat-providing animal and fish species. Model experiments, mimicking the processed foods, were performed in binary and ternary mixtures after mechanical grinding and prolonged autoclaving. Finally, four types of the most popular finished meat products (meatball, streaky beacon, frankfurter, and burger) which are prevalent in the Malaysian food market were analyzed in order to verify the assay performance. The assay was sensitive enough to detect 0.0001 ng of swine DNA in pure formats and 0.01% (w/w) spiked pork in extensively processed ternary mixture of pork, beef, and wheat flour.     

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

Lupin and soya are members of the Leguminosae family which are recognised as some of the richest source of vegetable proteins. Lupin- and soya-containing products are available on the EU market and could cause severe adverse reactions in allergic individuals, even if consumed at low concentrations. In this context the development of methods for reliable detection of these allergens in food products is a useful tool for the surveillance of established legislation on food labelling within the EU. This work described the development of a duplex real-time PCR method allowing the simultaneous detection of traces of lupin and soya in processed food based on a specific TaqMan® probe designed on a mitochondrial tRNA-MET gene. A set of primers and probes was designed for the amplification of a 168 and 175 bp fragment of lupin and soya mitochondrial DNA, respectively. The performance of the method was established using lupin and soya flours and cookies baked from lupin- and soya-containing dough (different concentrations and baking times). The PCR platform yielded consistent and repeatable results. The specificity of the system was tested with DNA from 28 plant species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg per kg range. Both lupin and soya at a level of 2.5 mg per kg food matrix could be detected in cookies baked at 180 °C for 10 min. The method was successfully applied to bakery (e.g. bread) and vegetarian (e.g. non-meat sausages) food products that contain or may contain soya and/or lupin as ingredient or contaminant (according to the declaration on the product label).     

Pork quality from pigs fed on low glucosinate rapeseed meal: Influence of level in the diet,sex and ultimate pH

The appearance and eating quality of pork M. longissimus lumbrum from gilt and castrate pigs fed on diets containing a low glucosinate/low erucic acid rapeseed meal (cv. Tower) were assessed and compared with pork from pigs fed diets containing soya bean meal. At 90 kg liveweight, higher levels of either protein supplement, and therefore higher levels of dietary protein, gave slightly leaner meat. Meat from pigs fed rapeseed meal had more haem pigment and was slightly darker and redder than that from soya bean fed pigs. Differences in the quality of pork from the low glucosinate rapeseed meal and soya bean diets were small. Ratings for texture, juiciness and flavour of the lean varied little between the treatment groups. Suggestions that rapeseed meal as a protein supplement in diets might lead to inferior quality meat were not substantiated. The ultimate pH varied from 5.3 to 7.1 independently of the treatments and provided, for the first time, definitive relationships between pH and eating quality. Toughness and flavour were maximum and juiciness was a minimum after roasting meat of about pH 5.9. The ultimate pH had a larger effect on organoleptic quality than either sex or level of dietary inclusion of rapeseed or soya bean meals.     

An assessment of commercially available reagents for an enzyme-linked immunosorbent assay (ELISA) of soya protein in meat products

An enzyme-linked immunosorbent assay (ELISA) procedure based on a limited supply of specially prepared experimental immunoreagents and designed to measure levels of soya protein in raw or processed mixed meat products has already been published. This report describes a modified method with commercially available immunoreagents suitable for routine use; its response to a range of commercial soya ingredients has been checked using a particular soya protein isolate (Unisol, Unimills BV) as arbitrary standard. Individual soya components and possible cross-reacting food materials have also been investigated. Both the original and modified methods have been applied to a set of model beefburgers containing known levels of Unisol. There was good agreement between observed and calculated levels in raw and heat-set beefburgers; sterilised samples gave a decreased but linear response. The procedure is recommended for the examination of meat products in non-specialised laboratories.     

Identification of Puffer Fish of the Genus Lagocephalus: L. lunaris,L. spadiceus and L. inermis,Using Multiplex PCR

A multiplex PCR assay was developed to identify widespread poison-containing puffer fishes of the genus Lagocephalus: L. lunaris, L. spadiceus and L. inermis, using a novel KUGEN_ILSspec primer set which is specific for 12S ribosomal RNA (12S rRNA) and Cytochrome b (Cyt b) genes. KUGEN_ILSspec set is composed of fish positive-amplified primers that produced a 289-bp fragment while L. lunaris-, L. spadiceus- and L. inermis-specific primers produced 123-, 196- and 493-bp fragments, respectively. The sensitivity of this primer set was found to be high as it was capable of amplifying targeted DNA at concentrations as low as 1 ng/μL. Moreover, the primer set was shown to amplify intact DNA and species-specific fragments from heat-processed food. Consequently, multiplex PCR technique in combination with KUGEN_ILSspec primer set could offer an effective measure for detecting poisonous puffer fish contamination in both fresh and processed fish products, which will greatly aid in public health control and law enforcing endeavors.