Nested PCR detection of genetically modified soybean in soybean flour, infant formula and soymilk

Due to the Brazilian market introduction of the genetically modified (GM) crop Roundup Ready™ (RR) soybean, the ability to detect GM crops has become a legal necessity. In order to detect the presence of RR soybean, a polymerase chain reaction (PCR) amplification method was evaluated for the detection of RR in soybean mixtures and commercially available soy flour, infant formula and soymilk powder. To detect the presence of RR soybean, a nested PCR resulted in an amplicon of 169 bp, present for all soybean mixed samples containing 0.01-10% GM soybean and absent for 0% GM soybean. None of the analysed infant formulas showed a positive signal after the nested PCR; four out of six soy flour samples and 15 out of 25 soymilk powder samples were positive for the presence of RR soybean. Results show that the nested PCR method used is adequate to determine the presence of GM soybean in the presented products.     

A survey of the use of soy in processed Turkish meat products and detection of genetic modification

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.     

Detection of genetically modified organisms in processed meat products on the Serbian food market

The addition of soybean proteins to processed meat products has significantly increased in recent years due to the interesting functional and nutritional properties of these vegetable proteins. Since the Roundup Ready (RR) soybean is the only transgenic soybean line approved for market in EU this work was aimed at monitoring its presence in meat products on the Serbian food market. The extracted DNA was analyzed using duplex polymerase chain reaction (PCR) with primer pairs aimed at the lectin gene and 35S promoter. Samples positive for the presence of GM soybean were subjected to a real-time quantification of the percentage of RR soya. The results indicated that out of fifty processed meat products examined, twelve gave positive results with 35S promoter and all contained RR soya below 0.1%.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

Detection of genetically modified soybean DNA in refined vegetable oils

In this study, four different protocols were tested for their ability to extract DNA from blended refined vegetable oils: the in-house prepared Wizard and CTAB methods and the methods based on the use of the commercial kits Wizard^® Magnetic DNA purification system for food and Nucleospin^® for food. The performance of the extraction protocols was determined by end-point polymerase chain reaction (PCR) targeting the soybean lectin gene with primers suitable for the amplification of small fragments and confirmed by real-time PCR with specific hydrolysis probes. From the tested protocols, the Nucleospin method was the only one able to produce amplifiable DNA from refined vegetable oils. To verify the presence of Roundup Ready^® (RR) soybean, event-specific primers were used for end-point PCR assays. The amplification of trace amounts of RR soybean by real-time PCR confirmed the label statements of two samples. The results highlight the importance of the DNA extraction protocol and the critical choice of PCR primers on processed food matrices, such as refined oils. Considering the few reports and difficulties pointed out in the literature to obtain amplifiable DNA from refined vegetable oils, the present results can be a step forward in the traceability of refined oils regarding authenticity issues and genetically modified organism detection.     

Quantitative real‐time PCR assays to detect DNA degradation in soy‐based food products

BACKGROUND: Polymerase chain reaction (PCR)‐based DNA diagnostics have grown in importance for assessing food quality and safety. PCR diagnostic reliability and sensitivity depend on the quality of the DNA extractions, including the extent of DNA degradation and the presence of PCR inhibitors. RESULTS: An approach has been described that quantifies the extent of DNA degradation in soy food samples using anchored real‐time PCR (qPCR) assays that amplify target sequences ranging from < 100 to > 1000 bp, based on two endogenous soy sequences. DNA degradation was quantified for model foods produced in the laboratory (cooked soy meal, tofu) as well as purchased soy‐containing food products. Considerably less than 1% of the total DNA extracted from samples was available for amplification of the longest amplicons (830 and 1022 bp) from the most highly processed food products (e.g., soy‐based infant formula). CONCLUSION: The utility of anchored qPCR assays was demonstrated for characterizing the amount of DNA that is available for amplifying different‐length PCR products from a range of soy‐containing processed food products. This approach should be useful for estimating the amount of amplifiable DNA in food ingredients in cases where food processing has caused degradation of DNA. Copyright © 2009 Society of Chemical Industry     

Validation of end-point and real-time PCR methods for the rapid detection of soy allergen in processed products

This work describes the development and validation of two PCR methods, end-point and real-time PCR, for the detection of soy protein in a wide rage of foodstuffs. These techniques are reliable and sensitive, allowing detection of trace amounts of soybean in processed products. TaqMan real-time PCR was the simpler and more rapid process, with a higher potential for automation and, therefore, currently the most suitable screening method. To verify correct operation of the proposed methodology, ELISA was used for quantitative determination of soy protein. In addition, 35 meat, fish and bakery processed products, which could potentially contain soy but was not declared on the label, were tested for the presence of soy DNA using the proposed methods. The methodologies will be valuable in issues regarding the presence of soy protein in processed products, especially in verifying labelling and security regulations to protect consumer's rights.     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

酱油、烤鳗酱油DNA 提取及原料成分的荧光PCR 检测

用4 种预处理方法对酱油进行浓缩,后采用CTAB 沉淀法提取DNA,再用SYBR Green Ⅰ荧光PCR 分别检测人工添加的大米gos9 基因、酱油原料成分(大豆Lectin 基因和小麦Wx012 基因)。结果显示：仅人工添加大米DNA、用CTAB 沉淀液浓缩的预处理方法提取的酱油DNA 中,大米gos9 基因、大豆Lectin 基因和小麦Wx012 基因的检测结果均为阳性,表明这种方法最适于酱油DNA 的提取。将建立的DNA 提取方法用于3 份酱油、4 份烤鳗酱油同样有效,gos9 基因检测结果均为阳性。2 份酱油检出大豆成分,2 份烤鳗酱油检出小麦成分,1 份酱油和2 份烤鳗酱油检出大豆成分和小麦成分。     

挤压对大豆蛋白构象及其组织化结构的影响研究进展

随着挤压技术的发展,以大豆蛋白为主要原料经过挤压加工制成具有明显组织化结构的素肉制品技术以及蛋白组织化结构形成机制越来越受到学者的关注.近几年的研究表明大豆蛋白微观结构的变化对大豆素肉的宏观组织化结构具有至关重要的作用.本文对挤压过程中大豆蛋白构象及其对组织化结构影响的研究进行综述,概述大豆蛋白分子构成,并总结了挤压对...     

Detection and Quantification of 4(5)‐Methylimidazole in Cooked Meat

4(5)‐Methylimidazole (4‐MeI) is a nitrogen‐containing heterocyclic compound found in class III and IV ammoniated caramel colors, a group of additives widely used in the food industry. A suspected carcinogen and neurotoxin and efforts are underway to limit its presence in foods. Several methods have been developed to detect and quantitate 4‐MeI in different food matrices, including roasted coffee, beer, soft drinks, and soy sauce; however, no methods are available to measure 4‐MeI in cooked meat and meat products containing lipids and high levels of interfering nitrogen compounds, such as amino acids and peptides. A rapid method using 0.1 M sodium acetate buffer (pH 5) as an extraction solvent followed by derivatization with isobutylchloroformate and gas chromatograph mass‐spectrometry was developed to quantify 4‐MeI in cooked meat products with added caramel colors containing 4‐MeI. Selected ion monitoring mode was used to monitor 4‐MeI ions fragments. In the 8 commercial meat products tested, 4‐MeI levels ranged from 0.041 to 1.015 mg/kg, with recovery of 94.76% to 103.94%. In addition, a matrix‐matched calibration performed by analyzing a spiked cooked meat sample indicated no significant difference (P > 0.05), which means the meat matrix had no effect on the developed method. This method proved useful in analyzing 4‐MeI in meat products with added caramel color containing 4‐MeI.     

Detection of allergenic additives in processed meat products

Allergic responses to food components are an increasing problem all over the world. It is therefore important to protect people who are vulnerable to food allergens against accidental and unintended consumption of products containing allergic ingredients. The meat industry commonly uses various allergic additives in the production of processed products, such as legumes (soy, peas, beans), milk and egg preparations, cereals containing gluten (wheat, rye, barley and oats), and spices (celery and mustard). These meat additives have specific technological properties, which help to create a texture or flavor profile, or affect the nutritional value, although some of them, such as soy, mustard, milk and egg white proteins, can cause severe allergic reactions. The aim of this paper is to discuss the application of various recently established methods of detection of allergenic additives in processed meat products – for instance cold cuts and sausages. The new methods are based mainly on protein, DNA, and isoflavones or phytic acid analysis. The article also characterizes the latest trends in the development of research on methods that would enable quick and reliable identification of targeted allergens in meat products. © 2018 Society of Chemical Industry     

Effect of Chemically Modified Soy Proteins and Ficin-tenderized Meat on the Quality Attributes of Sausage

ABSTRACT: The purpose of this investigation was to use ficin‐tenderized meat and cysteine‐modified soy proteins in the production of bologna and to evaluate the effect of these modifications on water‐holding capacity (WHC), emulsion stability (ES), texture, and protein solubility. The effect of ficin on meat protein was also evaluated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Results indicated that both ficin‐tenderized meat and modified soy proteins substantially improved WHC, ES, and other quality factors. SDS‐PAGE results showed the disappearance of several protein bands in ficin‐treated meat. Solubility of meat proteins increased when ficin was used for meat tenderization. The results of this study indicated that some quality attributes of meat products can be improved by enzymatic and chemical modification of protein sources in the manufacture of meat products.     

Analysis of Lunasin in Commercial and Pilot Plant Produced Soybean Products and an Improved Method of Lunasin Purification

Abstract: Lunasin is a bioactive peptide present in soybean. It is important to quantify lunasin concentration in soy products to assess its potential impact as functional food. The objectives of this study were to analyze lunasin in commercial soymilk products and implement an efficient method to isolate and purify it from defatted soybean flour. Defatted soybean flour was suspended in water, and the extract was loaded in a pre‐equilibrated diethylaminoethyl column and bound proteins eluted using a step gradient of salt. Most lunasin was eluted from the column at 0.2 to 0.4M NaCl as quantified by immunoassays and purified using ultracentrifugation and ultrafiltration techniques. Lunasin purity was ≥90% and a standard curve was used to quantify its concentration in soymilk products. Concentration of lunasin in soy products, including organic soymilk, soy protein shakes, and soy infant formulas, ranged from 1.78 to 9.26 mg lunasin/100 g product. The concentration per serving ranged from 1.59 ± 0.01 to 22.23 ± 0.74 mg lunasin with variability depending on brand and size per serving. Steam‐ground‐cooked soy had the highest concentration of lunasin (22.23 ± 0.74 mg/serving), similar to some commercial products. Ground‐cooked soymilk contained roughly half the concentration of lunasin (14.39 ± 1.4 mg/serving). Soy infant formulas that used soy protein isolate revealed lower concentrations of lunasin (P < 0.05). It was concluded that all soymilk products analyzed contained lunasin, and a more efficient method to isolate lunasin with higher purity was developed. Practical Application: Soy foods have shown to play a role in cardiovascular health prevention. The quantification of lunasin in commercially available soy products can add to the already existing health claim for soy foods and encourage consumers to include soy products in their diets.     

Quantification of Human IgE Immunoreactive Soybean Proteins in Commercial Soy Ingredients and Products

ABSTRACT:  The objective of this study was the detection and quantification of human IgE immunoreactive soybean proteins in commercially available soy ingredients and products. Optimum dilutions of primary antibody and antigens as well as detection sensitivity were determined for the implementation of a sandwich ELISA method using plasma from soy sensitive subjects (IgE ranging from 0.35 to 98.7 IU/mL). Human IgE immunoreactivity of commercial soybean ingredients showed that the plasma of subjects with strong allergic reaction to soybean presented proportionally higher immunoreactive response. Soy protein isolate and soy protein concentrate contained less immunoreactive proteins than soy flour and grits. As expected, a hypoallergenic soybean product presented the lowest IgE immunoreactivity. Hydrolyzed and fermented soy ingredients showed negligible human IgE immunoreactivity when proteins and peptides were < 20 kDa. The IgE immunoreactivity of soymilk samples ranged from 3.4 to 68.9 ng IgE/mg extracted protein. Tofu contained about 20-fold higher IgE immunoreactivity than soymilk products (median 171 ng IgE/mg extracted protein). Furthermore, soy cheese products presented twice the IgE immunoreactivity than tofu products (median 359 ng IgE/mg protein). Meat analogues presented considerably high extracted protein concentration (median 67.9 mg/g product). The findings of the current investigation demonstrate sandwich ELISA as a reliable immunochemical method with good repeatability, sensitivity, and low detection limit to quantify IgE immunoreactive proteins in soy ingredients and products. Quantitative measurement of specific IgE is likely to become an increasingly valuable tool for soybean industry to comply with food labeling for manufacturers, thus protecting soy-sensitive consumers.     

Iron Availability from Soy, Meat and Soy/Meat Samples in Anemic Rats With and Without Prevention of Coprophagy

The biological availability of iron from samples of soy proteins (nontextured, extruded and spun), meat (chicken and beef) and spun soy/meat combination products was compared to ferrous sulfate using a hemoglobin regeneration bioassay. Compared to ferrous sulfate (55%) iron availability from the various soy proteins ranged from 29-57%, for the meat samples 32-39% and for soy/meat combination products 61-92%. There was no significant improvement in iron availability by fortification with ferrous sulfate or ascorbic acid. Prevention of coprophagy in the anemic rats during the bioassay using aluminum anal cups produced varying degrees of reduction in iron availability for various samples and this effect clearly needs further investigation.     

Dietary purines in vegetarian meat analogues

BACKGROUND: The meat alternatives market offers a wide range of products resembling meat in taste, flavour or texture but based on vegetable protein sources. These high protein–low purine foods may find application in a low purine or purine‐free diet, which is sometimes suggested for subjects with increased serum urate levels, i.e. hyperuricaemia. RESULTS: We determined purine content (uric acid, adenine, guanine, hypoxanthine, xanthine) in 39 commercially available meat substitutes and evaluated them in relation to their protein content. Some of the products contained a comparable sum of adenine and hypoxanthine per protein as meat. Analysis of variance showed an influence of protein source used. Mycoprotein‐based products had significantly higher contents (2264 mg kg^?1) of adenine and hypoxanthine per kg of 100% protein than soybean‐based products (1648 mg kg^?1) or mixtures consisting of soybean protein and wheat protein (1239 mg kg^?1). CONCLUSION: Protein‐rich vegetable‐based meat substitutes might be generally accepted as meat alternatives for individuals on special diets. The type of protein used to manufacture these products determines the total content of purines, which is relatively higher in the case of mycoprotein or soybean protein, while appearing lower in wheat protein and egg white‐based products. These are therefore more suitable for dietary considerations in a low‐purine diet for hyperuricaemic subjects. Copyright © 2010 Society of Chemical Industry     

PCR detection of soy ingredients in bread

Bread may contain soy ingredients to variable extends. The nature of this soy may be genetically modified, or the ingredient may cause allergic reactions in sensitive patients. PCR is a powerful tool to detect the presence of soy in food products. Major prerequisite for a PCR analysis is DNA of good quality and quantity. The persistence of soy DNA during bread making was evaluated using different amounts of different soy ingredients. Agarose gel electrophoresis of the samples taken during the baking process reveal that DNA is degraded, but subsequent PCR detection remains possible. Results, however, greatly depend on the type of ingredient and the amount present. Lower detection limits were obtained for full-fat and defatted soybean flours than those for toasted soybean flour and soy fibre samples. Samples taken at different places in the bread, however, reveal that sampling strategy may influence the PCR results.     

PCR法检测大豆加工食品中的转基因成分

通过分子生物学手段,以聚合酶链式反应(PCR)技术为基础,建立了检测大豆加工食品中转基因成分的方法。实验分别采用CTAB法和试剂盒(Kit)法对大豆锅巴、豆浆、豆奶粉、豆腐、豆腐丝等五种大豆加工食品中的DNA进行了提取,用内标基因Lectin对此两种方法的提取效果进行了比较,并以提取的DNA为模板,利用不同的引物分别对目标基因35S和NOS进行了PCR扩增和琼脂糖凝胶电泳检测。结果表明:Kit法的DNA提取效果优于改良CTAB法,上述五种大豆加工食品中均检测出35S启动子和NOS终止子,且均含有转基因成分。     

Effect of Sequestering Intrinsic Iron on the Electron Paramagnetic Resonance Signals in Powdered Soy Proteins

This investigation examined iron in powdered soy protein products using electron paramagnetic resonance (EPR) spectroscopy, and the effect that selectively binding free iron in isolated soy protein (ISP) had on the occurrence of metastable radicals in powdered soy proteins. EPR analyses of soybean defatted flour, commercial ISP and laboratory ISP samples revealed a peak at g = 4.3 characteristic of high‐spin ferric iron in a rhombic‐coordinated environment. Commercial ISP samples examined contained higher levels of the rhombic ferric iron than laboratory‐prepared ISP samples. During the first 6 wk of storage the primary singlet EPR signal at g = 2.0049 in the commercial ISP samples approximately doubled, and the laboratory prepared samples increased by about 9‐fold. The EPR signal was initially about 4‐times higher in the freshly prepared commercial samples compared to the corresponding laboratory ISP. Laboratory ISP samples prepared with added deferoxamine to sequester endogenous iron exhibited a large increase in the high‐spin ferric iron EPR signal at g = 4.3. ISP treated with deferoxamine also exhibited a multiple‐line EPR signal at about g = 2.007, instead of the typical singlet signal at g = 2.0049. The power at which the signal amplitude was half‐saturated also changed from about 1 mW in the control ISP to about 20 mW in the deferoxamine treated ISP. The multiple‐line EPR spectrum from the ISP treated with deferoxamine increased during storage over a 6‐wk period by about 6‐fold. The observed changes in EPR line‐shape, g‐value, and power saturation with the deferoxamine treatment indicate that the primary free‐radical signal in powdered ISP samples may be from stabilized tyrosine radicals with spin densities distributed over the aromatic ring.