Detection of genetically modified organisms obtained from food samples

Genetially modified organisms (GMOs) were explored in food samples obtained from November 2000 to March 2003 in the Tokyo area by using PCR and real-time PCR techniques. The existence of Roundup Ready Soybean (RRS) was surveyed in processed foods derived from soybeans, such as tofu, boiled soybean, kinako, nama-age, abura-age, natto, miso, soymilk and yuba. RRS was detected in 3 of 37 tofu, 2 of 3 nama-age, 2 of 3 yuba and 3 of 3 abura-age samples. The CBH351 in 70 processed corn foods, NewLeaf Plus and NewLeaf Y in 50 processed potato foods, and 55-1 papaya in 16 papayas were surveyed. These GMOs were not detected among the samples. Qualitative and quantitative analyses of RRS and genetically modified (GM) corn were performed in soybean, corn and semi-processed corn products such as corn meal, corn flour and corn grits. RRS was detected in 42 of 178 soybean samples, and the amount of RRS in RRS-positive samples was determined. The content was in the range of 0.1-1.4% in identity-preserved soybeans (non-GMO), and 49.8-78.8% in non-segregated soybeans. On the other hand, GM corns were detected in 8 of 26 samples. The amount of GM corn in GM corn-positive samples was in the range of 0.1-2.0%.     

CP4-EPSPS蛋白抗体的制备及其夹心ELISA定量检测方法的建立

为了快速高效地检测转Cp4 epsps基因抗除草剂大豆,本研究利用已制备的CP4-EPSPS蛋白免疫实验动物,获得5株CP4-EPSPS蛋白鼠单克隆抗体及3份羊抗血清,建立了CP4-EPSPS蛋白双抗夹心酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)检测方法,同时利用该方法对结荚期的Cp4 epsps转基因抗除草剂大豆的根、茎、叶、种子进行定量检测。结果显示,最佳检测条件为捕获抗体1D12,工作浓度1.25 μg/mL,包被酶标板,4 ℃静置过夜,检测样品20 ℃孵育45 min,检测抗体8092,工作浓度2.5 μg/mL,20 ℃孵育30 min;CP4-EPSPS蛋白在模拟大豆环境中最低检测限为3.6 ng/mL,检测范围为7.5~125 ng/mL;重复性变异系数小于15%。利用上述检测条件,在转Cp4 epsps基因抗除草剂大豆的根、茎、叶、种子中均检测到CP4-EPSPS蛋白的表达,且各组织部位表达量差异显著,其中CP4-EPSPS蛋白在叶片中表达量最高,茎、种子、根中CP4-EPSPS蛋白表达量逐渐降低。     

Four new SYBR<Superscript>®</Superscript>Green qPCR screening methods for the detection of Roundup Ready<Superscript>®</Superscript>, LibertyLink<Superscript>®</Superscript>, and CryIAb traits in genetically modified products

SYBR^®Green qPCR methods for the detection of the Roundup Ready^® “CP4-EPSPS”, LibertyLink^® “PAT” and “BAR,” and the Bacillus thuringiensis “CryIAb” traits as present in genetically modified organisms (GMO) were developed. Their specificity, sensitivity, and PCR method efficiency were determined. All methods are specific and generate amplicons of 108, 73, 109, and 69 bp, respectively, for “CP4-EPSPS,” “CryIAb,” “PAT,” and “BAR” targets. They perform well at low target levels and can detect down to 5 copies of their respective targets extracted from a sample. The PCR efficiency of the methods ranges between 91 and 109%. Due to their trait-specific nature, these methods allow an efficient screening between the different GMO. In this way, the number of possible GMO candidates present in a sample can be reduced what results in lower global costs due to limiting of further the number of analytical identification steps. The application of these methods in CoSYPS GMO analysis is illustrated using two GEMMA proficiency test samples and a reference material from the GM rapeseed event RF3. This set of SYBR^®Green qPCR trait-specific methods represents a very interesting novel set of GMO analysis methods allowing cost-effective identification of GM materials in products.     

Development and application of a selective detection method for genetically modified soy and soy-derived products.

A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.     

Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products

An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10-10⁶ copies of each target.     

Quantitative competitive PCR for the detection of genetically modified soybean and maize

 The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques. Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more than 1% RRS were determined by QC-PCR using various amounts of standard DNA. Received: 13 January 1998 / Revised version: 4 March 1998     

Detection of genetically modified soybean and its product tou-kan by polymerase chain reaction with dual pairs of DNA primers

Since genetically modified (GM) crops and foods began to appear in market, the detection of these GM foods has become an important issue. Efficient and reliable methods are urgently needed to analyze the GM content of a large quantity of foods. However, most foods are processed through heating or cooking, and their proteins are denatured. Therefore, DNA rather than protein is the major target for detecting GM foods. In this report, polymerase chain reaction (PCR) was performed with dual sets of DNA primers, which co-amplified the soybean-specific lectin gene and the transgenic petunia transit peptide sequence (CTP) in the 5′ end of the 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene within the same reaction. PCR with these two sets of primers reacted specifically with lectin gene and CTP, and can detect soybean with GM content less than 1%. PCR detecting both lectin gene and CTP was also applied to a soybean-derived product tou-kan, a traditional oriental food. Results indicated that although DNA was partially degraded in tou-kan, this PCR method was still able to detect the presence of EPSPS gene. However, when the DNA within tou-kan was destroyed badly, neither lectin nor EPSPS genes were detected by the PCR suggested here. Finally, an examination procedure for the Roundup Ready soybean was suggested according to the results of PCR with dual pairs of DNA primers. The proposed PCR method has proved to be a reliable and efficient method for detecting the GM content of the processed foods from Roundup Ready soybean. An erratum to this article can be found at     

植物基替代蛋白的利用进展

未来食品对食品的营养健康属性和环境友好性都提出了更高的要求。在此背景下,以“植物肉”为代表的植物基食品引起了国内外的广泛关注。作为“植物肉”生产的主要原料,植物基替代蛋白的开发和利用对食品产业链上下游都有着重要的影响。本文首先介绍了植物基替代蛋白在“植物肉”中的应用现状,阐述了植物基替代蛋白新资源挖掘的必要性;然后总结了植物基替代蛋白的来源、制备手段和加工技术,重点分析了利用高水分挤压技术重构植物蛋白纤维化结构及其机理的研究进展和3D打印技术在蛋白质成型加工方面的研究现状;最后总结了植物基替代蛋白高效利用面临的机遇和挑战,为未来植物基食品的发展方向提供了参考。     

大豆蛋白素肉风味影响研究进展

素肉制品是近年才刚确定行业标准的新型仿生食品,并在口感、风味等方面大有取代肉制品的趋势,在实际生产中以大豆蛋白为主要原材料的素肉制品最为普遍。大豆蛋白素肉是大豆蛋白通过挤压等技术生产的具有类似肉类口感和风味的植物蛋白仿肉制品,风味和口感是影响蛋白素肉能否被消费者接受的重要因素,其中风味是消费者接收到的第一信号,因此,风味是否被消费者接受决定了素肉的市场前景。本文首先介绍大豆蛋白素肉的风味来源,然后总结不同加工因素对大豆蛋白素肉风味物质的影响,并对大豆蛋白肉风味物质的保留机制进行论述,以期为大豆蛋白素肉风味研究开发提供基础。     

Genetically modified maize and soybean on the Egyptian food market

The results of a survey study on food samples produced from genetically modified soybean and maize collected from the Egyptian market are presented. Forty samples of soybean and 40 samples of maize products have been gathered randomly from markets in Cairo and Giza. The genetic modification was detected by polymerase chain reaction (PCR) using official detection methods according to section 35 of the German Foodstuffs Act. Samples were investigated for the presence of material derived from the following genetically modified organisms (GMOs) all of which are approved for food use in Europe: Roundup Ready soybean (RRS) and maize lines Bt176, Bt11, T25 and MON810. In addition, samples were examined in qualitative and quantitative analysis for the presence of material derived from the transgenic maize line StarLink (Aventis) which was approved for animal feed use exclusively in the US. Twenty % of 40 investigated soy samples contained Roundup Ready soybean; 15% of 40 maize samples tested positive for Bt176 and 12.5% positive for Bt11 maize. Furthermore, the presence of StarLink maize could clearly be demonstrated in four samples mixed with Bt176 and Bt11. The percentage of StarLink was less than 1% in quantitative analysis. The maize lines T25 and MON810 were not detected.     

Effects of feeding rations with genetically modified whole cottonseed to lactating Holstein cows

Two experiments were conducted to evaluate dry matter intake (DMI), milk yield, and milk composition from feeding rations that contained different sources of genetically modified whole cottonseed to Argentinean Holstein dairy cows. Twenty-four lactating multiparous Argentinean Holstein dairy cows were used in 2 experiments with a replicated 4 x 4 Latin square design, with cows averaging 565 kg body weight and 53 d in milk at the beginning of the experiments. Treatments in Experiment 1 were: Bollgard cotton containing the cry1Ac gene, Bollgard II cotton containing cry1Ac and cry2Ab genes, Roundup Ready cotton containing the cp4 epsps gene, and a control nongenetically modified but genetically similar cottonseed. In Experiment 2, two commercial sources, a parental control line, and the transgenic cotton containing both cry1Ac and cp4 epsps genes were used as treatments. All cows received the same total mixed ration but with different whole cottonseed sources. Cottonseed was included to provide 2.50 kg per cow daily (dry matter [DM] basis) or about 10% of the total diet DM. The ingredient composition of the total mixed ration was 32% alfalfa hay, 28% corn silage, 22% corn grain, 17% soybean meal, and 2% minerals and vitamins. In addition, genomic DNA was extracted from a subset of milk samples and analyzed by polymerase chain reaction followed by Southern blot hybridization for small fragments of the cry1Ac transgene and an endogenous cotton gene, acp1. No sample was positive for transgenic or plant DNA fragments at the limits of detection for the assays following detailed data evaluation criteria. The DMI, milk yield, milk composition, body weight, and body condition score did not differ among treatments. Cottonseed from genetically modified varieties used in these studies yielded similar performance in lactating dairy cows when compared to non-transgenic control and reference cottonseed.     

Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cry1Ab) after preheating

We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification. For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp. kurstaki strain HD-1. The former is expressed in GM-soybeans and the latter is expressed in GM-corns. Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF. Both proteins were rapidly digested within 60 sec. After preheating, the digestibility by SGF was slightly increased. Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF. The digestion time of both proteins by SGF was almost the same as that of the purified proteins. Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined. The digestion time of these proteins was 240 min or more. However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec. Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF. Digestion time of both proteins by SIF was almost the same as that of the purified proteins. From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating. Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.     

Serum testing of genetically modified soybeans with special emphasis on potential allergenicity of the heterologous protein CP4 EPSPS

Roundup Ready soy contains the CP4-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein. Serum IgE from two distinct populations of soy-allergic patients were recruited to determine their IgE-binding specificity. One population consisted of 10 adult patients from Europe, whose primary diagnosis was soy food allergy with some also having mite allergy. In addition, 6 primarily mite-allergic, 6 food-allergic (celery, carrot, milk, shrimp, walnut, and apple), and 5 non-allergic patients were tested. Another population consisted of 13 children from Korea, whose primary diagnosis was atopic dermatitis and secondarily soy and egg sensitization. In addition, 11 non-allergic patients were tested. Each patient population was extensively characterized with respect to clinical symptoms, specific IgE (CAP) scores, and total IgE. Immunoblots and ELISA assays were developed using serum IgE from these patients and soy extracts, CP4 EPSPS, rice extract, ovalbumin, rubisco, purified major peanut allergen Ara h 2, the putative soy allergen Gly m Bd 30k and mite allergen Der f 2 proteins as the intended targets. Immunoblot results indicated that soy-allergic patients bound soy extracts but did not specifically bind rubisco or CP4 EPSPS. ELISA results were in general agreement with the immunoblot results except that rubisco bound significant quantities of serum IgE from some patients. These results indicate that the CP4 EPSPS protein does not bind significant quantities of IgE from two geographically distinct sensitive populations and there is no evidence for an increased allergenic potential of this biotech protein.     

Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase

Microbial transglutaminase (MTG) isolated from Streptomyces mobaraensis has been available on a commercial scale for several years. MTG generates inter- and intramolecular cross-links between γ-carboxylamide groups of glutamine residues and ɛ-amino groups of lysine residues in proteins. Due to its great potential to improve various functional properties of proteins, MTG is mainly used to enhance texture, stability, and water binding. Application of MTG for the production of plant protein-based foodstuffs such as tofu, noodles, bread and bakery products, is still limited to raw materials from soybean and wheat. However, with the increasing demand for vegetarian foods, the utilisation of novel proteins as functional ingredients, e.g. from peas, lupins, sesame, and sunflower, seems promising. To open new horizons for MTG application, this review aims at demonstrating the actual potential of MTG in processing foodstuffs based on vegetable proteins. Particular focus was laid on novel plant protein sources suitable for cross-linking with MTG. Furthermore, strategies for improving texture and nutritive value of the proteins are discussed.     

Detection of genetically modified organisms in foreign-made processed foods containing corn and potato

Investigations of the validity of labeling regarding genetically modified (GM) products were conducted using polymerase chain reaction (PCR) methods for foreign-made processed foods made from corn and potato purchased in the Tokyo area and in the USA. Several kinds of GM crops were detected in 12 of 32 samples of processed corn samples. More than two GM events for which safety reviews have been completed in Japan were simultaneously detected in 10 samples. GM events MON810 and Bt11 were most frequently detected in the samples by qualitative PCR methods. MON810 was detected in 11 of the 12 samples, and Bt11 was detected in 6 of the 12 samples. In addition, Roundup Ready soy was detected in one of the 12 samples. On the other hand, CBH351, for which the safety assessment was withdrawn in Japan, was not detected in any of the 12 samples. A trial quantitative analysis was performed on six of the GM maize qualitatively positive samples. The estimated amounts of GM maize in these samples ranged from 0.2 to 2.8%, except for one sample, which contained 24.1%. For this sample, the total amount found by event-specific quantitative analysis was 23.8%. Additionally, Roundup Ready soy was detected in one sample of 21 potato-processed foods, although GM potatoes were not detected in any sample.     

激光诱导荧光结合磁分离检测CP4-EPSPS基因

建立一种激光诱导荧光结合磁分离的检测技术，用于在线检测CP4-5-烯醇丙酮酸酯-3-磷酸合酶基因（CP4-5-enol acetate-3-phosphate synthase gene，CP4-EPSPS）。以氨基磁纳米粒子（magnetic nanoparticles，MNPs）为载体修饰cDNA，构建捕获基因探针，并设计FAM荧光标记的信号基因探针（sDNA）。在目的基因CP4-EPSPS（tDNA）存在的情况下，cDNA和sDNA通过碱基互补配对原则与tDNA结合形成双链结构。该结构通过毛细管时被磁场富集分离，然后在激光诱导荧光检测器的作用下在线检测其荧光信号。通过对MNPs质量浓度、捕获探针浓度、牛血清白蛋白质量浓度进行考察，在优化的实验条件下，在1.0×10－11～2.0×10－7?mol/L范围内，CP4-EPSPS基因浓度与荧光峰面积呈良好线性关系，检出限为4.0×10－12?mol/L（RSN=3），该方法成功应用于转基因大豆的聚合酶链式反应扩增产物的检测。     

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%.     

利用PCR方法检测转基因大豆加工食品中的修饰基因

市场上的转基因产品以及深加工产品越来越多,其安全性引起全世界的极大争论,很多国家的检验检疫机构相继开展了转基因产品的检测工作。本文以转基因大豆类食品为材料,以聚合酶链式反应(PCR)方法为基础,选择适用于转基因食品安全性检验的核酸检测技术,针对抗除草剂Round up Ready (RR)大豆的插入基因进行PCR检测,建立适合转基因大豆类食品的检测方法．该方法简便快速、检测结果与标准及其他文献资料相符．     

羧甲基纤维素钠对挤压豌豆蛋白素肉品质的影响

为了提高豌豆蛋白素肉的表观品质,探究挤压过程中多糖与蛋白相互作用对素肉品质的影响,本研究采用高水分挤压法,制作羧甲基纤维素钠（carboxymethylcellulose sodium,CMC）与豌豆蛋白粉混合基素肉,分析CMC添加量（0%、2%、4%、6%、8%）对豌豆蛋白素肉的质构特性、色泽、微观结构和蛋白分子作用力的影响,以及素肉蛋白二级结构、分子作用力与表观结构（质构、色差）的相关性。结果表明,维持豌豆蛋白素肉结构的主要作用力是二硫键。与未添加CMC相比,添加量4%CMC通过促进豌豆蛋白素肉中二硫键的形成,将素肉的硬度提高了95%。同时,观察到豌豆蛋白素肉具有光滑完整的表面和均匀的结构。二级结构中的无规卷曲含量增加,促进明亮度增强,CMC改善了豌豆蛋白素肉的品质。     

Differences in quality and quantity of transgenic component in Roundup Ready soybean during bean curd preparation

Using 2.5% (w/w) transgenic Roundup Ready soybean as raw material, the effects of critical processing procedures on the fragment sizes of endogenous gene lectin and transgenic gene epsps and the quantity of transgenic component during bean curd preparation were analysed. The results showed that the different processing procedures differed in their impact on the degradation of endogenous and transgenic genes and that the transgenic content of the intermediate products and final product was consequently changed. Physical mechanical action such as jordaning caused both endogenous and transgenic genes to degrade but had more effect on the latter. The jordaning process degraded the DNA of endogenous gene from 1883 to 836 bp and that of transgenic gene from 1512 to 408 bp. The adding CaSO₄ process, predominantly a chemical reaction, had less influence on the transgenic gene than on the endogenous gene, the latter being degraded from 836 to 407 bp. The extrusion moulding process resulted in a significant degradation of DNA in transgenic gene from 408 to 190 bp but had no obvious effect on the endogenous gene. After the critical processing procedures of jordaning, syruping, adding CaSO₄ and extrusion moulding during bean curd preparation the transgenic content of the intermediate products and final product was 1.656, 0.435, 1.150 and 0.797% respectively. Copyright © 2007 Society of Chemical Industry