The effect of glyphosate on digestion and horizontal gene transfer during in vitro ruminal fermentation of genetically modified canola

BACKGROUND: The impact of glyphosate on ruminal fermentation, selective pressure on ruminal bacteria and horizontal transfer of the gene encoding 5‐enolpyruvylshikimate‐3‐phosphate synthase (epsps) to ruminal bacteria was studied using batch culture with glyphosate‐tolerant (Roundup Ready^®) canola meal as substrate. RESULTS: A glyphosate concentration × time interaction (P < 0.05) occurred when glyphosate (0–100 mmol L^?1) was included in in vitro ruminal incubations with a diet containing 150 g kg^?1 Roundup Ready^® canola meal (Experiment 1). Glyphosate at 50 and 100 mmol L^?1 inhibited fermentation. In Experiment 2, epsps fragments were detected in plant debris for up to 16 h of incubation using primer sets that amplified three fragments (62, 108 and 300 bp) of DNA spanning the transgenic construct. Persistence was affected by fragment size but not by glyphosate concentration (0, 10 or 60 mmol L^?1). Extensive polymerase chain reaction assays provided no evidence of acquisition of epsps by feed‐ or fluid‐associated bacteria during fermentation. A glyphosate concentration × time interaction (P < 0.05) was observed for all fermentation parameters measured, and glyphosate caused a general inhibition of fermentation. CONCLUSION: The presence of glyphosate did not increase selective pressure for gene transfer of DNA encoding glyphosate resistance from Roundup Ready^® canola meal to ruminal bacteria. Copyright © 2007 Society of Chemical Industry     

如何做好能力验证样品中转基因成分检测

目的 能力验证是利用实验室间比对来确定实验室检测或校准能力的活动, 是提高检验机构内部质量控制最有效的手段之一。方法 通过参加2012 “CNAS T0659大豆粉中转基因成分定性检测”能力验证获得满意结果, 对实验室参加能力验证过程中应注意的问题做简要分析。结果与结论 标准方法的选择, DNA提取效率, 以及实验过程的质量控制等都是影响能力验证结果的重要因素。     

食用大豆油中转基因成分的检测

食用油脂中转基因成分的检测是当前食品检验工作的一个主要方面。针对食用油脂中DNA含量极低、DNA序列片段短、破坏严重的特点,建立了食用油脂中DNA提取方法,通过实时荧光PCR可检测出大豆内源基因(Lectin)以及外源抗除草剂基因EPSPS,为食用油脂进行核酸类生物性检测提供了一种简捷有效的方法。     

转基因大豆油的安全性研究--动物实验

以转基因大豆油为原料,对其在急性毒性实验、长期毒性实验、病理组织学检查等几方面进行安全性的研究,结论显示转基因大豆油在中长期内食用是安全的,但还需要对慢性毒性实验以及不同加工工艺下的产品进行进一步的验证.     

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.     

Advances in the in vitro digestion and fermentation of polysaccharides

In vitro digestion models are widely used to study the structural changes, digestion and release of food components under simulated gastrointestinal conditions. As compared to the in vivo digestion tests, the in vitro digestion reflects the digestion and utilisation of food after ingestion and has the advantages of being time consuming, inexpensive, reproducible and free from moral and ethical restrictions. This study reviewed the current research studies on the in vitro simulated digestion of polysaccharides conducted in the last 5 years and focused on the oral, gastric and intestinal digestion models, with the aim of providing a basis for the further testing of changes in the content, structure and active ingredients of polysaccharides before and after digestion.     

PCR法定性检测大豆食用油中转基因成分

针对大豆食用油中核酸含量低且被严重破坏、DNA难以提取的问题,对CTAB法进行优化,有效从食用油中提取到可用于PCR扩增反应的DNA模板。同时定性检测出大豆内源基因lectin,外源调控序列启动子CaMV35S、终止子Nos及目的基因CP4-EPSPS序列,成功建立了基于PCR技术在大豆食用油中快速检测转基因成分的方法。     

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.     

Effects of water-soluble soybean polysaccharide on rheological properties,stability and lipid digestibility of oil-in-water emulsion during in vitro gastrointestinal digestion

Water-soluble soybean polysaccharide (SSPS) is a naturally occurring emulsifier. SSPS was used as the sole emulsifier to stabilize an oil-in-water (O/W) emulsion. The effects were investigated of different SSPS concentrations (3–20% (w/w)) on the lipid digestibility, rheological properties and stability of O/W emulsions during in vitro digestion model. The droplet size of the emulsions tended to increase during the oral phase because the emulsions were unstable and droplets coalesced, except with a SSPS concentration of 20% (w/w). The presence of SSPS markedly reduced the free fatty acid (FFA) content after its stabilized O/W emulsion passed through in vitro gastrointestinal digestion. The amount of FFA significantly decreased as the concentration of SSPS increased due to SSPS stabilization film on oil droplet surface and high viscous system. SSPS may be an attractive alternative ingredient to control the lipid digestibility of emulsions for various food products.     

广州市售豆制品转基因成分的检测与分析

为了解广州市市售豆制品的转基因情况,对广州市售散装豆制品和预包装豆制品分别进行抽样检验.采用改良十六烷基三甲基溴化铵（CTAB）法提取大豆DNA,利用核酸定性PCR、实时荧光PCR及环介导等温扩增技术（LAMP）对外源基因CaMV5S,NOS和EPSPS进行检测.调查共抽检豆制品207份,研究结果表明,在检测的159份散装豆制品中,87份检出转基因成分;在48份预包装豆制品中,18份检出转基因成分.散装豆制品无任何食品标签,而预包装豆制品均无转基因食品标识.     

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     

转基因大豆DNA检测芯片的研究

为提高对转基因大豆的监督检测能力，研制了转基因大豆DNA检测芯片。根据转基因大豆(Roundup Ready)中所转入的外源基因，选择CaMV35S启动子、NOS终止子、NOSIEPSPE基因和内源Lectin基因设计特异性引物，采用多重PCR法对待测样品进行扩增，通过缺口平移法合成DIGdUTP标记杂交探针，并制备基因芯片。在对PCR反应和扩增产物与芯片杂交条件进行优化的同时，比较了芯片检测的特异性和重复性，并对检测的灵敏度进行测试。结果表明，该方法具有较好的特异性和重复性，检测灵敏度可达0．5％，由于采用了多重PCR技术，一次可同时检测多个基因，提高了检测的准确性和效率。     

大豆转基因成分测定能力验证结果与分析

目的进一步提高实验室对转基因成分的检测水平,提升检验人员的专业素养。方法根据能力验证计划作业指导书中推荐使用的GB/T 19495.5-2004《转基因产品检测核酸定量PCR检测方法》中提供的3种方法分别对样品进行检测。结果样品013和083为GTS-40-3-2品系转基因大豆,142为非转基因大豆。能力验证获得满意结果。结论转基因成分检测应重点关注DNA提取效率和实验过程质控等因素。     

Analysis of physiochemical composition and antioxidant properties between hulls of the genetically modified glyphosate-tolerant soybean and northeast soybean

In this study, the physiochemical and antioxidant properties of the soybean hulls from the genetically modified glyphosate-tolerant soybeans (line 40-3-2) and local cultivar northeast soybeans were investigated. The levels of fat, total phenolic, total extractable pectin and soluble dietary fiber in northeast soybeans hulls were less than that in glyphosate-tolerant soybeans hulls, respectively. The antioxidant capacity of total phenolic, water soluble pectin, and soluble dietary fiber showed that DPPH free radical scavenging activities of glyphosate-tolerant soybeans hulls were 118.23, 57.34 and 197.22 μg AAE/g, which were 2.3, 1.2 and 9.4 times of northeast soybeans hulls, respectively (p < 0.05), and FRAP of glyphosate-tolerant soybeans hulls were 401.67, 747.51 and 328.53 μg AAE/g, which were 1.8, 8.7 and 4.8 times of northeast soybeans hulls (p < 0.05). Glyphosate-tolerant soybeans hulls extract showed the stronger antioxidant activity, which was positively correlated with total phenolic content (r = 0.890, p = 0.001). It provides evidence on developing value-added utilization of hulls, soybean processing by-products, as nutraceuticals or functional food ingredients.     

The impact of non- and genetically modified soybean diets in aorta wall remodeling

The aim of this study was to evaluate the influence of nongenetically modified soybean (non-GMS) and genetically modified soybean (GMS) meal on growth and cardiometabolic parameters in rats. Thirty male Wistar rats were divided into 3 groups (n= 10): non-GMS, GMS, and control group (CG). All animals received water and an isocaloric diet ad libitum for 455 d. Blood was drawn by cardiac puncture, and serum was separated for subsequent biochemical analyses (total cholesterol, triacylglycerols, insulin, glucose, and testosterone). The aorta was quickly harvested and fixed; the body fat mass was removed and weighed. Non-GMS and GMS had a growth index (GI) similar to CG but with a lower body weight (P < 0.05) and a lower amount of body fat mass (P < 0.05). Total cholesterol, triacylglycerol, glucose concentrations, and aortic tunics were reduced (P < 0.05) in non-GMS and GMS compared to CG. Non-GMS and GMS are able to reduced serum cholesterol, triacylglycerols, glucose, and aortic remodeling in aged rats. No differences were observed between non-GMS and GMS in all parameters.     

聚合酶链式反应方法检测豆制品中转基因成分的研究概述

大豆作为种植极广的农作物,在人们的生活中占据了重要位置,能加工成多种多样的产品,满足日常需要。 随着生物技术的 快速发展,转基因大豆以其高产量、低成本的优势迅速占领市场。 由于转基因产品的安全性未知,各国对其用途都有严格规定和监 管。该文从我国大豆制品的市场状况、核酸的残留、现行检测标准及实验过程中存在的问题等方面进行了总结和讨论,以期对转基因 产品的市场监管检测体系能够有所裨益。     

Effect of rabadi fermentation on phytic acid and in vitro digestibility of barley

Rabadi, an indigenous fermented food, was prepared by mixing cereal flour with buttermilk, allowing it to ferment at 30, 35 and 40 °C for 6, 12, 18, 24 and 48 h and cooking the fermented mixture for 0.5 h with continuous stirring. Two types of rabadi were prepared i.e. autoclaved and unautoclaved. In autoclaved type of rabadi cereal flour was mixed with water, autoclaved (0.103 MPa = 15 psi for 15 min), cooled, mixed with buttermilk and fermented. As this type of rabadi was precooked prior to fermentation, hence, the fermented product did not require cooking afterwards, while in unautoclaved rabadi, barley flour and buttermilk were mixed, fermented and then cooked prior to consumption. Phytic acid was reduced drastically at all the temperatures and periods of fermentation in both autoclaved and unautoclaved type of rabadi; greater reduction occurred at higher temperature and duration of fermentation. A significant improvement in the in vitro digestibility of starch and protein was observed; maximum improvement was noticed when fermentation was carried out at 40 °C for 48 h in both the types of rabadi. Phytic acid had a significant (P < 0.05) negative correlation with digestibility (in vitro) of proteins and starch of barley flour rabadi.     

In vitro fermentation vessel type and method alter fiber digestibility estimates

Selected vessel types and conditions used for in vitro fermentation were compared to evaluate their effects on determinations of neutral detergent fiber (NDF) digestibility (NDFD) in 2 replicate 48-h fermentations. Treatments included 50-mL polyethylene centrifuge tubes with gas-release valves (treatment 1); 50-mL polyethylene centrifuge tubes with continuous gassing with CO₂ (treatment 2); 50-mL polyethylene centrifuge tubes sealed, oriented horizontally, and shaken continuously parallel to the long axis of the tube, with manual gas release (treatment 3); 125-mL Erlenmeyer flasks with continuous gassing with CO₂ (treatment 4); and 125-mL serum vials sealed with stoppers and crimp seals with (treatment 5) or without (treatment 6) manual gas release. Goering and Van Soest medium and blended ruminal inoculum from 4 lactating cows were used. Substrates were alfalfa hay, corn silage, ryegrass hay, and soyhulls. Gas was released and measured in treatments 3 and 6 at 3.0, 5.5, 9.0, 11.5, 23.5, 29.5, and 47.5 h by using a syringe with a hypodermic needle. Vessels from each treatment were harvested at 0, 6, 12, 24, 30, and 48 h for NDF analysis, with NDFD calculated as 1 - [(residual NDF, g - residual NDF in fermentation blank, g)/sample NDF, g]. Medium pH did not decline below 6.3 for any treatment. Average values for NDFD for 24 through 48 h were 0.576, 0.639, 0.688, 0.668, 0.679, and 0.681 for treatments 1 through 6, respectively (standard error of the difference = 0.008). The lowest NDFD was noted for treatment 1, which differed from all other treatments; treatments 3, 4, 5, and 6 did not differ by treatment or by the interaction of treatment and substrate. Treatments 1 and 2 gave lower NDFD values than the other treatments, but these differences were not consistent and differed by substrate, with alfalfa showing the fewest differences among treatments and soyhulls the most. Net energy of lactation values for substrates, as predicted from differences in 48-h NDFD, were 7 to 15% lower for treatment 1 than for the average of all other treatments. Slopes of the gas production per gram of substrate dry matter curves differed between treatments 3 and 5. In conclusion, measured NDFD was altered by fermentation treatment, with polyethylene tubes + gas-release valves giving the lowest values. Consequently, NDFD values may not be comparable across fermentation methods, but the effect will vary among feedstuffs. The combination of methods used for sealing, gassing, or agitating vessels may have a greater impact on NDFD than does vessel type.     

Monitoring genetically modified soybean along the industrial soybean oil extraction and refining processes by polymerase chain reaction techniques

In the present work, the extraction and detection of DNA along a complete industrial soybean oil processing chain was described to monitor the presence of Roundup Ready^® (RR) soybean. The analysed samples comprised all the steps prior to industrial oil extraction, namely, raw, cracked, laminated and expanded seeds, and the defatted flour as a sub-product. The samples collected at the refining unit included the crude oil, degummed/neutralised, washed, bleached and deodorised oil, as final product. The amplification of soybean lectin gene by end-point polymerase chain reaction (PCR) was successfully achieved in all the steps of extraction and refining processes, until the fully refined soybean oil. The amplification of RR soybean by PCR assays using event-specific primers was also achieved for all the extraction and refining steps, except for the intermediate steps of refining (neutralisation, washing and bleaching) possibly due to sample instability. The real-time PCR assays using specific probes confirmed all the results and proved that it is possible to detect and quantify genetically modified organisms in the fully refined soybean oil. To our knowledge, this has never been reported before and represents an important accomplishment regarding the traceability of genetically modified organisms in refined oils.     

热处理对过氧自由基氧化米糠蛋白体外胰蛋白酶消化性质及消化产物抗氧化性的影响

选用不同浓度的2,2’-盐酸脒基丙烷(2,2’-azobis (2-amidinopropane) dihydrochloride,AAPH)在有氧条件下热降解生成的过氧自由基氧化米糠蛋白,再通过95℃水浴处理氧化米糠蛋白,研究热处理对过氧自由基氧化米糠蛋白体外胰蛋白酶消化性质及消化产物抗氧化性的影响。结果表明:随着AAPH浓度的增加,过氧自由基氧化米糠蛋白体外胰蛋白酶消化率、初始消化速率、消化产物分子质量分布在500～1 500 u的肽含量、消化产物清除ABTS~+·、·OH、O~-_2·能力和还原能力均先上升后下降,消化产物清除DPPH·能力和金属螯合能力先不变后下降;而热处理后,氧化米糠蛋白体外胰蛋白酶消化产物金属螯合能力先上升后下降,ABTS~+·清除能力和还原能力先不变后下降。同未热处理相比,热处理显著提高了相同氧化程度下米糠蛋白体外胰蛋白酶消化率、初始消化速率以及消化产物抗氧化性。表明过氧自由基氧化会改变米糠蛋白体外胰蛋白酶的消化性质,而热处理可以改善相同氧化程度下米糠蛋白体外胰蛋白酶的消化率和消化产物的抗氧化性。