Detection of GMO in food products in Brazil: the INCQS experience

Regulations for the use and labeling of genetically modified organism products and derived ingredients are being implemented worldwide, what demands reliable and accurate methods to detect genetically modified organisms (GMO) in raw materials and food products. This study aimed at monitoring products derived from GMO in the Brazilian market using detection methods for the presence of Roundup Ready soybean, Bt176 and MON 810 maize. The results demonstrate for the first time the presence of GM-soy in Brazilian food products, reinforcing the need for the development of accurate quantitative methods in routine analyses.     

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified soya and maize.     

Monitoring of genetically modified soybean events in sausage products in South Korea

The use of genetically modified (GM) ingredients in various types of food products has increased worldwide. In this study, qualitative real-time PCR assays targeting five GM soybean events (RRS, MON89788, A2704-12, A5547-127, and MON87701) were performed to monitor their use in sausage samples. Thirty sausage samples containing soybean ingredients on their label were collected from Korean food markets and analyzed. The endogenous lectin gene of soybean was used as an internal positive control. Real-time PCR showed that the threshold cycle (Ct) values ranged from 28.62 to 33.90 for the 30 sausage samples. Of the 30 sausage samples, 19 samples were negative for the presence of GM soybeans. In the positive samples, the results revealed three GM soybean events [Roundup Ready Soybean (RRS), A2704-12, and/or MON89788] in 11 of the 30 food samples tested. These results demonstrate that sausage products contain different GM soybean events.     

SIGMO: A decision support System for Identification of genetically modified food or feed products

Since their introduction in 1994, more and more genetically modified (GM) crops are grown worldwide and introduced in food or feed products. In the European Union (EU), the production, trade and marketing of GM products is strictly regulated, but the situation is becoming more complex due to the increasing number and complexity of GM crops, and asynchronic approval procedures with the major GM crop producing countries. Importers and traders are obliged to assess their respective supply chains for the potential presence of authorised and unauthorised GM organisms (GMOs), where wrong decisions may lead to substantial economic losses. This article presents a decision support system SIGMO aimed at guiding producers and traders with the assessment of the likelihood that their products may comprise authorised or unauthorised GM materials. The assessment is based on traceability data about the product (nature and origin of the raw materials, transportation aspects), as well as analytical results of the presence of GMOs in the final product or its ingredients. The approach uses a combination of data-driven and model-driven decision support. SIGMO is composed of (1) a data base providing data about GMO crop species produced and approved in counties worldwide, (2) a multi-attribute model for the assessment of GMO presence in food/feed products, and (3) an on-line user interface. SIGMO helps producers and traders to better comply to valid EU GMO regulations and to better control their products and supply chains in terms of the unintended presence of (unauthorised) GMOs in a cost-effective way.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. An indicator based electrochemical disposable genosensor for the voltammetric detection of NOS-terminator, a genetic element present in GMOs is described as a possible substitute method for the common technique of gel electrophoresis and fluorescent image analysis. The biosensor relies on the immobilization of the 25-mer single stranded oligonucleotides (probe) related to NOS-terminator DNA sequence and the relative binding of this sequence with the polymerase chain reaction (PCR) amplified samples from certified reference material (CRM) of Roundup Ready soybean (Fluka) at a screen printed electrode (SPE). The extent of hybridization between the probe and target DNA is determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), as the hybridization indicator. The difference between the MB signals, obtained from the hybrid modified and probe modified SPEs, is used to detect GMOs from PCR amplified DNA samples. Numerous factors affecting the hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

The implications for mandatory GM labelling under the Consumer Protection Act in South Africa

The South African Consumer Protection Act 68 of 2008 was passed into law, which among other things, mandates the labelling of genetically modified (GM) ingredients in packed goods. The aim of this study was to evaluate the potential impact of mandatory GM food labelling in terms of the Consumer Protection Act by determining what products currently on the market would be implicated. A total of 46 food products from different companies was selected and sampled randomly with an emphasis on those containing canola, maize and soybean, since GM varieties for these crops have been approved in South Africa in terms of the Genetically Modified Organisms (GMO) Act of 1997. The products were screened for the presence of genetic modification and, if positive, quantified. Genetic modification was detected in 50% of products tested, including seven out of 14 (50%) products labelled to indicate an absence of genetic modification. The results from this study indicate that at a 5% GM labelling threshold, 19 out of the 46 products tested would need to be labelled for their GM content. Of the 14 products labelled to indicate an absence of GM, five exceeded the 1% threshold for non-GM labelling. Many companies are concerned about the cost effectiveness of mandatory labelling. However, the Consumer Protection Act makes a provision for a “may contain” clause that if used relieves the producer of the financial considerations to verify the GM content of a product. Although the Consumer Protection Act does not make any provision for monitoring, consumers or consumer groups have recourse against companies in contravention of the Consumer Protection Act through the provision of the National Consumer Commissioner (NCC).     

Validation of a DNA biochip for species identification in food forensic science

Authentication of food product ingredients by food control agencies relies on analytical laboratory methods for animal species identification. Such methods must be validated if their results are to be used as acceptable evidence in a court of law. Following the validation guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), we carried out a validation process for the Low Cost and Density (LCD) Array (MEAT 5.0 version) kit for food forensics based on a DNA biochip technology that simultaneously detects 24 animal species. Mixtures of the animal species the kit is designed to detect were prepared at different concentrations and tested on raw/pasteurized, and heated meat and milk matrices. The LCD Array showed high specificity and high sensitivity and it appeared to be robust and repeatable. As such, it provides a useful tool for detecting common adulterants in food items of animal origin.     

Detection strategies for food authenticity and genetically modified foods

Analytical methods for authenticity testing have been described for all types of food and can give us important indications for analytical strategies to be developed for the detection and quantitation of genetically modified foods. Transgenic plants contain newly introduced traits or marker genes that are expressed and should be detectable by DNA or protein-based methods. Recent literature clearly favours PCR as the method of choice for the identification of GMO-derived food. Quantitative competitive PCR is an important extension of this method allowing to control labelling and maximum limits to be set for genetically modified crops in non-GMO-crops.     

Effect of polyphosphates on the quality of frozen light salted cod (Gadus morhua L.) fillets

Frozen light salted fillets have been subject to discussion concerning their definition as foodstuff. EU has recently placed these products in the frozen category. The aim was to investigate how a commercial polyphosphate blend affected the quality of light salted fillets. Fresh and previously frozen fillets of cod were injected with a brine containing 0, 4.5, 9 or 18 g/L of Carnal 2110. Quality, chemical characteristics and phosphate residues were analyzed in fillets. Compared to previously frozen, light salted fillets from fresh raw materials had a whither and less yellow color while significantly lower yields and oxidation levels were registered. No differences were found in drip loss during thawing. Addition of polyphosphate contributed to increased whiteness of both frozen and thawed fillets from both raw material groups, while no effects on yields or drip loss were detected. Polyphosphate reduced oxidation only in the frozen raw material group. Total phosphate contents were higher in fillets from fresh than frozen raw materials and residues were mainly detected as monophosphate. The choice of raw material will affect the end quality and yields and results suggest that polyphosphate can improve light salted fillet quality and therefor may be defined as a food additive.     

Evaluation of LabChip technology for GMO analysis in food

Ongoing technological advances permit the development of new scientific strategies for bio-analysis. The work described here sought to compare a traditional agarose slab gel electrophoresis method combined with digital image analysis against a new microfluidic capillary electrophoresis system (LabChip^TM, Agilent Technologies) to assess their relative performance in the quantitative analysis of polymerase chain reaction (PCR) products. The comparative strategy exploited methodology for providing quantitative genetically modified organism (GMO) determinations in food samples and demonstrated that the LabChip^TM system offered improvements in quantitation accuracy, objectivity and ease of use.     

Detection of sixteen genetically modified maize events in processed foods using four event-specific pentaplex PCR systems

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.     

Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed

A diverse range of genetic elements has been used to develop genetically modified organisms (GMOs) over the last 18 years. Screening methods that target few elements, such as the Cauliflower Mosaic Virus 35S promoter (P-35S) and Agrobacterium tumefaciens nopaline terminator (T-nos), are not sufficient to screen GMOs. In the present study, a multiplex PCR system for all globally commercialized GM soybean events was developed to easily trace the events. For this purpose, screening elements of 24 GM soybean events were investigated and 9 screening targets were selected and divided into three individual triplex PCR systems: P-35S, ribulose-1,5-bisphosphate carboxylase small subunit promoter of Arabidopsis thaliana, T-nos, T-35S, pea E9 terminator, open reading frame 23 terminator of A. tumefaciens, proteinase inhibitor II terminator of potato, acetohydroxy acid synthase large subunit terminator of A. thaliana, and the revealed 3′ flanking sequences of DP-305423-1. The specificity of the assays was confirmed using thirteen GM soybean events as the respective positive/negative controls. The limit of detection of each multiplex set, as determined using certified reference materials of specific GM events, ranged from 0.03 to 0.5%, depending upon target. Furthermore, 26 food samples that contained soybean ingredients, which were purchased from the USA, China, Japan, and Korea, were analyzed, 17 of which contained one or more GM soybean events. These results suggest that the developed screening method can be used to efficiently track and identify 24 GM soybean events in food and feed.     

Food safety challenges associated with traditional foods in German-speaking regions

The “German-speaking region” in Central Europe is characterized by a large variety of regional food specialities and long tradition in craftsman's skills and experience in safe small-scale food processing and preservation. There is also increasing interest in these traditional products in other countries. Hence, this paper discusses the properties of characteristic food products, and outlines the steps critical for their safety. Such foods include fermented milk products (in particular cheeses), meat and fish products, fermented vegetables, and baked goods such as sourdough breads and spiced cookies. Data analysed show, among others, that (1) hard cheeses made from raw milk are regarded as safe, due to effective hurdles which eliminate foodborne pathogens during production and ripening, and semi-hard cheeses made from raw milk – which are generally ripened for more than 60 days – exhibit only a low health risk if Good Manufacturing Practice (including mastitis control) and effective HACCP systems (e.g. control of starter activity) are implemented; (2) Listeria monocytogenes is not a hazard specific to products from raw milk since it may grow on the surface of smear- and mold ripened cheeses after recontamination, and can be effectively controlled by monitoring systems including environmental samples; (3) semi-dry and dry sausages have a favourable record of safety whereas, in the manufacture of some undried, spreadable types, salmonellae (especially in pork sausages) and Shiga toxin-producing Escherichia coli (STEC; especially in sausages containing meat from ruminants) are frequently reduced only by one log cycle or less. Hence, the safety of these products critically depends on the quality of the raw material. This stresses the need of implementing Good Manufacturing Practice also in traditional processes.     

Challenges for methods to detect genetically modified DNA in foods

Qualitative detection methods for genetically modified (GM) DNA sequences in foods have evolved fast during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, in future, quantitative results about the fraction of GM material in a composite food will be needed and the fast increasing number of GM foods on the market demands the development of more advanced multi-detection systems. Other challenges and problems might arise from the decreasing relevance of methods which screen for sequences commonly found in GMOs, the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardisation procedures and the need to up-date continuously databases comprising commercially available GM foods and the respective detection strategies.     

硅藻土的改性处理及合成NaY分子筛的初步研究

对长白山硅藻土进行了改性处理,应用X射线衍射（XRD）、傅里叶变换红外光谱（FT-IR）、扫描电镜（SEM）表征手段考察了长白山硅藻土和改性硅藻土经不同温度热处理后的结构变化。并对改性硅藻土原位晶化合成NaY分子筛进行了初步的探索。结果表明,长白山硅藻土转变为方英石的相变温度为1100℃,改性硅藻土的相变温度为950℃。这种物相转变温度的差异,主要与Na等杂质元素的存在有关。利用改性硅藻土通过原位晶化方法合成出了结晶度较高的NaY分子筛。     

Reform of food regulation in Australia and New Zealand

Australia and New Zealand agreed to establish a joint system for developing food standards in 1996. The establishment of the joint system provided the opportunity to reform the food regulatory requirements for composition, labelling and contaminants, culminating in the development of the Australia New Zealand Food Standards Code. In addition to the development of the joint food standards system, several other regulatory reforms have occurred over the same period. These include the establishment in Australia of a national food safety regulatory system, and in both countries, a shift towards the pre-market safety assessment and approval of certain whole foods, such as genetically modified foods. The impact of the regulatory reforms is subject to an ongoing program of evaluation.     

Survey of undeclared allergenic pistachio (Pistacia vera) in commercial foods by hydrolysis probe real-time PCR

Tree nut allergies represent an important health problem in industrialized countries. Among these, pistachio (Pistacia vera) kernels which are consumed as snack foods and used as ingredients in confectionery, chocolates, meat products, and ice-cream industries have been reported to cause IgE-mediated allergic reactions. Trace amounts of undeclared pistachio allergens can cause serious health risks for food-allergic consumers. In order to provide an appropriate method for the detection of pistachio in food products, a real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of pistachio was developed. The sensitivity was investigated on spiked wheat flour samples with defined raw and heat-treated pistachio contents (0.1–100,000 mg kg⁻¹). The real-time PCR detected pistachio in these mixtures down to the lowest investigated spike level of 0.1 mg kg⁻¹. In addition, analysis of different retail samples from the market was performed to demonstrate the suitability of the assay in the food industry. The real-time PCR results obtained from the analysis of 229 commercial food products revealed 29 that didn't declare pistachio or traces on the label but were found to contain pistachio. The presented real time PCR method is useful for relatively fast, highly selective, and sensitive detection of pistachio in food samples.     

Microbial composition of turbid rice wine (Makgeolli) at different stages of production in a real processing line

The aim of this study was to examine for changes in microbial populations during the production of turbid rice wine (also known as Makgeolli) at different production plants and to identify critical points for the control of microbial quality. Samples from raw ingredients (water, rice and wheat flour), materials from different production stages (steamed ingredients, the base of fermentation, primary and secondary fermentation stages at different time points), and the final rice wine products (non–sterilized and sterilized) were analyzed. The microbiological content of samples was assessed by quantitative (aerobic plate counts, lactic acid bacteria, fungi, acetic acid bacteria, coliforms and Bacillus cereus) and qualitative (Escherichia coli and eight foodborne pathogens) analyses. Aerobic plate counts for rice and wheat flour were relatively low (3.1 and 1.9 log CFU/g, respectively), as were those for lactic acid bacteria (1.6 and 2.1 log CFU/g), and fungi counts (2.2 and 0.7 log CFU/g). All counts increased to 7.8–7.9 log CFU/ml in the base of fermentation after the addition of Nuruk and Koji, and these counts were maintained at 8–9 log CFU/ml during fermentation. Acetic acid bacteria were not detected in the ingredients, but were isolated from the base of fermentation (1.2–2.8 log CFU/ml). Heat sterilization reduced aerobic pate counts significantly from 8.4 to 2.1 log CFF/ml, and lactic acid bacteria, fungi and acetic acid bacteria were reduced to non-detectable or negligible levels. Isolated microorganisms were considered as autochthonous to the environment or were artificially introduced with the starter culture. B. cereus was widely distributed throughout the manufacturing process, and may have been introduced from the raw material (present in 100% rice and 41.7% wheat flour samples). B. cereus counts were not significantly affected by sterilization, suggesting that it may exist at low levels as spores in the final products. No coliforms and other pathogens were detected in any samples. The raw materials, the base of fermentation, and sterilization stage were identified as important points for the control of microbial quality during the fermentation process.     

Improving the control of food production in catering establishments with particular reference to the safety of salads

Food production in four school kitchens was checked in order to improve food safety by establishing a self-regulated control system based on good manufacturing practices (GMPs) and as an introduction to hazard analysis and critical control points (HACCP). A form, which referred to different aspects such as the cleanliness of the installations, personnel hygiene and the prevention of cross-contamination, was used to obtain the necessary data. Furthermore, foods thought to be of high risk were periodically collected for microbiological analysis. Samples for microbiological examination were taken from cutting boards, tables, machines, knives and ingredients (on-line sampling). We used the results as a basis to train foodhandlers to improve the safety of salad preparation, in accordance with GMPs, observing that hygiene improvement depended on chlorine levels in the rinsing water. We also designed several controls for raw materials, cold storage, freezers and available chlorine levels in water. At the end of the study period, we observed a decrease in microbial populations of examined samples, which indicated that the knowledge of hygiene practices on the part of foodhandlers represents a critical control point, as defined by the EC Directive 93/43/EEC on Hygiene of Food Stuffs.