Traceability of genetically modified Roundup Ready soybean: A case study on sampling and analytical uncertainty along processing chain

A study on the fate of Genetically Modified (GM) Roundup Ready soybean (RRS) was undertaken on the following products: flour, protein flour, lecithin, crude and refined oil, broken grain, hull and expander of an industrial soybean manufacturing plant, with the aim to evaluate the possible effects of processing on the reliability of control plans. A sampling control plan was applied to all the products of the industrial manufactory plant. The best sampling point was identified based on the lowest impact of the analytical and sampling uncertainty.The best “fit for purpose” sampling point for the accurate evaluation of the Genetically Modified Organism (GMO) concentration measurement was identified in the processed products, e.g. flour and protein flour, thanks to the homogeneity on RRS in the batch and the better yield and quality of the extracted DNA.This study presents a practical approach to assess the two main factors that affect the reliability of the control plans: analytical and sampling uncertainty. The work was undertaken on GM soybean derived products, nevertheless the conclusions we reached could be also applied to verify compliance with GMO labelling threshold.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.     

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.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Prevalence of Listeria spp. in ready to eat foods (RTE) from Algiers (Algeria)

The aim of this study was to establish the occurrence of Listeria spp., especially Listeria monocytogenes in ready to eat RTE food marketed in Algiers (Algeria).A total of 227 samples were collected from different producers and retailers.All samples were analyzed using a conventional cultivation method AFNOR V08-055.Out of 227 samples tested, 21 (9.3%) tested positive for Listeria spp. among them, 6 (2.6%) tested positive for L. monocytogenes. L. innocua was the most common Listeria species found being detected in 11 samples (4.8%), although both Listeria ivanovii and Listeria welshimeri were detected in 3 (1.3%) and 1 (0.4%) food samples respectively.The study of the antimicrobial sensitivity of Listeria monocytogenes strains showed no resistance.The study has enabled us to detect these contaminants in a wide range of RTE foods, to suggest that contamination likely occurs after heat treatment, and to assess the danger represented by this category of food for populations at risk.     

Detection of Norovirus and Feline Calicivirus in spiked molluscs subjected to heat treatments

Aim of the study was the evaluation of the effect of heat treatment in shellfish experimentally contaminated with human Norovirus (NoV). Feline Calicivirus (FCV), often used as surrogate for human NoV, was examined in parallel to test for virus infectivity after treatment. The experiments were performed subjecting suspensions and spiked mussels to heat treatment (60 °C and 80 °C) for various times. Analysis by rRT-PCR showed a limited reduction of NoV (less than 1 log RT-PCR units ml⁻¹) and FCV (0.3-1.2 log TCID₅₀ ml⁻¹) both in virus suspensions and in spiked mussels, with the exception of NoV suspensions treated at 80 °C (reduction of 3 log RT-PCR units ml⁻¹). Cell culture assay showed that the infectious FCV in suspensions decreased of 4 log after 3 min of treatment while a lower reduction (2 log) was obtained in spiked samples treated for 15 min. Data showed that mussel matrix plays a protective role against heat inactivation of viruses. Despite the efficacy of rRT-PCR for viral nucleic acid detection, its inability to provide information on virus infectivity, represents a limit to the evaluation of product safety. Caution should therefore be used in the evaluation of efficacy of heat treatments on virus-contaminated foods and when the judgment of food product safety is based exclusively on rRT-PCR results.     

Real-time PCR with internal amplification control for the detection of Cronobacter spp. (Enterobacter sakazakii) in food samples

Cronobacter spp. (Enterobacter sakazakii) is an opportunistic pathogen and is linked with life-threatening infections in neonates. The organism has been isolated from a wide variety of foods and environments. In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Cronobacter spp. in foods. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and 67 bacterial strains, including four strains of Cronobacter spp., were evaluated. All Cronobacter strains were successfully identified, however no cross-reactivity was observed with non-Cronobacter strains. Detection limit of the assay in pure culture and formula infant without enrichment was 1.2 × 10³ CFU/ml (1.2 × 10¹ CFU/assay). After 24 h enrichment in broth, as few as 10⁰ CFU/ml or g of Cronobacter could be detected in artificially contaminated food samples (infant formula, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of 10⁸ CFU/ml Salmonella typhimurium in another analysis. A total of 92 food samples were analyzed for the presence of Cronobacter, out of which two pork samples were found as positive by real-time PCR, whereas only one was detected by the ISO standard method. The adjusted real-time PCR assay can be adopted to rapidly detect Cronobacter spp. in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.     

Effectiveness of combined Pulsed Electric Field (PEF) and Manothermosonication (MTS) for the control of Listeria innocua in a smoothie type beverage

The combination of novel, non-thermal technologies for preservation purposes is a recent trend in food processing research. The objectives of the current study were (i) to optimise PEF or MTS treatment conditions which would achieve a maximum reduction of up to 3 log cycles of Listeria innocua in a milk based smoothie, when these technologies were applied individually, and (ii) to investigate possible additive or synergistic effects of the combined technologies. Microbiological analysis was performed by inoculating the smoothie with L. innocua and enumerating populations pre- and post-processing. All technologies applied within combinations significantly reduced L. innocua in the smoothie, when compared to untreated controls (p ≤ 0.0001). The sequence in which the MTS and PEF were applied was found to have a significant impact on the level of microbial reduction achieved (p ≤ 0.05). The sequence of MTS followed by PEF was the most effective in inactivating L. innocua achieving a mean reduction of 5.6 log cfu/ml, thereby exceeding the 5 log cycles minimum requirement specified by the United States Food and Drug Administration (US FDA). Significantly (p ≤ 0.05) lower reductions of 4.2 log cfu/ml were achieved when the PEF + MTS sequence combination was applied. The combination of MTS + PEF achieved inactivation comparable to thermally treated samples (p > 0.05). This study has shown the combination MTS + PEF is a promising hurdle preservation approach to control undesirable microorganisms in milk based smoothie beverages.     

Identification of the wedge clam Donax trunculus by a simple PCR technique

The wedge clam Donax trunculus is an important bivalve commercial species in Portugal which can be easily mistaken with other three morphologically similar species (Donax semistriatus, Donax vittatus and Donax variegatus) that have a lower market price. This may lead fish sellers to make false claims about the authenticity of their products in order to get higher profits. To overcome this problem it is important to develop analytical techniques that can be used to test the authenticity of the species that is being sold. In this study we present two DNA extraction methodologies and a simple PCR method for the accurate identification of D. trunculus based on the amplification of the nuclear marker 5S rDNA. The PCR amplification results showed that this method is reliable to differentiate D. trunculus and D. variegatus from the remaining Donax species, since fragments of D. trunculus were about 275-300bp while D. variegatus were about ∼450 bp a little lower molecular weight than DNA fragments of the other two species (∼500 bp).     

Influence of sunflower oil based nanoemulsion (AUSN-4) on the shelf life and quality of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C

The influence of nanoemulsion (AUSN-4) on the microbiological, proximal, chemical, and sensory qualities of Indo-Pacific king mackerel (Scomberomorus guttatus) steaks stored at 20 °C was studied for a time period of 72 h. AUSN-4 treatment showed initial reduction (P > 0.05) in the heterotrophic, H₂S and lactic acid bacterial populations in 12 h, followed by a gradual increase in their respective populations. Irrespective of treatments, reduction in total carbohydrate, protein, and fat contents were observed in all samples with an increase in storage time (h), with AUSN-4 treated steaks having the lowest reduction. AUSN-4 treatment significantly (P < 0.05) decreased the values of chemical indicators of spoilage throughout the storage period. Organoleptic evaluation revealed that AUSN-4 treated steaks showed an extension of shelf life of 48 h, when compared with control and antibiotic treated samples, respectively. Based on the results obtained in our present study we conclude that sunflower oil based nanoemulsion preservative technique is able to extend the shelf life and maintain the quality of S. guttatus steaks during storage.     

Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures

Natural antimicrobials are being more and more considered as alternative approach for controlling growth of microorganisms in food. The objective of this study was to evaluate the pomegranate extract’s (PE) potential to be used as a natural preservative in ready to eat meats. Listeria monocytogenes was the main target. In a preliminary assessment with the disk diffusion method PE showed inhibitory effect against all five tested species, in the following order of increasing sensitivity: L. monocytogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli and Staphylococcus aureus.No viable cells of L. monocytogenes were detected after incubation in BHI broth in presence of 7.5% v/v of the liquid PE (or 24.7 mg dry PE/ml). This concentration was considered as the Minimal Bactericidal Concentration (MBC) of the tested PE. Two pure components commonly found in PE, namely gallic and ellagic acids were also tested in BHI broth, however they did not show considerable inhibition of L. monocytogenes. PE in a concentration equal to the measured MBC was tested against L. monocytogenes in meat paté at different temperatures. At 4 °C during 46 days the extract inhibited the growth in meat paté by 4.1 log CFU/g compared to the control, which had reached log 9.2 CFU/g already on the 18th day. Inhibition was less pronounced at higher temperatures. The results indicate that the PE has a potential to be used as a natural preservative in meat products.     

Authentication of meat and commercial meat products from common pigeon (Columba livia) woodpigeon (Columba palumbus) and stock pigeon (Columba oenas) using a TaqMan real-time PCR assay

A species-specific real-time polymerase chain reaction (PCR) assay using TaqMan^® probes has been developed for the identification of meat and meat products from common pigeon (Columba livia), woodpigeon (Columba palumbus) and stock pigeon (Columba oenas). The method combines the use of species-specific primers and TaqMan^® probes that amplify small fragments (amplicons < 200 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141 bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species, demonstrated the suitability of the assay for the detection of the target DNAs. The PCR assay reported in this work could be useful in inspection programs to verify the correct labelling of raw and heat-treated pigeon meat products.     

Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.     

Comparison of intense pulsed light- and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157:H7

The purpose of this study was to compare the degree of microbial inactivation and cell damage induced by intense pulsed light (IPL) and short-wavelength ultraviolet (UVC) in Listeria monocytogenes and Escherichia coli O157:H7. The viability of the food-borne pathogens treated with IPL and UVC (254 nm) decreased exponentially with treatment time. Particularly dramatic reductions in L. monocytogenes and E. coli O157:H7 were observed for IPL treatments at energy densities of 376 and 455 W/m², with an approximately 7-log reduction for a treatment time of 60-180 s. Also, a 4-log reduction of L. monocytogenes and a 5-log reduction of E. coli O157:H7 were achieved with UVC irradiation for 1200 s. The types and amounts of IPL- and UVC-induced DNA damage in both microorganisms were determined and compared. DNAs from cells irradiated with either IPL or UVC accumulated double-strand breaks (DSBs), single-strand breaks, and cyclobutane pyrimidine dimers, and with a similar pattern; however, more DSBs were detected following UVC than following IPL in both types of microorganism. Transmission electron microscopy observations of IPL- and UVC-induced cell damage clearly indicate that bacterial cell structures were destroyed by IPL treatment but not by UVC treatment.     

Oxidative Desulfurization of Dibenzothiophene Catalyzed by VO(acac)2 in Ionic Liquids at Room Temperature

A simple extraction and catalytic oxidative desulfurization (ECODS) system composed of VO(acac)₂, 30% H₂O₂, and 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim]BF₄) has been found to be suitable for the deep removal of dibenzothiophene (DBT) in model oil at room temperature. The optimal conditions were as follows: [n(H₂O₂)/n(DBT)/n(catalyst) = 100:20:1], model oil = 5 mL, ionic liquid [IL] = 1 mL, T = 30°C, t = 2 hr. With the ECODS system, the sulfur removal of DBT could reach 99.6%, which was superior to that of the simple extraction with IL (15.6%) or oxidation without catalyst (17.1%). The IL could be recycled five times without a significant decrease in activity.     

湿H2S环境中Q345R(HIC)的氢扩散规律及性能

目的 模拟湿H₂S环境,采用氢渗透技术对Q345R(HIC)、SA516 Gr70N+316L复合钢板及316L在湿H₂S环境下的氢扩散规律及性能进行了研究。方法 采用电化学渗氢技术、拉伸、冲击试验和断口分析等方法,比较了不同pH值环境条件下材料中的原子氢浓度、有效扩散系数、强度、韧性和断口形貌等。结果 随着溶液pH值的降低,Q345R(HIC)中原子氢浓度(C₀)、氢有效扩散系数(D_eff)等均升高,pH值=3时,材料中的C₀可达17 mol/m³,D_eff可达2.22×10^-10 m²/s。大量原子氢的渗入,使材料产生位错钉扎效果,发生了氢致硬化现象,材料内部出现了氢损伤白点特征,316L未发生氢损伤。结论 316L复合层可有效避免扩散氢对基材SA516 Gr70N或Q345R的损伤,对于Q345R(HIC),随着溶液pH值的降低,材料中渗入的原子氢越多,材料性能损伤越大。