Protection of maize (Zea mays) and soybeans (Glycine max) using Aframomum danielli

When maize and soybeans were stored under ambient conditions (26ǃ °C/RH 75LJ%) using the ground material of the spice Aframomum danielli (family Zingiberaceae), mouldiness and insect infestation were controlled for 15 months. After eight weeks of storage, chemical compositions of maize and soybeans (moisture content, mc 10.0%) treated with 4.0%, 6.0% and 8.0% ground powder of A. danielli did not differ significantly (P=0.05) from those of untreated (control) samples. However, chemical compositions of samples maintained at 15.0% and 20.0% mc respectively were different from those of control samples (P>0.05). At 10.0% mc, maize and soybean samples treated with different concentrations of A. danielli powder were preferred to treated samples maintained at 15.0% and 20.0% respectively. The preservative capability of the powder of A. danielli was associated with phytochemical components tentatively identified as alkaloids. Viability of maize and soybeans was not affected by treatment with A. danielli powder. The powder of A. danielli has some protective effects on liver cells because it exerted no hepatotoxic effect on test albino rats as serum enzyme levels of glutamate oxaloacetate transmaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) were lowered by 67.0%, 86.3% and 49.7% respectively when compared with control albino rats not fed with A. danielli powder in their rations (meals).     

Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods should preferably enable detection of several different GMOs. Here we present a simple, robust, qualitative, nineplex PCR method for event-specific detection of maize T25, GA21, TC1507, MON863, MON810, NK603, construct specific detection of BT176, BT11 and detection of the endogenous hmga maize reference gene. PCR is carried out with primers labelled with fluorescent groups and the amplicons are detected using fluorescence capillary electrophoresis. Using mixtures of DNA from different certified reference materials, the detection limit was determined to approximately 0.1% for each GMO. Good agreement was observed in 85 of 88 determinations when eleven food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. Discrepancies were only observed for one GMO at or close to the detection limit. The presented method is therefore suitable for screening purposes for food and feed containing the most common maize GMOs.     

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.     

Quantitative,multiplex ligation-dependent probe amplification for the determination of eight genetically modified maize events

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a robust, quantitative, sensitive, nine-plex ligation-dependent probe amplification method, GMO-MLPA, for event-specific detection of maize TC1507, MON810, NK603, MON863, BT176, T25, GA21, construct-specific detection of BT11, and detection of the endogenous hmga maize reference gene. Ligated probes are amplified by PCR. Amplicons are detected using capillary electrophoresis. Specific GMO signals are normalised relative to the signal from the endogenous hmga gene and quantified by comparing with known standard curves. The method is suitable for quantification in the 0–2% range. Agreement was obtained in 149 of 160 determinations when 11 known mixtures of GMO and 9 food and feed samples were analysed using the GMO-MLPA method and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is, therefore, suitable for quantification purposes for food and feed containing the most common maize GMOs.     

5'-Nuclease PCR for quantitative event-specific detection of the genetically modified Mon810 MaisGard maize

Genetically modified maize is grown extensively in the world today. MaisGard (Monsanto, Yieldgard in the USA) is a genetically modified maize harbouring the Mon810 transformation event. European Community legislation requires that genetically modified organisms (GMOs) be approved before they are placed on the market. Labelling is required when more than 1% of any ingredient of a food originates from a GMO. There is consequently a need for specific, quantitative methods for detection of genetically modified foods. We have determined the DNA sequence of the 5'-flanking region of the Mon810 insert using ligation mediated PCR. A primer probe set overlapping the junction was designed and used in a quantitative, event-specific Taqman 5'-nuclease assay. Mon810 DNA was quantified relative to endogenous maize zein gene DNA. The results were expressed as the percentage of genetically modified Mon810 maize DNA relative to the total content of maize DNA.     

PCR方法对转基因食品定性检测的研究

转基因食品的检测一般基于聚合酶链式反应（PCR）方法，对35S启动子和NOS终止子进行筛选。本实验以转基因大豆、玉米（购自Fluka）为参照物，转基因含量为0％，1％，2％，5％的样品均获得正确识别。以上介绍的方法能对转基因原材料及加工成品实施高精度、高灵敏的检测。     

Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0–2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5′-sequence and a GMO specific 3′-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is therefore suitable for quantification purposes for food and feed containing the most common maize GMOs.     

Differential scanning calorimetric studies on structured lipids from coconut oil triglycerides containing stearic acid

Triglycerides from coconut oil contain high levels of lauric acid. They were replaced by incremental amounts of stearic acid by interesterification reactions catalyzed by immobilized lipase (IM 60 from Rhizomucor miehei). The reactions were carried out in organic solvents such as hexane. Maximum incorporation of stearic acid was observed by 4 h at 37vv°C or by 2 h at 60vv°C when triglycerides to fatty acid (stearic acid) ratio was maintained at 1v:Ң. The stearic acid level in coconut oil triglycerides was increased from an initial value of 2% to 60% under these conditions. The stearic acid replaced lauric, myristic, and palmitic acids in unmodified triglycerides. A major portion of stearic acid incorporated was found in positions 1 and 3 of triglycerides. Differential scanning calorimetry indicated that stearic acid enrichment increased the solid fat content and also the higher melting polymorphs in modified lipids. The studies also indicated that low melting polymorphic forms of coconut oil triglycerides are converted to higher melting forms by stearic acid enrichment. The modified lipids thus obtained can find use in various food applications.     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography–tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC–APCI–MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5–4.0 µg kg^?1 for NIV, 2.8–5.3 µg kg^?1 for DON, 0.4–1.7 µg kg^?1 for HT-2 and 0.4–1.0 µg kg^?1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^?1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.     

Screening of genetically modified organisms and specific detection of Bt176 maize in flours and starches by PCR-enzyme linked immunosorbent assay

Polymerase chain reaction (PCR)-enzyme linked immunosorbent assays (ELISAs) targeting either the 35S promoter or the Bt176 specific junction sequence were developed to screen for the presence of genetically modified organisms (GMOs) and specifically detect Bt176 maize in flours and starches. Two additional PCR-ELISA assays were developed to validate the results: one, based on the detection of the maize alcohol dehydrogenase 1 promoter specifically detected the presence of maize, and the other, based on the detection of a conserved sequence of plants ( 26S ribosomal RNA gene), validated the extracted DNA amplification. The PCR-ELISA assays developed here were highly specific and found to be as sensitive as the reference Southern hybridisation assay. The PCR-ELISA tests were at least 6 times more sensitive than gel electrophoresis and allowed 0.1% GMOs to be detected in Bt176, Bt11, Mon810 maize and Roundup Ready soybean. The PCR-ELISA tests are a method of choice for GMO screening and identifying Bt176 maize in flours and native starches. They may offer a cheaper alternative to the expensive real-time PCR assays and may be useful in laboratory GMO monitoring.     

Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis

The identification of both approved and non-approved genetically modified organisms (GMOs) is an integral part of GMO biosafety legislation in many countries. One aspect that may affect PCR-based detection of a GMO lies within the analysis of its genetic stability, as sequence alterations or DNA instabilities may impede quantification by PCR. Genetic stability can be analyzed using various methods, yet many of these methods have distinct disadvantages, including low sensitivity. In this study, high resolution melting (HRM) analysis and real-time PCR with Scorpion primers were used as a method to analyze the 3′ end of RR soybeans (RR 40-3-2) in a large number of samples (n = 1,034). No evidence for the occurrence of mutation events was found, implying that the nucleotide sequence of this region is unlikely to be unstable and is well suited as a target for the quantification of RR soybeans. Additionally, and as a preparative work for an optimization of the method, a 174 bp region of the first intron of the Adh1 gene was analyzed in several varieties of maize with different GMO events using the same approach. The results show that 2 alleles are present. In further experiments, the different alleles were cloned into plasmids to generate homozygous plasmids from heterozygous templates in order to generate for a more precise analysis. The overall methodological aim of these studies was to compare HRM analysis with Scorpion primer PCR. Both methods were capable of differentiating between the 2 homozygous and heterozygous alleles. For a better discrimination, however, we conclude that it is most reliable to consider the results of both methods. This dual approach is assumed to be an effective tool as an accurate, high-throughput means of the screening of GMOs for potential genetic instabilities that may interfere with the detection and identification of specific GM events.     

Inter-laboratory Testing of GMO Detection by Combinatory SYBR®Green PCR Screening (CoSYPS)

Combinatory SYBR®Green real-time PCR Screening (CoSYPS) is an efficient, sensitive approach for detecting complex targets such as genetically modified organisms (GMOs) in food and feed products. GMO analysis for legal purposes has become increasingly complex and costly due to the diversity in recombinant targets present in the different GMOs. For this reason, screening for the presence of GMOs is in general the first step in the detection of GM material in a product. CoSYPS allows detecting the large majority of globally commercial GMOs using SYBR®Green real-time PCR methods for six GM targets (P35S, Tnos, CryIAb, CP4-EPSPS, PAT and BAR) combined with species-specific PCR methods (e.g., maize, soy, rapeseed). Here, the results of an inter-laboratory trial on seven samples with different GMO mixtures at different levels are presented. In total, 13 laboratories participated in the trial and the currently most frequently used PCR analysis platforms are represented. The inter-laboratory study clearly demonstrates that PCR methods used in CoSYPS form a very robust GMO screening system. Sensitivity, specificity, positive and negative predictive values are for all PCR methods higher than 95 % for all samples. Together, these results show that the SYBR®Green real-time PCR methods used in CoSYPS are effectively applicable to different PCR platforms and amendable to configuration into a sensitive high-throughput GMO screening and decision support tool.     

A Rapid Size-Exclusion Solid-Phase Extraction Step for Enhanced Sensitivity in Multi-Allergen Determination in Dark Chocolate and Biscuits by Liquid Chromatography–Tandem Mass Spectrometry

Enhanced sensitivity for the simultaneous determination of five nut allergens in biscuit and in dark chocolate complex matrices was obtained by introduction of a rapid size-exclusion solid-phase extraction-based step before liquid chromatography–electrospray ionization-tandem mass spectrometry (LC-ESI-MS²) analysis. A very fast and efficient separation (<12 min) of marker peptides with selected reaction monitoring detection was obtained. Limits of detection in the 0.1–1.3 mg nut/kg and 5–15 mg nut/kg ranges for biscuit and dark chocolate samples as well as high recoveries (84(±6)–106(±4)% for biscuits and 98(±5)–108(±6)% for dark chocolate) proved the excellent capabilities of the exploited sample treatment method combined with the LC-MS² analysis. Good precision in terms of intra- and inter-day repeatability was calculated, being always lower than 19 % (n?=?75). Linearity was demonstrated up to four and three orders of magnitude for biscuit and dark chocolate, respectively. Finally, the validated method was successfully applied to the investigation of hidden nut trace allergens in commercially available biscuits and chocolates of different brands aiming to ascertain possible discrepancies between allergen content and food allergen labelling.     

Extraction and purification of free cholesterol from some egg-containing food by on-line supercritical fluid extraction – solid-phase extraction

Two methods for the extraction and determination of free cholesterol in some egg-containing food samples were compared: the classical Soxhlet method and a supercritical carbon dioxide extraction (at 338 bar and 40vv°C) coupled on-line with solid-phase extraction (SPE with a solid trap packed with an ODS stationary phase). Egg noodles, biscuits and sweet snacks were homogenised and added to the internal standard and anhydrous sodium sulphate. Part of each sample was extracted by on-line supercritical fluid extraction - solid-phase extraction (SFE-SPE) and the remaining part was extracted by the classical method using petroleum ether. A further purification after the Soxhlet extraction was performed by SPE. Free cholesterol was determined in both the off-line Soxhlet-SPE fraction and the on-line SFE-SPE extract with capillary gas chromatography after silylation. The two extraction methods gave similar results. However, on-line SFE-SPE represents a valid alternative to the Soxhlet method because it shows important advantages, such as a short analysis time and low solvent consumption.     

Effect of Soybean Cooking Temperature on the Texture and Protein Digestibility of Miso

Miso samples were prepared with soybeans cooked for 10 min at different temperatures (up to 100°C). Appreciable changes in the amount of 0.2 M trichloroacetic acid-soluble nitrogen (TCA-soluble N) and the pattern of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of fermented miso samples were observed in the temperature range between 90°C and 100°C. No further changes in the TCA-soluble N and SDS-PAGE pattern of miso were observed when the cooking time was prolonged up to 3 hr at 100°C. On the other hand, a 2 hr cooking period at 100°C was required to achieve a level of softness for cooked soybeans to give an acceptable texture of miso.     

Duplex real-time PCR assay using SYBR Green to detect and quantify Malayan box turtle (Cuora amboinensis) materials in meatballs,burgers, frankfurters and traditional Chinese herbal jelly powder

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected species widely used in exotic foods and traditional medicines. Currently available polymerase chain reaction (PCR) assays to identify MBT lack automation and involve long targets which break down in processed or denatured tissue. This SYBR Green duplex real-time PCR assay has addressed this research gap for the first time through the combination of 120- and 141-bp targets from MBT and eukaryotes for the quantitative detection of MBT DNA in food chain and herbal medicinal preparations. This authentication ensures better security through automation, internal control and short targets that were stable under the processing treatments of foods and medicines. A melting curve clearly demonstrated two peaks at 74.63 ± 0.22 and 78.40 ± 0.31°C for the MBT and eukaryotic products, respectively, under pure, admixed and commercial food matrices. Analysis of 125 reference samples reflected a target recovery of 93.25–153.00%, PCR efficiency of 99–100% and limit of detection of 0.001% under various matrices. The quantification limits were 0.00001, 0.00170 ± 0.00012, 0.00228 ± 0.00029, 0.00198 ± 0.00036 and 0.00191 ± 0.00043 ng DNA for the pure meat, binary mixtures, meatball, burger and frankfurter products, respectively. The assay was used to screen 100 commercial samples of traditional Chinese herbal jelly powder from eight different brands; 22% of them were found to be MBT-positive (5.37 ± 0.50–7.00 ± 0.34% w/w), which was reflected through the Ct values (26.37 ± 0.32–28.90 ± 0.42) and melting curves (74.63–78.65 ± 0.22°C) of the amplified MBT target (120 bp), confirming the speculation that MBT materials are widely used in Chinese herbal desserts, exotic dishes consumed with the hope of prolonging life and youth.     

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%.     

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography-tandem mass spectrometry

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC-APCI-MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5-4.0 µg kg^-1 for NIV, 2.8-5.3 µg kg^-1 for DON, 0.4-1.7 µg kg^-1 for HT-2 and 0.4-1.0 µg kg^-1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg^-1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.     

Effect of temperature and time during steam treatment on the protein quality of full-fat soybeans from different origins

Soybeans (Glycine max) of Argentinian and Chinese origin were steam-processed at 102°C, 118°C and 136°C for various durations with the use of a laboratory-scale steam toaster. Samples of raw and processed soybeans were analysed for dry matter (DM), crude protein (CP), trypsin inhibitor activity (TIA), protein dispersibility index (PDI) and reactive lysine content. Chinese raw soybeans showed higher levels of CP, TIA and PDI, and a lower level of reactive lysine than Argentinian raw soybeans (366 vs 357 g kg⁻¹, 20.6 vs 15.2 mg g⁻¹, 87.6 vs 85.6% and 17.4 vs 19.4 g kg⁻¹, respectively). Both the TIA and PDI of the two soybean samples were decreased following a logarithmic pattern with the lengthening of the heating time when beans were steam heated at various temperatures. The reduction rates for TIA and PDI, however, were different between the two origins of soybeans. Chinese soybeans required a longer time or a higher temperature to reduce their TIA to a safe level in comparison with Argentinian soybeans. In the case that the beans were heated at 136°C, the difference in PDI between Chinese and Argentinian soybeans was smaller. It is concluded that the two soybeans origins need different processing conditions to improve their protein properties. © 1998 SCI.     

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8 × 10¹ and 3.4 × 10¹ CFU ml⁻¹ were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10° CFU g⁻¹, ten and six were positive by the respective methods. Moreover, 10° CFU 10 g⁻¹ was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.