Applicability of the quantification of genetically modified organisms to foods processed from maize and soy

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.     

Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays)

Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.     

Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis

Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.     

Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products

Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.     

Isoflavones in retail and institutional soy foods.

A national sampling plan was developed to select the most widely used isoflavone-containing foods in the United States. Foods were selected based on their retail volume and sampled in five geographical areas representing seven metropolitan areas. Isoflavones were analyzed from composite samples, raw and cooked, and reported by brand. Quality control measures were evaluated throughout the study. Isoflavone levels ranged from 1 microg/g in soy sauces to 540 microg/g in tempeh. Soymilk and tofu represented the major portion of soy foods evaluated. These data will appear in the electronic version of USDA Handbook No. 8 of Food Composition Data in 1999.     

Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.     

Changes of astringent sensation of soy milk during tofu curd formation

The effect of isoflavone on soy milk and tofu astringency was investigated, and no consistency was found between an undesirable astringent taste and isoflavone contents. Isoflavone-enriched extract (approximately 39% isoflavones) showed no astringency. Soybean foods having high amounts of isoflavones showed less astringency. About 80% of isoflavones exist freely in both soy milk and tofu, but 55% of phytates (which play an important role in the formation of the tofu curd network) exist freely in the soy milk, and 6-13%, on the basis of coagulation, existed freely in the tofu curds. A 1% potassium phytate solution at pH 7 showed the very same astringency as soy milk; however, calcium phytate at the same concentration and pH showed no undesirable sensation. Thus, it is assumed that the astringent characteristics caused by phytic ions in soy milk are lost upon conversion of phytic ions to their insoluble salt forms during soy milk coagulation.     

Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863

Qualitative and quantitative analytical methods were developed for the new event of genetically modified (GM) maize, MON863. One specific primer pair was designed for the qualitative polymerase chain reaction (PCR) method. The specificity and sensitivity of the designed primers were confirmed. PCR was performed on genomic DNAs extracted from MON863, other GM events, and cereal crops. Single PCR product was obtained from MON863 by the designed primer pair. Eight test samples including GM maize MON863 were prepared at 0.01 approximately 10% levels and analyzed by PCR. Limit of detection of the method was 0.01% for GM maize MON863. On the other hand, another specific primer pair and probe were also designed for quantitative method using a real-time polymerase chain reaction. As a reference molecule, a plasmid was constructed from a taxon-specific DNA sequence for maize, a universal sequence for a cauliflower mosaic virus (CaMV) 35S promoter used in most genetically modified organisms, and a construct-specific DNA sequence for the MON863 event. Six test samples of 0.1, 0.5, 1.0, 3.0, 5.0 and 10.0% of GM maize MON863 were quantitated for the validation of this method. At the 3.0% level, the bias (mean vs true value) for MON863 was 3.0%, and its relative standard deviation was 5.5%. Limit of quantitation of the method was 0.5%. These results show that the developed PCR methods can be used to qualitatively and quantitatively detect GM maize MON863.     

Oxalate and phytate of soy foods

The consumption of foods made from soybeans is increasing because of their desirable nutritional value. However, some soy foods contain high concentrations of oxalate and/or phytate. Oxalate is a component of calcium oxalate kidney stones, whereas phytate is an inhibitor of calcium kidney stone formation. Thirty tested commercial soy foods exhibited ranges of 0.02-2.06 mg oxalate/g and 0.80-18.79 mg phytate/g. Commercial soy foods contained 2-58 mg of total oxalate per serving and 76-528 mg phytate per serving. Eighteen of 19 tofu brands and two soymilk brands contained less than 10 mg oxalate per serving, defined as a low oxalate food. Soy flour, textured vegetable soy protein, vegetable soybeans, soy nuts, tempeh, and soynut butter exhibited greater than 10 mg per serving. The correlation between oxalate and phytate in the soy foods was significant (r = 0.71, P < 0.001) indicating that oxalate-rich soy foods also contain higher concentrations of phytate. There also was a significant correlation, based on molar basis, between the divalent ion binding potential of oxalate plus phytate and calcium plus magnesium (r = 0.90, P < 0.001) in soy foods. Soy foods containing small concentrations of oxalate and moderate concentrations of phytate may be advantageous for kidney stone patients or persons with a high risk of kidney stones.     

Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing

Roundup-Ready soybeans have been genetically modified to resist the effects of the herbicidal glyphosate and have become the most prevalent transgenic crop in the world. In this work, Roundup-Ready soybeans were used as raw material to study the effects of critical processing procedures such as grinding, cooking, blending, homogenization, sterilization, and spray-drying on the length of DNA fragments of an endogenous gene (lectin) and an exogenous gene (epsps) examined in material from three soybean foods of bean curd, soy milk, and soy powder and from samples taken during their processing. The results showed that various processing procedures caused degradations of both the endogenous and exogenous genes to different degrees. In the grinding procedure, endogenous gene DNA was degraded from 1883 to approximately 836 bp, and exogenous gene DNA was degraded from 1512 to approximately 408 bp. In the blending and squeeze-molding procedures, exogenous gene DNA was also degraded from about 408 to 190 bp, but there was no obvious action on the endogenous gene. After the endogenous and exogenous genes had been degraded to some degree, such as 836 and 408 bp, respectively, they were not evidently affected by cooking procedure at 100 degrees C for 15 min. However, the endogenous gene was further considerably degraded from around 836 to 162 bp in the sterilization procedure at 121 degrees C for 30 s. The effect of the homogenization step on endogenous and exogenous genes was similar to that of the cooking procedure. The coagulation procedure, principally a biochemical reaction, did not greatly affect the exogenous gene but did affect endogenous gene, reducing DNA size from about 836 to 407 bp. Furthermore, the spray-drying procedure, a process of physical shearing, high temperature, and sudden high pressure, distinctly caused degradation of both the lectin and epsps genes, rapidly decreasing the sizes from about 836 to 162 bp for the endogenous gene and from about 408 to 190 bp for the exogenous gene.     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

Quantification of genetically modified soybean by quenching probe polymerase chain reaction

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.     

小麦抗秆锈突变系龙辐03D51的筛选及其抗病性的遗传分析与RAPD标记

以龙6239幼胚为外植体,利用辐射诱变和组织培养相结合的方法,选育出突变系龙辐03D51,经秆锈接种鉴定,发现其对优势小种21C3CPH免疫,而亲本龙6239对21C3CPH高度感染。遗传分析表明,抗病性由显性单基因控制。该突变系还具有其亲本的优质、高产特点,已成为优异的后备品系。在RAPD检测中,所用的60个随机引物中有3个引物在龙辐03D51和其亲本龙6239中具有多态性,引物E07、E11、E17在龙辐03D51中分别扩增出380bp、700bp和600bp的特异带,初步认为这些谱带可能与秆锈抗性有关。     

Quantification of the group B soyasaponins by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the isolation and quantitative determination of the group B soyasaponins, including 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins alphag, betag, and betaa, and their non-DDMP counterparts, soyasaponins V, I, and II, respectively, with formononetin used as the internal standard. The limits of quantification for soy products were 0.11-4.86 micromol/g. The within-day and between-days assay coefficients of variation were <9.8 and < 14.3%, respectively. The group B soyasaponin concentrations in 46 soybean varieties ranged from 2.50 to 5.85 micromol/g. Soy ingredients (soybean flour, toasted soy hypocotyls, soy protein isolates, textured vegetable protein, soy protein concentrates, and Novasoy) and soy foods (commercial soy milk, tofu, and tempeh) contained the group B soyasaponins from 0.20 to 114.02 micromol/g. There was no apparent correlation between isoflavone and soyasaponin concentrations in the soy products examined.     

Identification of sex-specific DNA markers in betel vine (Piper betle L.)

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in male and female plants of Piper betle L. Two bulks of DNA were made drawing one each from male and female, by pooling an equal volume of DNA samples from each group of individual contributing to the bulk analysis. Fifty different random decamer primers were screened with the two bulks to identify markers associated with sex expression of which only four primers were found to be associated with sex expression. These four primers were then tested with individual plant DNA samples where sex-associated RAPD markers were identified. A ~1,400 and ~850?bp fragment from the primer OPA04 and OPN 02 respectively was found to be present in all the male individuals and absent in all the female plants. In another primer, a ~980?bp amplification product from the primer OPC 06 was present only in the female individuals. A common primer OPA 08 showed both male and female specific markers of 650 and 1,200?bp respectively. Thus, the three male- specific RAPD markers OPA04₁₄₀₀, OPA08₆₅₀ and OPN02₈₅₀ and two female-specific markers OPA08₁₂₀₀ and OPC06₉₈₀ can reliably differentiate the male and female plants of P. betle L. Ploidy comparison also showed the differences in male and female plants.     

Elimination of trypsin inhibitor activity and beany flavor in soy milk by consecutive blanching and ultrahigh-temperature (UHT) processing

Soy foods contain significant health-promoting components but also may contain beany flavor and trypsin inhibitor activity (TIA), which can cause pancreatic disease if present at a high level. Thermal processing can inactivate TIA and lipoxygenase. Ultrahigh-temperature (UHT) processing is relatively new for manufacturing soy milk. Simultaneous elimination of TIA and soy odor by UHT processing for enhancing soy milk quality has not been reported. The objective was to determine TIA in soy milk processed by traditional, steam injection, blanching, and UHT methods and to compare the products with commercial soy milk products. Soybean was soaked and blanched at 70-85 degrees C for 30 s-7.5 min. The blanched beans were made into base soy milk. The hexanal content of the base soy milk was determined by gas chromatography to determine the best conditions for further thermal processing by indirect and direct UHT methods at 135-150 degrees C for 10-50 s using the Microthermics processor. Soy milk was also made from soaked soybeans by traditional batch cooking and steaming methods. Eighteen commercial products were selected from the supermarket. Residual TIA in soy milk processed by the traditional and steam injection to 100 degrees C for 20 min was approximately 13%. Blanching could inactivate 25-50% of TIAs of the raw soy milk. The blanch conditions of 80 degrees C and 2 min were selected for UHT processing because these conditions produced blanched soy milk without hexanal, indicating a complete heat inactivation of lipoxygenases. The TIA decreased with increased temperature and time of UHT heating. The maximal trypsin inhibitor inactivation was achieved by UHT direct and indirect methods with residual activities of approximately 10%. Some commercial soy milk products contained high TIAs. The results are important to the food industry and consumers. Kinetic analysis showed that heat inactivation (denaturation) of TIA, under the continuous processing conditions of the Microthermics processor, followed first-order reaction kinetics, and the activation energy of the inactivation was 34 kJ/mol.     

Development of a multiplex polymerase chain reaction method for simultaneous detection of eight events of genetically modified maize

In this study, we developed a novel multiplex polymerase chain reaction (PCR) method for simultaneous detection of up to eight events of genetically modified (GM) maize within a single reaction. The eight detection primer pairs designed to be construct specific for eight respective GM events (i.e., Bt11, Event176, GA21, MON810, MON863, NK603, T25, and TC1507) and a primer pair for an endogenous reference gene, ssIIb, were included in the nonaplex(9plex) PCR system, and its amplified products could be distinguished by agarose gel and capillary electrophoreses based on their different lengths. The optimal condition enabled us to reliably amplify two fragments corresponding to a construct specific sequence and a taxon specific ssIIb in each of the eight events of GM maize and all of nine fragments in a simulated GM mixture containing as little as 0.25% (w/w) each of eight events of GM maize. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM maize.     

Quality and stability of edible oils enriched with hydrophilic antioxidants from the olive tree: the role of enrichment extracts and lipid composition

Phenolic extracts from olive tree leaves and olive pomace were used to enrich refined oils (namely, maize, soy, high-oleic sunflower, sunflower, olive, and rapeseed oils) at two concentration levels (200 and 400 μg/mL, expressed as gallic acid). The concentration of characteristic olive phenols in these extracts together with the lipidic composition of the oils to be enriched influenced the mass transfer of the target antioxidants, which conferred additional stability and quality parameters to the oils as a result. In general, all of the oils experienced either a noticeable or dramatic improvement of their quality-stability parameters (e.g., peroxide index and Rancimat) as compared with their nonenriched counterparts. The enriched oils were also compared with extra virgin olive oil with a natural content in phenols of 400 μg/mL. The healthy properties of these phenols and the scarce or nil prices of the raw materials used can convert oils in supplemented foods or even nutraceuticals.     

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.