日本食品化学学会誌（日食化誌）、Vol. 23(3), 2016 141 N o t e 日本食品化学学会誌、Vol. 23(3), 141-148(2016) Japanese Journal of Food Chemistry and Safety (JJFCS) Wheat DNA fragmentation of commercial processed foods (Received June 22, 2016) (Accepted July 29, 2016) Taira Miyahara a), Nao Miyake a), Kotoha Sawahuji a), Kazumi Kitta b), Kosuke Nakamura c), Kazunari Kondo c), Yoshihiro Ozeki a) a) Tokyo University of Agriculture and Technology b) National Food Research Institute, National Agriculture and Food Research Organization c) National Institute of Health Sciences Abstract Recent advances in plant biotechnology have established transgenic wheat lines, which are almost ready to be cultivated for commercial production in the field. Wheat flours are used as ingredients in many food products. Here, in order to detect genetically modified wheat in processed foods, the yield and fragmentation of genomic DNA prepared from processed foods were investigated. Qualitative PCR using primer sets that gave 96-755 bp PCR products at ca. 100 bp intervals showed that in fermenting processes by yeast and baking processes for breads and buns, including steaming and frying, DNA fragmentation of less than 430 bp did not occur. Amplicons longer than 755 bp were found in all noodles, but roasting and retort processes to produce stews caused severe degradation of genomic DNA leading to fragmentation and reduced yields. A Japanese traditional sweet, kuzumochi, which is processed by Lactobacillus fermentation with kneaded flours for a year, gave amplicons shorter than 323 bp. These results indicated that PCR detection methods for transgenes in wheat processed foods should be established using primer pairs that target DNA sequences shorter than 200 bp. Keywords : DNA fragmentation, wheat flour, wheat processed food, qualitative PCR Ⅰ Introduction qualitative regulation by the Food Sanitation Act, and quantitative regulation for authorized GM crops and materials Many genetically modified (GM) crops have been developed for processed foods should mandate labeling by Food Labeling over the years worldwide. Novel traits can be introduced by Law in the case of unintended contamination over 5% of transgenes into major GM crops, among these are herbicide total weight4). Therefore, the establishment of qualitative and tolerance, insect and virus resistance. Furthermore, the genetic quantitative detection methods for GM crops and foods have alteration of nutrient composition, addition of new nutrients, been required. Generally, the detection methods are by PCR and enhancement of environmental stresses such as drought analysis and specific PCR primers designed to amplify target resistance have also been developed recently1-3). A mandatory genomic DNA sequence shorter than 200 bp in length for real- safety assessment of GM crops used for foods should be time PCR and microarray analysis5-8). The raw materials of undertaken in each country, and unauthorized GM crops GM crops are cooked by physical, chemical, and biological and foods produced with GM crops prohibited from entering treatments to produce processed foods. These treatments are commercialized markets before a safety assessment. In known to denature, degrade, and fragment genomic DNA. order to prevent importation and circulation of unauthorized The different degrees of the treatments, from shallow to GM crops and foods in markets, detection methods should deep processing, result in different levels of genomic DNA be established internationally. In Japan, the zero-tolerance degradation and fragmentation. Genomic DNA of crop for unauthorized GM crops and foods is obligated through ingredients such as soybean, corn, and tomato are lightly Corresponding author: Taira Miyahara, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan 142 Jpn. J. Food Chem. Safety, Vol. 23(3), 2016 fragmented by boiling and steaming at 100°C and frying in small pieces then frozen in liquid nitrogen. Both cooked stew oils at 160-180°C, but heavily fragmented by autoclaving and retort packaged stew were dropped as 1 g samples into and microbial fermentation to lengths shorter than 100 bp. liquid nitrogen. These frozen samples were ground with a This means that some genomic DNA from processed foods mortar and a pestle followed by thawing and genomic DNA are unable to be detected by PCR using primer sets that was extracted in extraction buffer (10 mM Tris-HCl, pH amplify over 200 bp target DNA sequences9-11). Thus, it is 7.5; 150 mM NaCl; 10 mM EDTA; 1% (v/v) SDS). Samples important to investigate the degree of DNA fragmentation of powdered flour, wheat starch, and wheat gluten, 1 g of in ingredients by different processing methods to know how each, were poured into the extraction buffer. Samples in the detectable transgenes of GM crops in processed foods are by extraction buffer were incubated at 60°C for 10 min, then PCR. Determining the degree of fragmentation in processed centrifuged 8,400 × g for 10 min. DNA was purified from foods will be useful in designing primers for detecting the water-soluble supernatant using a DNeasy Plant Mini Kit contamination of unauthorized GM crop materials and for (Qiagen, Hilden, Germany) according to a Ministry of Health, quantifying the amount of authorized GM crops in total Labour, and Welfare announcement17). The concentration ingredients. and quality of the extracted DNA were measured using a In the case of wheat, detection primers for species-specific U-008OD spectrophotometer (Hitachi, Tokyo, Japan). The genes and taxon-specific genes have already been designed yields of DNA prepared from each sample were calculated by and some information concerning DNA fragmentation in absorbance at 260 nm (A260) and the purity was estimated by 12-16) breads were reported . Wheat is a common ingredient used the ratio of A260 and absorbance at 280 nm (A280) derived from throughout the world, and cooking and processing methods for aromatic amino acids of contaminated proteins. A ratio of food production vary in different food cultures and countries. A260/A280 in the range 1.5-2.0 was considered to be a suitable In this report, we investigate genomic DNA yields from wheat quality for PCR analysis18). Three individual DNA extracts processed foods and the fragmentation of genomic DNA in from each sample were carried out by qualification PCR and several varieties of processed breads and buns, noodles, and electrophoresis on the same day to prevent contamination from other foods in the commercial market. fine powder and aerosol of other samples. The extracted DNA samples were analyzed by electrophoresis using an Agilent 2100 Bioanalyzer system (Agilent Technologies, CA, USA). Ⅱ Materials and Methods 3. PCR amplification of genomic DNA 1. Processed foods samples In order to design primer sequences to give several lengths For the control of processed foods, homemade bread was of genomic DNA fragments, wheat endogenous gene Waxy-D1 prepared from bread flours using a home-bakery machine (GenBank accession no. AF113844 as cDNA sequence) was (SD-BMS106, Panasonic, Osaka, Japan) according to a amplified by PCR using the specific primers F and R (Table general recipe. Udon was prepared with soft flours according 1) with the genomic DNA prepared from bread flour as a to a Japanese traditional recipe which included boiling. template. The reaction mixture of 10 μL consisted of 5 μL of The vegetable bread, steamed bun, roll, corn mayonnaise SapphireAmp Fast PCR Master Mix (Takara Bio Inc., Shiga, bread, fried bread, croissant, muffin, English muffin, and Japan), 60 ng of the template DNA, and 0.2 μM of the primers. doughnut were purchased from markets. Noodles as somen, The PCR proceeded using a T-100 Thermal Cycler (Bio-Rad, fried noodles, Chinese noodles, snack noodles and yakisoba, Tokyo, Japan) as follows: 94°C for 2 min, followed by 40 Chinese foods of Chinese dumpling (manju) and wonton, cycles of denaturing at 98°C for 5 s, annealing at 55°C for 10 s others of retort packaged stew, kuzumochi (different from the and extension at 72°C for 20 s. The amplified DNA fragment cake made from root starch of Pueraria lobata), and wheat was cloned into pMD20 (Takara Bio Inc.) and the nucleotide starch and wheat gluten as food additives were also purchased sequence confirmed using an ABI prism 3100 Genetic from markets. The stew was cooked using condensed stew Analyzer (Applied Biosystems, CA, USA). According to the mix (blocked) according to the recommended recipe on the nucleotide sequence, the reverse primers, R1-R7, were designed package without any meat and vegetables. to give amplified DNA fragments of ca. 100 bp interval lengths as shown in Table 1 and Fig. 1. The qualitative PCR conditions 2. Extraction of DNA for fragmentation of genomic DNA in processed foods using Breads were roughly torn into small blocks and 5 g mixed these primers were as follows: after 94°C for 2 min incubation, samples from the surface and inside were frozen in liquid 40 cycles of denaturing at 98°C for 5 s, annealing at 58°C nitrogen. Noodles, buns of Chinese dumpling and the dough for 10 s and extension at 72°C for 5 s. The lengths of PCR of wonton and kuzumochi, 1 g of each, were chopped into amplicons were evaluated by agarose gel electrophoresis. 日本食品化学学会誌（日食化誌）、Vol. 23(3), 2016 143 Table 1. PCR primer list for the Waxy-D1 gene value of less than 43 and exponential amplification plots were Amplicon scored as positive. If the Ct value could not be obtained, the Primer Nucleotide sequence length (bp) reaction was scored as negative. Reactions with a Ct value of F 5'-GCACCGTCCTCGGCATCA-3' - less than 43, but without exponential amplification as judged R 5'-CCTGTAGATGCCATTGGACTGGTAGTT-3' 1151 by visual inspection of the respective ΔRn plots and multi- R1 5'-CGGTCCTCATGCCGAGAG-3' 96 component plots were scored as negative. R2 5'-CCGACGAACACGAGGTTCAT-3' 211 R3 5'-ATGAACATTATGAGAAGACAGTGGT-3' 323 R4 5'-GCGTCCTTGTACTGGTCGTA-3' 430 Ⅲ Results and Discussion R5 5'-CAGAAATGTGCAGGAGCATG-3' 515 R6 5'-GTCACCTTCTCCAGGAAGCA-3' 654 1.