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 , 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. 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 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 , 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. Quality of extracted DNA from processed foods R7 5'-ACCTGGAAATTGCAACCAGC-3' 755 Homemade bread was baked using a home bakery and the samples were drawn at each processing stages; dry flour, fresh dough, dough after yeast fermentation, and after baking (Table 2). The yields of genomic DNA from the fresh dough of bread were reduced by about one-fourth of dry flour. Since the fresh dough was ground in liquid nitrogen, the extraction efficiency from powdered dough in the buffer might not be so different Fig. 1. Oligonucleotides design for wheat endogenous as similar dry flour. Thus, this decreased yield might not only reference gene, Waxy-D1 be due to DNA degradation but also it is possible that gluten PCR products in the range of 96-755 bp were amplified generated during the kneading process might disturb the using primers indicated by arrows. Closed black rectangles genomic DNA extraction and purification process. indicate exons, dots are introns. The numbers in brackets are amplicon length. Table 2. Yield of genomic DNA during homemade bread processing

4. Real-Time PCR analysis Yield Percentage A260/A280 Real-time PCR assays were performed using ABI PRISMTM (µg/g weight) of the yield* 7900 Sequence Detection System (Thermo Fisher Scientific Dry flour 117.5 ± 43.50 1.68 ± 0.14 100 Inc., MA, USA) in 25 μL reaction mixture consisted of 5 μL Fresh dough 30.5 ± 3.85 1.70 ± 0.13 26 sample DNA solution (50 ng, 187.5 ng and 375.5 ng), 12.5 μL Fermented dough 25.0 ± 3.60 1.73 ± 0.04 21 FastStart Universal Probe Master (ROX) (Roche Diagnostics Bread 27.3 ± 1.25 1.66 ± 0.14 23 K. K., Tokyo, Japan), 0.5 μM forward and reverse primers, * dry flour as 100%. Values are mean ± SD of 3 trials. and 0.2 μM probe. The PCR conditions were as follows: 2 min at 50°C, 95°C for 10 min followed by 45 cycles of 15 s at 2. Primers designed to evaluate DNA fragmentation 95°C and 1 min at 60°C. The proline-rich protein (PRP) gene Waxy-D1 has been widely used as a reference gene for (accession no. X52472) was used as an endogenous reference quantitative PCR in common wheat (Triticum aestivum) gene of common wheat (Triticum aestivum) and detected using but not durum wheat (Triticum durum) because this gene is the following primers and probe as described previously13). lacking in its genome. In contrast, PRP gene was suitable as Forward primer: 5′-GCACCCATGATGAGTACTACTATTCT an endogenous reference gene for both common and durum GTA-3′ (PRP8F) wheat13). To evaluate DNA fragmentation by qualitative PCR, Reverse primer: 5′-TGCAAACGAATAAAAGCATGTG-3′ we tried to design primer pairs to amplify various length (PRPds6R) fragments for both Waxy-D1 and PRP that amplified stepwise Probe: 5′ FAM-CTGTGCACATGACTCAGTTGTTCTTTCGTG- from 100-800 bp. Our trials failed to produce specific primer TAMRA-3′ (PRP-Taq5) pairs for PRP to generate objective length amplicons (data All primers and probes were diluted with an appropriate not show). Thus, Waxy-D1 was chosen as an endogenous volume of distilled water and stored at −20°C until use. Results gene for the template of primers. Waxy genes encoding a were analyzed using SDS 2.1 sequence detection software (Life granule-bound starch synthase, consist of a gene family in Technologies) for ABI PRISMTM 7900 Sequence Detection common wheat, including Wx-7A (accession no. AB019622), System. The baseline was set to cycles 3 through 15. The ΔRn Wx-4A (accession no. AB019623), and Wx-7D (accession threshold for plotting the cycle threshold (Ct) values was set no. AB019624)19). The nucleotide sequence of the fragment to 0.2 during exponential amplification. Reactions with a Ct amplified by primers F and R was corresponding to Wx-7D, 144 Jpn. J. Food Chem. Safety, Vol. 23(3), 2016

which is a single copy gene in the common wheat genome20), Table 3. PRP gene copy numbers during homemade bread and identical to registered Waxy-D1 sequence. The nucleotide processing sequence of the single amplicons derived from the designed A primer sets of F and R1-R7 giving rise to 96-755 bp was Percentage of 50 ng DNA using Ct value Copy number confi rmed for specifi city by sequencing (Table 1 and Fig. 1). copy number* Dry fl our 29.05 ± 0.21 7956.97 100 3. Qualitative and quantitative PCR to evaluate Fresh dough 29.69 ± 0.34 5269.26 66 DNA fragmentation of homemade bread Fermented dough 29.69 ± 0.17 5268.17 66 An amplicon of 755 bp was detected in all samples Bread 33.31±1.30 516.33 6 following the homemade bread process, but the intensity of B that found in bread was faint compared to samples before Percentage of 187.5 ng DNA using Ct value Copy number baking suggesting that severe fragmentation of genomic DNA copy number* could occur during the baking process, even though a 755- Dry fl our 27.18 ± 0.21 26458.13 100 bp amplified product was detectable (Fig. 2). Similar results Fresh dough 27.97 ± 0.19 15872.59 60 were reported with bread containing soybean flours from Fermented dough 27.96 ± 0.16 15965.07 60 which more than 500 bp length amplicons of genomic DNA Bread 31.48 ±1.20 1674.27 6 were detected by qualitative PCR in all bread processing C 16) stages including after baking . In order to determine the Percentage of 375.5 ng DNA using Ct value Copy number degree of genomic DNA fragmentation, a quantitative PCR copy number* to amplify 117 bp of PRP from genomic DNA was performed Dry fl our 26.00 ± 0.22 56294.45 100 (Table 3). The copy number of PRP in the fresh dough Fresh dough 26.92 ± 0.09 31133.29 55 ± was 66% of that in dry flour at using 50 ng genomic DNA. Fermented dough 26.87 0.11 32283.01 57 Bread 30.32 ±1.25 3517.13 6 Furthermore, it was noticed that copy number of PRP did *dry fl our as 100% not decrease after fermentation by yeast. The similarity to Three different amounts of genomic DNA were analyzed (A, B, and C). copy number decreasing after fresh dough preparation but Values are mean ± SD of 3 trials. not after yeast fermentation was observed at using 187.5 and 375.5 ng genomic DNA. We have already shown that deep fermentation by Bacillus subtilis var. natto, Aspergillus oryzae and A. sojae caused severe fragmentation of the soybean genomic DNA in natto, , and soy sauce10, 21). Our results showed that a remarkable fragmentation of wheat genomic M1234 567 DNA did not occur in the short fermentation process by A yeast (Saccharomyces cerevisiae) during bread production. Contrary to the fermentation process, the baking process B drastically decreased PRP copy number (Table 3). These results were also supported by agarose gel electrophoresis (Fig. 3). Genomic DNA fragments of dry fl our were visualized C predominantly around 7,000 bp, but that of fresh dough, dough after fermentation and in bread were predominantly around 300 bp or less. The results considered that the baking process  D caused severe degradation of genomic DNA but there still remained enough genomic DNA of at least 755 bp length to be detected by qualitative PCR. This fact indicated that genomic DNA of longer than 211 bp in length could be detected by Fig. 2. Qualitative PCR amplifi ed products of genomic DNA qualitative PCR, which could be enough to evaluate the copy during homemade bread processing number of genomic DNA by quantitative PCR. Therefore, we DNAs were prepared from A, wheat fl our; B, fresh bun just investigated DNA fragmentation of other processed foods in after kneading; C, bun after yeast fermentation; D, bread. the market by qualitative PCR. Lane 1, 96 bp using F and R1 primers; lane 2, 211 bp using F and R2; lane 3, 323 bp using F and R3; lane 4, 430 bp using F and R4; lane 5, 515 bp using F and R5; lane 6, 654 4. Extraction yield of the genomic DNA from bp using F and R6; lane 7, 755 bp using F and R7. M, 100 bp processed foods in the market DNA ladder marker. Flours are used not only for breads but also noodles, white 日本食品化学学会誌（日食化誌）、Vol. 23(3), 2016 145

sauce for stew, and food additives. In these products, fl ours are mixed with several other materials and baked, fried, boiled, roasted, and fermented. The manufacturing processes for these market products were summarized in Table 4, but their detailed processing methods are not publicly disclosed. The

results showed that many samples achieved the ratio A260/A280 over 1.5, indicating little protein contamination in the genomic DNA preparations. While handling the steam bun, muffin and corn mayonnaise bread, a thick oil layer was observed when the extraction buffer was added, which might interrupt recovery of the aqueous phase after centrifugation and allow L 1 2 3 4 5 6 7 8 9 10 11 12 protein contamination. The processed foods from which genomic DNA could be extracted with a high yield had a low oil content in their dough. Fig. 3. Electrophoresis of genomic DNA during homemade bread processing Genomic DNA were loaded 37.5 ng in each lane. Sample 5. Fragmentation of genomic DNA from processed no. 1-3, dry fl our; 4-6, fresh dough; 7-9, dough after yeast foods in the market fermentation; 10-12, bread; L, Ladder 50-10380 bp. Evaluation of DNA fragmentation in each sample by

Table 4. The yield and quality of genomic DNA prepared from wheat processed foods

Process

Sample Yield (µg/g) A260 A260/A280 Bake Fry Steam Dry Ferment Other Homemade bread 27.30 ±1.25 1.17 ± 0.93 1.66 ± 0.14 ○ × × × ○ a) Roll (A) 7.67 ± 2.33 1.79 ± 0.40 1.56 ± 0.06 ○ × × × ○ a) Steamed bun (B) 3.44 ± 0.40 1.77 ± 0.30 1.18 ± 0.10 × × ○ × ○ a) Fried bread (C) 4.20 ±1.20 1.70 ± 0.46 1.54 ± 0.06 ○ ○ × × ○ a) Croissant (D) 3.07 ± 0.59 2.42 ± 0.37 1.60 ± 0.12 ○ × × × ○ a) Vegetable bread (E) 4.49 ±1.05 3.29 ± 0.47 1.62 ± 0.05 ○ × × × ○ a) Muffi n (F) 6.57 ± 0.31 2.84 ± 0.45 1.17 ± 0.03 ○ × × × × Doughnut (G) 6.97 ±1.33 1.43 ± 0.11 1.51± 0.07 × ○ × × × Pie (H) 7.15 ±1.16 1.49 ± 0.36 1.58 ± 0.08 ○ × × × × Corn mayonnaise bread (I) 1.03 ± 0.43 1.72 ± 0.22 1.26 ± 0.09 ○ × × × ○ a) English muffi n (J) 3.54 ± 2.34 1.83 ± 0.97 2.03 ± 0.20 ○ × × × ○ a) Udon (K) 18.94 ± 8.44 1.89 ± 0.84 1.74 ± 0.06 × × × × × Boiled Yakisoba (L) 15.92 ± 2.88 1.59 ± 0.29 1.54 ± 0.07 × × ○ × × Chinese noodle (M) 108.13 ±17.28 2.16 ± 0.35 1.82 ± 0.08 × × × × × Lye water Fried noodle (N) 35.40 ±18.57 0.98 ± 0.10 1.65 ± 0.09 × ○ × × × Lye water Snack noodle (O) 9.41± 2.56 0.94 ± 0.26 1.78 ± 0.05 × ○ ○ × × Non-fried noodle (P) 25.21±14.60 1.30 ± 0.80 1.63 ± 0.05 × × ○ ○ × Lye water Chinese dumpling (Q) 45.38 ± 2.99 0.91± 0.06 1.70 ± 0.02 × × × × × Frozen Fried wonton (R) 43.05 ±11.95 0.86 ± 0.50 1.71± 0.07 × ○ × × × Stew cooked with mix (S) 5.25 ± 0.67 0.53 ± 0.07 1.68 ± 0.11 ○ b) × × × × Retort packaged stew (T) 6.20 ±1.78 0.62 ± 0.18 1.51± 0.06 ○ b) × × × × Autoclaved Kuzumochi (U) 12.72 ±1.48 1.27 ± 0.15 1.79 ± 0.10 × × ○ × ○ c) Wheat starch (V) 5.38 ± 0.98 0.54 ± 0.10 1.80 ± 0.05 × × × ○ × Wash with water Wheat gluten (W) 100.73 ±19.55 2.01± 0.39 1.88 ± 0.01 × × × ○ × Wash with water

Values are mean ± SD of 3 trials. The alphabets in bracket behind the samples consist with that of fi gure 4. a) fermented with yeast for several minutes b) fl our was roasted c) fermented with Lactobacillus for one year 146 Jpn. J. Food Chem. Safety, Vol. 23(3), 2016

qualitative PCR using the primer sets gave rise to stepwise Heating processes, boiling, baking, and frying did not amplicons of ca. 100 bp intervals (Fig. 4). In steamed bun and give severe genomic DNA fragmentation as amplicons were vegetable bread 755 bp amplicons could be observed (Fig. longer than ca. 500 bp. These processes were also confi rmed 4B and E). Doughnut, pie, and English muffin gave short for mild fragmentation in genomic DNA of soybean and corn amplicons of up to 430 bp (Fig. 4G, H and J). In contrast, materials10, 11). The roasting process to produce white sauce all noodles gave 755 bp amplicons regardless of the process. for stew, similar to kinako powder production by roasting These results suggested that neither the addition of alkaline soybean grains, gave severe fragmentation of genomic DNA lye water for elastic noodle dough nor boiling at 100°C caused to amplicons shorter than 515 bp (Fig. 4S). A food process to severe fragmentation of wheat genomic DNA (Table 4). This produce canned soybeans and retort packed boiled-soybeans might occur because noodles have a large surface area so (Nimame) sold in markets at room temperature, involving that processing is concluded after a short time compared with sterilization by autoclave at 121°C for a short time also gave breads, therefore, genomic DNA of noodles was not degraded severe DNA fragmentation of amplicons less than 195 bp10). into short fragments. However, with retort stew also sterilized by autoclave, we Short-term fermentation by yeast did not give rise to could detect an amplicon of 430 bp as a faint band (Fig. 4T). a serious degradation of genomic DNA as shown by the Food sterilization by autoclave over a short time can avoid loss quantitative PCR in homemade bread. In general recipes, of taste, color, and fl avor, but can cause severe fragmentation yeast fermentation is ended in less than 4 h for bread baking. of genomic DNA. In contrast, the Japanese traditional cake “Kuzumochi” Common commercial processed foods made from wheat is processed by Lactobacillus fermentation for about one flour maintained genomic DNA longer than 211 bp length year, thus, genomic DNA was fragmented into amplicons even after fermentation by yeast or Lactobacillus, baking, shorter than 300 bp (Fig. 4U). Fragmentation of genomic boiling, frying, and roasting. We demonstrated that the DNA was moderate in bread fermented by yeast, but long- official qualification and quantification detection methods term fermentation by Lactobacillus brought severe DNA for transgene DNA derived from wheat GM materials in fragmentation, which was the same for miso and soy sauce10). processed foods might be approved because amplicons

Breads and buns A M 1 2 3 4 5 6 7 B M 1 2 3 4 5 6 7 C M 1 2 3 4 5 6 7 D M 1 2 3 4 5 6 7 E M 1 2 3 4 5 6 7

F M 1 2 3 4 5 6 7 G M 1 2 3 4 5 6 7 H M 1 2 3 4 5 6 7 I M 1 2 3 4 5 6 7 J M 1 2 3 4 5 6 7

Noodles K M 1 2 3 4 5 6 7 L M 1 2 3 4 5 6 7 M M 1 2 3 4 5 6 7 N M 1 2 3 4 5 6 7 O M 1 2 3 4 5 6 7 P M 1 2 3 4 5 6 7

Chinese dim sum Stew Q M 1 2 3 4 5 6 7 R M 1 2 3 4 5 6 7 S M 1 2 3 4 5 6 7 T M 1 2 3 4 5 6 7

Kuzumochi Food additives U M 1 2 3 4 5 6 7 V M 1 2 3 4 5 6 7 W M 1 2 3 4 5 6 7

Fig. 4. Qualitative PCR for genomic DNAs prepared from commercial processed foods [Breads and buns] A, roll; B, steamed bun; C, fried bread; D, croissant; E, vegetable bread; F, muffin; G, doughnut; H, pie; I, corn mayonnaise bread; J, English muffi n; [Noodles] K, Udon; L, Yakisoba; M, Chinese noodles; N, fried noodles; O, snack noodles; P, non-fried noodles; [Chinese dim sum] Q, bun a part of Chinese dumping; R, a pastry of fried wonton; [Stew] S, stew cooked with stew mix; T, retort packaged stew [Kuzumochi] U, Kuzumochi; [Food additives] V, wheat starch; W, wheat gluten. M and 1-7 in each panel is the same as Figure 2. 日本食品化学学会誌（日食化誌）、Vol. 23(3), 2016 147

of about 100 bp genomic DNA length remained in these (2013). processed foods shown here. 9) Nakamura, K., Yamada, C., Akiyama, H., Takabatake, R., Kitagawa, M., Kitta, K., Kawakami, H., Teshima, R.: Evaluation of tomato DNA fragmentation and PCR Ⅳ Acknowledgments amplicon size for detection of tomato DNA in processed products. Jpn. J. Food Chem. Safety, 17, 123-129 (2010). This study was supported in part by a grant from The Tojuro 10) Ogasawara, T., Arakawa, F., Akiyama, H., Goda, Y., Iijima Foundation for Food Science and Technology and The Ozeki, Y.: Fragmentation of DNAs of processed foods Japan Food Chemical Research Foundation. made from genetically modified soybean. Jpn. J. Food Chem., 10, 155-160 (2003). 11) Ogasawara, T., Arakawa, F., Watanabe, T., Akiyama, H., Ⅴ References Hino, H., Maitani, T., Goda, Y., Ozeki, Y.: Genomic DNA fragmentation of genetically modified corn during food 1) Harada, H., Maoka, T., Osawa, A., Hattan, J., Kanamoto, processing. Jpn. J. Food Chem., 11, 137-143 (2004). H., Shindo, K., Otomatsu, T., Misawa, N.: Construction 12) Iida, M., Yamashiro, S., Yamakawa, H., Hayakawa, K., of transplastomic lettuce (Lactuca sativa) dominantly Kuribara, H., Kodama, T., Furui, S., Akiyama, H., Maitani, producing astaxanthin fatty acid esters and detailed T., Hino, A.: Development of taxon-specific sequences of chemical analysis of generated carotenoids. Transgenic common wheat for the detection of genetically modified Res., 23, 303-315 (2014). wheat. J. Agric. Food. Chem., 53, 6294-6300 (2005). 2) Wang, W.S., Zhao, X.Q., Li, M., Huang, L.Y., Xu, 13) Imai, S., Tanaka, K., Nishituji, Y., Kikuchi, Y., Matsuoka, J.L., Zhang, F., Cui, Y.R., Fu, B.Y., Li, Z.K.: Complex Y., Arami, S., Sato, M., Haraguchi, H., Kurimoto, Y., molecular mechanisms underlying seedling salt tolerance Mano, J., Furui, S., Kitta, K.: An endogenous reference in rice revealed by comparative transcriptome and gene of common and durum wheat for detection of metabolomic profiling. J. Exp. Bot., 67, 405-419 (2016). genetically modified wheat. Food Hyg. Saf. Sci., 53, 203- 3) GMOs List (http://www.mhlw.go.jp/file/06-Seisakujouhou- 210 (2012). 11130500-Shokuhinanzenbu/0000071167.pdf) (Apr. 25, 14) Test Results (http://www.maff.go.jp/j/press/seisan/boeki/ 2015), Ministry of Health, Labour, and Welfare, Japan. pdf/130705-01.pdf) (Jul. 5, 2013), Ministry of Agriculture, 4) Q&A for food labeling, chapter 3 (http://www.caa.go.jp/ Forestry and Fisheries, Japan. foods/pdf/syokuhin737.pdf) (Dec. 2011), Consumer Affairs 15) Pauli, U., Linger, M., Zimmermann, A., Schrott, M.: Agency, Japan. Extraction and amplification of DNA from 55 foodstuffs. 5) Bettazzi, F., Lucarelli, F., Palchetti, I., Berti, F., Marrazza, Mitt. Lebensm. Hyg., 91, 491-501 (2000). G., Mascini, M.: Disposable electrochemical DNA-array 16) Gryson, N., Messens, K., Dewettinck, K.: PCR detection for PCR amplified detection of hazelnut allergens in of soy ingredients in bread. Eur. Food Res. Technol., foodstuffs. Anal. Chim. Acta., 614, 93-102 (2008). 227, 345-351 (2008). 6) Arlorio, M., Cereti, E., Coïsson, J.D., Travaglia, F., 17) Notification No. 3 (Jly. 3, 2015), Director-General, Martelli, A.: Detection of hazelnut (Corylus spp.) in Department of Food Safety, Ministry of Health, Labour processed foods using real-time PCR. Food Control, 18, and Welfare, Japan. (http://www.mhlw.go.jp/topics/yunyu/ 140-148 (2007). other/2013/dl/130703-01.pdf) (Jul. 3, 2013) 7) Noguchi, A., Akiyama, H., Nakamura, K., Sakata, K., 18) Gryson, N.: Effect of food processing on plant DNA Minegishi, Y., Mano, J., Takabatake, R., Futo, S., Kitta, K., degradation and PCR-based GMO analysis: a review. Teshima, R., Kondo, K., Nishimaki-Mogami, T.: A novel Anal. Bioanal. Chem., 396, 2003-2022 (2010). trait-specific real-time PCR method enables quantification 19) Murai, J., Taira, T., Ohta, D.: Isolation and characterization of genetically modified (GM) maize content in ground of the three Waxy genes encoding the granule-bound starch grain samples containing stacked GM maize. Eur. Food synthase in hexaploid wheat. Gene, 234, 71-79 (1999). Res. Technol., 240, 413-422 (2015). 20) Chao, S., Sharp, P.J., Worland, A.J., Warham, E. J., 8) Nakamura, K., Akiyama, H., Takahashi, Y., Kobayashi, Koebner, R. M.D., Gale, M. D.: RFPL-based genetic T., Noguchi, A., Ohmori, K., Kasahara, M., Kitta, K., maps of wheat homoeologous group 7 chromosomes. Nakazawa, H., Kondo, K., Teshima, R.: Application of a Theor. Appl. Genet., 78, 495-504 (1989). qualitative and quantitative real-time polymerase chain 21) Kakihara, Y., Matsufuji, H., Chino, M., Takeda, M.: reaction method for detecting genetically modified papaya Extraction and detection of endogenous soybean DNA line 55-1 in papaya products. Food Chem., 136, 895-901 from fermented foods. Food Control, 17, 808-813 (2006). 148 Jpn. J. Food Chem. Safety, Vol. 23(3), 2016

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小麦加工食品におけるゲノム DNA 断片化の調査 （2016 年 6 月 22 日受付） （2016 年 7 月 29 日受理）

宮原 平 a)、三宅奈穂 a)、澤藤ことは a)、橘田和美 b)、中村公亮 c)、近藤一成 c)、小関良宏 a)

a) 東京農工大学大学院工学研究院生命機能科学部門 b) 農業・食品産業技術総合研究機構食品総合研究所 c) 国立医薬品食品衛生研究所生化学部

キーワード : DNA 断片化、小麦粉、小麦加工食品、定性 PCR

概 要 小麦加工食品から公定法に従いゲノム抽出を行った結果、生地に油分の多く含まれる製品ではゲノム DNA の収量が低下する ものの、PCR により解析が可能な純度は維持できることが確認された。また焼き、蒸し、揚げなどの熱をかける工程でゲノム DNA の断片化が進行しやすく、酵母による短時間の発酵ではほとんど断片化は起こらないことが示された。一方で乳酸菌によ る長時間の発酵ではゲノム DNA は 200 bp ほどまで断片化していることも明らかとなった。これらの結果より、小麦穀粒および粉 砕物を対象とした遺伝子組換え小麦検知の公定法は、PCR により検知する遺伝子のサイズを 100 bp 程度に設計していることか ら、一般的に市場に流通している小麦加工食品にも適用できる可能性を示した。

連絡先： 〒 184-8588 東京都小金井市中町 2-24-16 東京農工大学 宮原 平