Diarrheagenic Escherichia coli strains harboring the astA gene which encodes for the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) have been implicated in several foodborne outbreaks in Japan even in the absence of any other specific virulence marker of each pathovar. The polymerase chain reaction (PCR) assay is a critical tool used for detection and many astA specific primer sets have been developed though their specificity and sensitivity for astA detection directly from food samples have not been evaluated. Herein, four distinct PCR primer sets and three enzymes were evaluated in enriched food cultures to optimize astA detection. The Yamamoto & Echeverria PCR method yielded clear, easily interpretable results with high intensity of PCR product or no products. Combining this primer set with the Quick Taq HS DyeMix enzyme resulted in astA-specific amplicons without non-specific products from food cultures, indicating the superiority of this system in detecting astA in food samples. Furthermore, this primer set demonstrated the highest consistency with the E. coli harboring astA-isolation results. Subsequently, this system exhibited high specificity and sensitivity with a ≤5 log CFU/mL detection limit. These findings suggest that combining the Yamamoto & Echeverria primer set and the Quick Taq HS DyeMix offers an effective tool for detecting astA in food samples. We anticipate this PCR assay will enhance the detection and subsequent isolation of E. coli strains harboring astA from food products.

Safety assessments are necessary for genetically modified foods in many countries, including Japan. Stabilities during pepsin, trypsin, or pancreatin digestion are a key criterion for assessing the allergenic potential of newly expressed proteins (NEPs). In digestibility tests, NEPs produced by heterologous expression systems were frequently used. Polyhistidine tags (His-tags) are primarily/often used to purify recombinant proteins. Studies of His-tags’ influences remain limited on the susceptibility of a protein to pepsin/trypsin digestion, although His-tags can affect protein folding and stability. In this study, we compared the digestibility of the natural peanut allergenic proteins Ara h 1 and Ara h 2 to the recombinant Ara h 1 protein with N-terminal His-tag and recombinant Ara h 2 protein with C-terminal His-tag, respectively. Peptides after the proteolysis were then analyzed using liquid chromatography–tandem mass spectrometry to determine the proteolytic cleavage sites. Differences were detected in the C-terminal region after pepsin cleavage of the His-tag extension of Ara h 1 and Ara h 2 proteins. No differences were observed in other cleavage sites between the natural and recombinant Ara h 1 and Ara h 2 proteins. The N-terminal region of Ara h 1 and Ara h 2, at which the epitopes recognized by most patients allergic to peanut were located, was equally resistant to pepsin digestion regardless of the natural or recombinant forms. In this study, an unintended short protein isoform was detected in the recombinant Ara h 2 samples. This short recombinant isoform may be misfolded, and it showed reduced susceptibility to pepsin digestion relative to natural full-length Ara h 2. In this short Ara h 2 isoform, newly paired disulfide bonds may make it more rigid. Recombinant proteins with His-tags can provide nearly comparable results to the corresponding natural proteins in protease digestions and thus offer information useful for safety assessment.

Beauvericin (BEA) and enniatins (ENNs) are cyclic depsipeptide mycotoxins mainly produced by Fusarium species. To investigate their presence in retail foods in Japan, we developed an analytical method for the simultaneous determination of BEA, enniatin A (ENNA), enniatin A₁ (ENNA₁), enniatin B (ENNB), and enniatin B₁ (ENNB₁). Five mycotoxins were extracted from food samples using a mixture of acetonitrile and water, and then purified using a C18 cartridge. LC-MS/MS was used to quantify the purified mycotoxins. This method was validated in an inter-laboratory study. Eight laboratories participated in the study, and three spiked and two naturally contaminated wheat samples were analyzed. The ranges of the mean recoveries of BEA, ENNA, ENNA₁, ENNB, and ENNB₁ were 92‒94, 94‒96, 97‒98, 98‒99, and 98‒100%, respectively. The relative standard deviations for repeatability and reproducibility in spiked and naturally contaminated samples ranged from 2.1 to 5.7% and from 6.2 to 15.3%, respectively. After the application of the method to the analysis of these five mycotoxins in other foods was confirmed by recovery tests, 658 food samples including cereals, bean products and dry fruits were analyzed using the developed analytical method. BEA, ENNA, ENNA₁, ENNB, and ENNB₁ were detected in 23%, 7%, 17%, 40% and 33% of all samples, respectively, at >1.5 µg/kg. About the result of BEA, the highest mean level in 12 food groups was shown in soybean flour samples (13 µg/kg). Among the four ENNs, the positive rate of ENNB was the highest in all food groups. ENNB was mainly detected in rye flour and wheat flour, and the mean ENNB levels of rye flour and wheat flour were 987 and 49 µg/kg, respectively. Our results are useful for the risk assessment of BEA and ENNs in retail foods in Japan.

Puffer fish and products containing puffer fish are highly regulated and restricted in the United States due to the potential presence of the alkaloid toxins tetrodotoxins (TTX) and saxitoxins (STX). Imported and domestic puffer fish are regulated under the U.S. Code of Federal Regulations (21 CFR Part 123 - Fish and Fishery Products) which identifies Hazard Analysis Critical Control Point (HACCP) processes for the control of specific hazards including natural toxins. Additional restrictions are placed on puffer fish depending on the source. The only approved source of imported puffer fish is allowed through an Exchange of Letters between the U.S. Food and Drug Administration and the Japanese Ministry of Health, Labour, and Welfare restricting imported products to the meat, skin, and testicles of Takifugu rubripes. Additional restrictions are placed on domestic puffer fish through specific state bans. Despite these efforts, puffer fish poisoning cases still occasionally occur. Illnesses from imported products have mainly been due to TTX in the meat of illegally imported Lagocephalus lunaris, while illnesses from domestically sourced products have been due to STX in the meat of Sphoeroides nephelus harvested from the Atlantic coast of Florida.

Food Safety Commission of Japan (FSCJ) conducted a risk assessment regarding the use of so-called “cattle-derived MBM” as raw material in feed intended for chickens, pigs, and others in response to a request of the Ministry of Agriculture, Forestry and Fisheries (MAFF). As far as the current risk mitigation measures against BSE are implemented, BSE prions are highly unlikely to be accumulated in the cattle, sheep, and goat parts which would be used as raw materials of feed for chickens, pigs, and others. There are negligible occurrence of cattle-derived MBM to be fed to cattle and other ruminants, as long as the Japanese current risk mitigation measures against feeding cattle-derived MBM to ruminants continue to be abided. Furthermore, oral transmission of BSE prions to chickens, pigs, and others is unlikely to occur, based on accumulated scientific findings. The risk of human infection with BSE is considered to be highly unlikely. FSCJ thus concluded negligible adverse human health-effects of foods from chickens, pigs, and others, in Japan’s circumstances where cattle-derived MBM is used as raw materials for feed these specified animals.