Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi) Vol. 58(2017) No. 3

Omphalotus japonicus is a poisonous mushroom that grows in Japan. It can be mistaken for edible mushrooms (Shiitake, Hiratake and Mukitake), and if ingested, it causes food poisoning within 30 min to 1 hr. We established a rapid detection method using PCR-RFLP to identify O. japonicus by restriction digestion of the amplified ITS region. By using Sau96I, Bpu10I, SfcI or DrdI/HincII as a restriction enzyme, it was possible to rapidly identify and discriminate O. japonicus based on the fragment length. This study also provided a short PCR-RFLP system comprising amplification and digestion of a short 200-bp DNA fragment within the ITS region. The system could identify and discriminate O. japonicus after in vitro gastric digestion of native and heated mushroom samples as a model of food poisoning. In addition, a confirmatory assay using real-time PCR was developed to achieve more sensitive detection of O. japonicus.

Following the previous report, a rapid dialysis method was developed for the extraction and purification of four artificial sweeteners, namely, sodium saccharide (Sa), acesulfame potassium (AK), aspartame (APM), and dulcin (Du), which are present in various foods. The method was evaluated by the addition of 0.02 g/kg of these sweeteners to a cookie sample, in the same manner as in the previous report. Revisions from the previous method were: reduction of the total dialysis volume from 200 to 100 mL, change of tube length from 55 to 50 cm, change of dialysate from 0.01 mol/L hydrochloric aqueous solution containing 10% sodium chloride to 30% methanol solution, and change of dialysis conditions from ambient temperature with occasional shaking to 50℃ with shaking at 160 rpm. As a result of these revisions, the recovery reached 99.3–103.8% with one hour dialysis. The obtained recovery yields were comparable to the recovery yields in the previous method with four hour dialysis.

In this study, species-specific identification of five toxic mushrooms, Chlorophyllum molybdites, Gymnopilus junonius, Hypholoma fasciculare, Pleurocybella porrigens, and Tricholoma ustale, which have been involved in food-poisoning incidents in Japan, was investigated. Specific primer pairs targeting internal transcribed spacer (ITS) regions were designed for PCR detection. The specific amplicons were obtained from fresh, cooked, and simulated gastric fluid (SGF)-treated samples. No amplicons were detected from other mushrooms with similar morphology. Our method using one-step extraction of mushrooms allows rapid detection within 2.5 hr. It could be utilized for rapid identification or screening of toxic mushrooms.

An analytical method for the determination of residues of 3 phenicol drugs (chloramphenicol, thiamphenicol and florfenicol) in Ayu (Plecoglossus altivelis) by LC-MS/MS was developed. We used the whole body of Ayu, including the bones and internal organs, in addition to muscle. Phenicols were extracted with 90% acetonitrile and an aliquot of the crude extract was cleaned up on a Florisil column (2 g), followed by defatting with n-hexane. The acetonitrile extract was evaporated and the solvent was replaced with phosphate buffer, then the extract was purified on a hydroxylated styrene–divinylbenzene copolymer column (200 mg). Finally, sample solution was passed through a deproteination cartridge filter with a lipid removal function. Chloramphenicol was quantitated by means of a calibration curve corrected with salogate standard (chloramphenicol-d₅) and thiamphenicol and florfenicol were quantitated based on absolute calibration curves. This method was validated in accordance with the notification of the Ministry of Health, Labour and Welfare of Japan. As a result of the validation study, the trueness, repeatability and within-laboratory reproducibility were 85–103, 5–13 and 8–13%, respectively. This method is useful for inspecting residues of 3 phenicol drugs in whole body of Ayu efficiently. Moreover, when chloramphenicol and thiamphenicol are detected by this method, the quantitated value is applicable to decide the compliance of the sample with the specifications and standards of the Food Sanitation Law.

An analytical method for the determination of nonvolatile amines (putrescine, cadaverine, histamine, tyramine, and spermidine) in foods was developed, using an improved dansyl derivatization technique. The five amines were extracted from food with 1% trichloroacetic acid. Three milliliter of extract was applied to a polymer-based strong cation exchange resin mini-column, which was washed with 5 mL of water, and eluted with 5 mL of 1 mol/L potassium carbonate solution. The eluate was dansylated, then 5 mL of toluene was added with shaking. The toluene layer was evaporated. The residue was taken up in 1 mL of acetonitrile and shaken with 1 mL of 5% proline in 1 mol/L potassium carbonate solution. The upper acetonitrile layer was collected, filtered, and subjected to HPLC. The limits of quantitation for putrescine and cadaverine in the samples were both 0.2 μg/g; those of spermidine, tyramine, and histamine were 0.8, 2.0, and 5.0 μg/g, respectively. The average recoveries of the five amines from nine foods exceeded 80%.

We evaluated the effectiveness of new cleanup agents (S-NH₂ and S-Si) compared with other previously reported cleanup agents (octadecylsilane, graphitized carbon, aminopropyl and silica gel) for removal of interfering substances such as catechin and caffeine prior to analysis of pesticide residues in tea. S-NH₂ and S-Si were highly efficient in removal of catechin and caffeine, respectively. Recoveries of 80 pesticides using S-NH₂ and S-Si were tested, and more than 70% of pesticides showed recovery greater than 70%. These results indicate that S-NH₂ and S-Si agents will be useful for analysis of pesticide residues in tea.

Extraction and clean-up methods were examined for the analysis of acidic tar dyes in various high-protein foods. 1% Aqueous ammonia followed by ethanol, 1% aqueous ammonia–ethanol (1 : 1) mixture, and 1% aqueous ammonia–tetrahydrofuran (1 : 1) mixture were used in sequence for boiled fish paste (kamaboko), pounded fish cake (hanpen), and sausage. The sausage extract was centrifuged at low temperature to solidify and remove the contained fat. Salted cod roe with red pepper was extracted twice with 1% aqueous ammonia–ethanol (1 : 1) mixture, followed by extraction with 1% aqueous ammonia–tetrahydrofuran (1 : 1) mixture. A divinylbenzene–N-vinylpyrrolidone copolymer column was used for the clean-up of xanthen dyes. In the case of clogging-prone samples, the same type of large-particle-size column was used. A polyamide column was used for clean-up of the other dyes. When each dye was added at 5 μg/g in the foods, recoveries from kamaboko, hanpen, and sausage ranged from 76 to 102%, and the average recovery from the two types of salted cold roe with red pepper ranged from 45 to 98%.

Stainless steel kitchenware and tableware on sale in Japan were investigated. Surface elemental composition ratios of 172 samples were analyzed by the fluorescence X-ray method. High levels of manganese (9.59–20.03%)were detected in 17 samples. This finding was confirmed by ICP analysis. Next, we conducted migration tests. Samples conformed to the Italian Specific Migration Limits. Moreover, lead and antimony were not detected in these samples, in accordance with the Japanese Food Sanitation Law.