Four-year surveillance for ochratoxin a and fumonisins in retail foods in Japan.

Between 2004 and 2007 we examined foods from Japanese retail shops for contamination with ochratoxin A (OTA) and fumonisins B(1), B(2), and B(3). A total of 1,358 samples of 27 different products were examined for OTA, and 831 samples of 16 different products were examined for fumonisins. The limits of quantification ranged from 0.01 to 0.5 microg/kg for OTA and 2 to 10 microg/kg for the fumonisins. OTA was detected in amounts higher than limits of quantification in wheat flour, pasta, oatmeal, rye, buckwheat flour and dried buckwheat noodles, raisins, wine, beer, coffee beans and coffee products, chocolate, cocoa, and coriander. OTA was found in more than 90% of the samples of instant coffee and cocoa, and the highest concentration of OTA, 12.5 microg/kg, was detected in raisins. The concentration of OTA in oatmeal, rye, raisins, wine, and roasted coffee beans varied remarkably from year to year. Fumonisins were detected in frozen and canned corn, popcorn grain, corn grits, cornflakes, corn soups, corn snacks, beer, soybeans, millet, and asparagus. The highest concentrations of fumonisins B(1), B(2), and B(3) were detected in corn grits (1,670, 597, and 281 microg/kg, respectively). All of the samples of corn grits were contaminated with fumonisins, and more than 80% of the samples of popcorn grain and corn snacks contained fumonisins. OTA and fumonisins were detected in several food products in Japan; however, although Japan has not set regulatory levels for these mycotoxins, their concentrations were relatively low.

[Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS].

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).

[Investigation of ochratoxin a, B and citrinin contamination in various commercial foods].

Ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in commercial foods were simultaneously determined and confirmed with high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The samples examined were made up of cereal, fruit, coffee, and cacao products. The limits of quantification (S/N> or =10) of OTA, OTB and CIT were 0.1 microg/kg or less. Aflatoxins (AF), deoxynivalenol (DON) and fumonisins were also surveyed. Of 157 samples examined, 44 were contaminated with OTA at levels of 0.11 to 4.0 microg/kg. At least 2 positive samples were labeled as domestics. In most positive samples, the OTA level was low, less than 1 microg/kg. The highest incidence of OTA was observed in cacao powder (10/12), followed by instant coffee (5/7), cocoa (5/8) and raisin (6/13). OTB was found in fruit and cacao products containing relatively high levels of OTA. Co-occurrence of OTA, CIT and DON was found in cereal products, and co-occurrence of OTA and AF was found in cacao products. Approximately 30% of naturally contaminated OTA in roasted coffee bean moved into the extract solution when brewed with paper filter.

Survey of Nitrate Ion Concentrations in Vegetables Cultivated in Plant Factories: Comparison with Open-Culture Vegetables.

A survey of nitrate-ion concentrations in plant-factory-cultured leafy vegetables was conducted. 344 samples of twenty-one varieties of raw leafy vegetables were examined using HPLC. The nitrate-ion concentrations in plant-factory-cultured leafy vegetables were found to be LOD-6,800 mg/kg. Furthermore, the average concentration values varied among different leafy vegetables. The average values for plant-factory-cultured leafy vegetables were higher than those of open-cultured leafy vegetables reported in previous studies, such as the values listed in the Standard Tables of Food Composition in Japan- 2015 - (Seventh revised edition). For some plant-factory-cultured leafy vegetables, such as salad spinach, the average values were above the maximum permissible levels of nitrate concentration in EC No 1258/2011; however, even when these plant-factory-cultured vegetables were routinely eaten, the intake of nitrate ions in humans did not exceed the ADI.

Occurrence of aflatoxins, ochratoxin A, and fumonisins in retail foods in Japan.

We conducted a survey of aflatoxin B1, B2, G1, and G2, ochratoxin A, and fumonisin B1, B2, and B3 contamination in various foods on the retail market in Japan in 2004 and 2005. The mycotoxins were analyzed by high-performance liquid chromatography, liquid chromatography-mass spectrometry, or high-performance thin-layer chromatography. Aflatoxins were detected in 10 of 21 peanut butter samples; the highest concentration of aflatoxin B1 was 2.59 microg/kg. Aflatoxin contamination was not found in corn products, corn, peanuts, buckwheat flour, dried buckwheat noodles, rice, or sesame oil. Ochratoxin A was detected in oatmeal, wheat flour, rye, buckwheat flour, green coffee beans, roasted coffee beans, raisins, beer, and wine but not in rice or corn products. Ochratoxin A concentrations in contaminated samples were below 0.8 microg/kg. Fumonisins were detected in popcorn, frozen corn, corn flakes, and corn grits. The highest concentrations of fumonisins B1, B2, and B3 in these samples were 354.0, 94.0, and 64.0 microg/kg, respectively.

[Inter-laboratory study for validation of a Japanese official analytical method for determination of patulin in apple juice].

To validate a modified version of AOAC official method of analysis 995.10 as an official standard in Japan for determination of patulin in apple juice, an inter-laboratory study was performed in 11 laboratories using a non-contaminated sample, 2 naturally contaminated samples and 2 spiked samples of apple juice. For naturally contaminated apple juices, the relative standard deviations for repeatability and reproducibility were 3.2, 7.1% and 10.0, 21.7%, respectively. HORRAT values were 0.4, 0.9. The average recovery of patulin from spiked sample was 83.7%. The limit of quantification was calculated as 10 microg/kg. From these results, the method was thought to be suitable as an official standard for determination of patulin in apple juice in Japan.

A quantification and confirmation method of patulin in apple juice by GC/MS.

A sensitive and selective method for quantification and confirmation of patulin in apple juice by GC/MS was developed. By this method, patulin was precisely determined and confirmed down to the level of 1 and 5 microg/kg in samples, respectively. Patulin was extracted with ethyl acetate from a sample and then hexane was added to the concentrated extract solution. Significant amounts of insoluble impurities were filtered off, followed by further clean-up by solid-phase extraction with combined silica gel and Florisil cartridges. The filtration step in a low-polarity condition was very effective to remove the impurities in the sample extract solution. The eluate from the cartridges was evaporated to dryness under reduced pressure and patulin was determined and confirmed by GC/MS after derivatization with 2.5% N,O-bis(trimethylsilyl)trifluoroacetamide ethyl acetate solution. Patulin was determined in the selected ion monitoring mode (m/z 226) and confirmed in the SCAN mode (m/z 40-340). The recovery from apple juice spiked with 10-500 microg/kg ranged from 93.4 to 100%. The limits of detection and quantification were 0.1 (S/N = 3) and 1 microg/kg (S/N = 30) of patulin in samples, respectively. Levels down to 5 microg/kg of patulin in sample were readily confirmed.

[The problem and improvement of colorimetric method for determination of sulfite in foods containing sulfur compounds].

The modified Rankine colorimetric method for measuring sulfite added to food as a food additive has a low determination limit and is little influenced by interfering substances from foods. However, it can give erroneous results for foods containing Liliaceae Allium. So, four different methods, alkaline titration, a colorimetric method, ion chromatography and qualitative analysis with potassium iodate-starch paper, were examined. It was found that the sodium azide used in the colorimetric method forms sulfur dioxide during bubbling and heating. The proposed colorimetric method can be applied to food containing sulfur compounds, if sodium azide is omitted and 1% triethanolamine solution is used as an absorbent instead of 0.1 mol/L sodium hydroxide solution.

[Production of tyramine in "moromi" mash during soy sauce fermentation].

The concentrations of 7 non-volatile amines, tyramine (Tym), histamine (Him), phenethylamine (Phm), putrescine (Put), cadaverine (Cad), spermidine (Spd) and spermine (Spm) in the liquid part of "moromi" mash during soy sauce fermentation were studied. These amines, except for him and Cad, were detected during fermentation by the conventional production method in the laboratory. Put and Spd were detected at the beginning, and Tym, Phm and Spm appeared later; these 5 amines increased gradually during the fermentation. Put, Spd, Spm and Cad were present in the raw starting material for soy sauce; thus, Tym and Phm were produced by the fermentation. When "moromi" mash was added to liquid medium and cultivated, Tym was detected in some "moromi" mash and the other amines were not detected. Tym-producing bacterial strains were isolated from the liquid culture media of Tym-positive "moromi" mash. The Tym-producing strain was a gram-positive coccus. The conditions for production of amines by Tym-producing bacterial strains were examined. These strains grew and produced tyramine under various conditions, which may occur during soy sauce fermentation. Namely, Tym was produced at pH 5-10, at salt concentrations of less than 8%, under either aerobic or anaerobic conditions. During soy sauce fermentation, it is assumed that Tym would be produced by these strains during the early stages of soy sauce aging within a short period when the salt concentration and pH conditions are optimal for growth. Based on the bacteriological properties, the strains were identified as Enterococcus faecium. With the exception of Phm and Him, which did not exist in the starting raw material, non-volatile amines (including Put, Cad, Spd and Spm) were not produced and microorganisms producing them are not believed to be present during "moromi" fermentation.

Occurrence of four Fusarium mycotoxins, deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin, in wheat, barley, and Japanese retail food.

A survey of the contamination of wheat, barley, and Japanese retail food by four Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), and HT-2 toxin (HT-2), was performed between 2010 and 2012. A method for the simultaneous determination of the four mycotoxins by liquid chromatography-tandem mass spectrometry was validated by a small-scale interlaboratory study using two spiked wheat samples (DON was spiked at 20 and 100 µg/kg and ZEN, T-2, and HT-2 at 6 and 20 µg/kg in the respective samples). The recovery of the four mycotoxins ranged from 77.3 to 107.2%. A total of 557 samples of 10 different commodities were analyzed over 3 years by this validated method. Both T-2 and HT-2 were detected in wheat, wheat flour, barley, Job's tears products, beer, corn grits, azuki beans, soybeans, and rice with mixed grains. Only T-2 toxin was detected in sesame seeds. The highest concentrations of T-2 toxin (48.4 µg/kg) and HT-2 toxin (85.0 µg/kg) were present in azuki beans and wheat, respectively. DON was frequently detected in wheat, wheat flour, beer, and corn grits. The contamination level of wheat was below the provisional standard in Japan (1,100 µg/kg). The maximum contamination level of DON was present in a sample of a Job's tears product (1,093 µg/kg). ZEN was frequently detected in Job's tears products, corn grits, azuki beans, rice with mixed grains, and sesame seeds. A sample of a Job's tears product presented the highest ZEN contamination (153 µg/kg). These results indicate that continuous monitoring by multiple laboratories is effective and necessary due to the percentage of positive samples detected.

Validation of an HPLC analytical method coupled to a multifunctional clean-up column for the determination of deoxynivalenol.

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of naturally contaminated wheat were in the range 5.8-11.3% and 12.0-20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSDr, RSDR and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.

Degradation of Aflatoxins by Food Additives.

Degradation of four aflatoxins (AFB1, AFB2, AFG1 and AFG2) by food additives was investigated. Pure aflatoxins were degraded by treatment with solutions of acidic food additives (hydrochloric acid:HCl and sulfuric acid:H2SO4), alkaline food additives (sodium bicarbonate:NaHCO3, sodium carbonate:Na2CO3, sodium hydroxide:NaOH, sodium sulfite:Na2SO3, and sodium hypochlorite:NaOCl) and neutral food additives (potassium metabisulfite:K2S2O5, sodium bisulfite:NaHSO3, sodium hydrosulfite:Na2S2O4, hydrogen peroxide:H2O2, sodium chlorite:NaClO2, and ammonium peroxodisulfate:(ÑH4)2S2O8). The aflatoxins were treated with these neutral food additives under several conditions, and the effects of treatment temperature, time, and concentration of food additives on aflatoxin degradation were studied. Potassium bromate (KBrO3), potassium nitrate (KNO3), and sodium nitrite (NaNO2) had no effect on aflatoxins. Of the aflatoxin added to corn, 20% AFB, remained after treatment with the solution of NaHSO3 (0.5%, 48 h), but all of the AFB1 was completely degraded by NaClO2 (0.25%, pH 4, 48 h) and (NH4)2S2O8 (0.25%, 48 h) at 60°C. Of the aflatoxins added to butter beans, less than 20 and 5% of AFB1 remained after boiling treatment with a 2 and 0.5% solution of Na2S2O4, respectively. These findings suggested that aflatoxins can be degraded or removed by treatment with food additives during food processing.