Detection of soybean proteins in fermented soybean products by using heating extraction.

UNLABELLED: Soybean is used in processed foods worldwide. Because soybean can cause adverse reactions in some atopic patients, appropriate labeling regarding its content in processed foods is needed to better protect consumers. In the previous study, we developed a reliable sandwich Enzyme Linked Immunosorbent Assay (ELISA) method with high sensitivity and specificity for detecting soybean proteins by using antibody to Gly m Bd 30K, which was originally characterized as a vacuolar protein with a molecular mass of 34 kDa in soybean. The ELISA displayed satisfactory repeatability and reproducibility in an interlaboratory evaluation. However, it could not detect soybean protein in fermented soybean products. We therefore developed an extraction method combined with a heating process to inhibit soybean protein degradation by microbial proteolytic enzymes in fermented soybean products. This extraction method enables the sensitive detection of soybean protein in fermented soybean products such as natto and miso. It was able to detect with high-sensitivity soybean protein present at 10 µg/g levels in model processed foods. This method is suitable for quantifying soybean protein in processed foods without the degrading effects of microbial proteolytic enzymes. The present extraction method can be used sensitively to monitor labeling systems in a reliable manner and should be useful for the mandatory inspections required under Japanese regulations. PRACTICAL APPLICATION: The extraction and ELISA methods that we developed enable sensitive detection of soybean protein in soybean products, including fermented foods. These methods should be useful for reliable and sensitive monitoring of product labeling systems and should help to solve the problem of insensitive in soybean labeling of processed foods.

[Correlation of fat content and dioxins, total mercury and methyl mercury levels in tuna].

In this study, we analyzed the concentrations of mercury and dioxins in tuna with various fat contents (akami; the leaner meat, Chutoro; the belly area of the tuna along the side of the fish between the akami and the otoro. Otoro; the fattiest portion of the tuna) in wild and farmed bluefin tuna and farmed southern bluefin tuna. In the three kinds of tuna, average dioxins concentrations in Akami, chutoro and otoro were 1.7, 4.7 and 9.6 pg TEQ/g, respectively. The dioxins concentration in all three regions of tuna was in direct proportion to the fat content. In the farmed bluefin tuna, the dioxins concentration was almost the same as that of the wild tuna, but differed from that of the farmed southern bluefin tuna. Average total mercury concentration based on wet weight in akami was 0.42 µg/g, being higher than the values of 0.36 µg/g of chutoro and 0.31 µg/g of otoro, and in inverse proportion to the fat content. In all three regions, the total mercury concentration of the wild bluefin tuna was equal to that of the farmed tuna. The total mercury concentration in the latter was two to three times higher than that of the farmed southern bluefin tuna. If the Japanese intake is one fin of tuna (80 g) a day, the daily intake levels of dioxins and methyl mercury can be estimated as 0.48-37 pg TEQ/kg bw and 0.21-0.90 µg/kg bw, respectively.

Specific detection of buckwheat residues in processed foods by polymerase chain reaction.

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.

Specific detection of wheat residues in processed foods by polymerase chain reaction.

A sensitive qualitative detection method for wheat in foods using polymerase chain reaction (PCR) was developed. Trace amounts of wheat in commercial food products could be qualitatively detected by this method. The sensitivity of the proposed PCR method appears to be similar to that of ELISA. The present method should be very useful for detecting wheat residues in processed foods.

[Application of two immunochromatographic test kits to screening of four kinds of allergic substances in imported foods].

We tested for four kinds of allergic substances (egg, milk, wheat and peanuts) in 52 imported processed foods using immunochromatographic test kits (ITK). ELISA was also employed to confirm the effectiveness of the ITK. Among 92 data from 23 samples, allergic substances were detected in 9 cases with one kind of ITK, but not with the ELISA test. Among 116 data from 29 samples, 6 were negative with one kind of ITK and but positive with the other ITK. These results suggested that these 4 kinds of allergic substances in imported foods can be detected by using a double-check method with two kinds of ITK.

[Evaluation of immunochromatographic test kits for food allergens using processed food models].

It has been mandatory to label five allergenic substances (AS; egg, milk, wheat, buckwheat and peanut) in all processed foods, since April 2002 in Japan. Two kinds of ELISA kits have been provided as screening test kits for the Japanese official method. The kits have many advantages but some disadvantages, i.e., the kits are not necessarily suitable for daily monitoring in food manufacturing plants, because they require various analytical equipments and the use of complicated procedures. To overcome these drawbacks, we have developed other diagnostic kits based on immunochromatography that should enable more rapid and simple screening for food allergens. Then we examined the performance of these immunochromatographic test kits (IC kits) in terms of sensitivity, repeatability and cross-reactivity to AS proteins in 11 kinds of food models with various heating conditions and physical properties. We also examined processed food models including AS protein of constant concentration, using the IC kits and ELISA kits, and compared the results. The IC kits detected AS proteins at 5 microg/g in the extracts from processed food models, and provided highly reproducible results. Cross-reactivity among the AS proteins was not observed. The results obtained using the IC kits showed performance equivalent to that of the ELISA kits we examined in unheating processed food models including AS proteins of constant concentration. The IC kits should be more suitable for daily monitoring in food manufacturing plants.

Effects of cooking on concentrations of polychlorinated dibenzo-p-dioxins and related compounds in fish and meat.

We investigated the cooking-induced changes in concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) (dioxins) using mackerel and beef. The concentrations of dioxins (29 congeners) were determined by isomer specific analyses and were compared between uncooked and cooked samples. The cooking procedures examined in this study included grilling as a fillet, boiling as a fillet, and boiling as tsumire (small, hand-rolled balls) for mackerel and boiling as a slice, broiling as a slice, and broiling as a hamburger for beef. Three trials were carried out for each cooking method. Generally, concentrations of dioxins were reduced in every cooking trial. When nondetected congener concentrations were assumed to be half the limit of detection for mackerel, the maximum percentage reductions of total concentrations given as 2,3,7,8-tetraCDD equivalents (TEQ) were 31% in grilling as a slice, 14% in boiling as a slice, and 21% in boiling as tsumire under the conditions of this study. In contrast, for beef, the reductions were 42% in boiling as a slice, 42% in broiling as a slice, and 44% in broiling as a hamburger. These results suggest that ordinary cooking processes with heating undoubtedly reduce the dioxin content in animal products, and the reductions estimated should be considered when dioxin intake is evaluated using contamination data for individual food items.

Contamination levels of PCDDs, PCDFs and Co-PCBs in commercial baby foods in Japan.

To examine dioxin contamination in commercial baby foods in Japan, congener analyses of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs) were performed on 102 varieties of baby foods obtained from supermarkets in 2001-2002. The toxic equivalent quantity (TEQ) levels for dioxins in samples ranged from < 0.001 to 0.135 pg-TEQ/g wet weight when undetected or trace levels of congeners were taken as zero. Among 102 samples tested, 26 samples exceeded 0.010 pg-TEQ/g. The highest TEQ value was for "sardine, vegetables" (0.135 pg-TEQ/g), followed by "Japanese radish (daikon), sardine" (0.080 pg-TEQ/g). Thus, dioxins were detected at low levels in baby foods containing animal products such as fishes and/or dairy products.

Inhibition of human lanosterol synthase by the constituents of Colocasia esculenta (taro).

Ethanol extracts of lyophilized vegetables were tested for inhibition of human lanosterol synthase (hOSC) in order to find the compounds to suppress cholesterol biosynthesis. Of 130 samples tested, twelve samples showed significant inhibition. Among them, Colocasia esculenta (taro) showed the highest inhibition (55% inhibition at 300 microg/ml). Examination of activity variation among eight taro cultivars indicated that "Aichi-wase" and "Yatsugashira" had the most potent activity for hOSC inhibition. In order to identify the active constituent of taro, ethanol extracts of "Aichi-wase" were partitioned with hexane and aqueous methanol, and fractionated by silica gel column chromatography. Inhibitory activity was concentrated in two major active fractions. Further purification of these fractions by preparative HPLC gave three monogalactosyldiacylglycerols and five digalactosyldiacylglycerols as active compounds that showed 28 to 67% inhibitory activities at the concentration 300 microg/ml.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (milk)].

Extracts of sausage, sauce, cookie, cereal and pasta sauce spiked with milk standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the three ELISA methods using a Milk Protein Casein ELISA Kit (Casein kit), a Milk Protein Beta-Lactoglobulin ELISA Kit (Beta-Lactoglobulin kit) and a FASTKIT Milk ELISA Kit (Milk ELISA kit) were mostly below 10%. Mean recoveries of the milk standard protein from the food extracts were over 40% in the three ELISA methods with a few excertions. The recoveries of milk standard protein from the sauce extract in Casein kit were improved by adjusting the extract to neutrality before the Casein kit assay. The recoveries of milk standard protein from cookie, cereal and pasta sauce were improved by the increasing the amount of antibody coated in the Milk ELISA kit. The detection limits of all the ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of milk protein levels in extracts of sausage, sauce, cookie, cereal and pasta sauce.

[Inter-laboratory evaluation studies for development of notified ELISA methods for allergic substances (wheat)].

Extracts of sausage, sauce, pasta sauce, fish paste and cereal spiked with wheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of both ELISA methods using a Wheat Protein ELISA Kit (Gliadin kit) and a FASTKIT Wheat ELISA Kit (Wheat ELISA kit) were mostly below 10%. Mean recoveries of the wheat standard protein from the food extracts were over 40% in the two ELISA methods except those from cereal extract determined using the Wheat ELISA kit. Repeatability relative standard deviations of wheat standard protein in the five food extracts were in the ranges of 16-26.9% and 3.7-36.2% for the Gliadin kit and the Wheat ELISA kit, respectively. Reproducibility relative standard deviations of wheat standard protein in the five food extracts were 21.6-38.5%, 29.7-53.8% for the Gliadin kit and the Wheat ELISA kit, respectively. The recoveries of wheat standard protein from the cereal extract were improved by the increasing the amount of antibody coated on the plate in the Wheat ELISA kit. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of wheat protein levels in extracts of sausage, sauce, pasta sauce, fish paste and cereal.

[Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (buckwheat)].

Inter-laboratory evaluation studies were conducted for the ELISA methods for allergic substances (buckwheat). Extracts of snack, bun and udon spiked with buckwheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate at 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Buckwheat Protein ELISA Kit (Buckwheat kit) and a FASTKIT Buckwheat ELISA kit (Buckwheat ELISA kit) were mostly below 10%. Mean recoveries of the buckwheat standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of buckwheat standard protein in three food extracts were in the ranges of 6.8-78.5% and 5.0-33.9% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. Reproducibility relative standard deviations of buckwheat standard protein in three food extracts were 11.9-69.5% and 16.5-34.1% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggest that the notified ELISA methods are reliable and reproducible for the inspection of buckwheat protein levels in extracts of snack, bun and udon.

[Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (peanuts)].

Inter-laboratory evaluation studies were conducted for ELISA methods for allergic substances (peanuts). Extracts of biscuit, sauce, chocolate and butter spiked with peanut standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Peanut Protein ELISA Kit (Peanut kit) and a FASTKIT Peanut ELISA kit (Peanut ELISA kit) were mostly below 10%. Mean recoveries of the peanut standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of peanut standard protein in four food extracts were in the ranges of 15.2-49.7% and 3.0-28.3% for the Peanut kit and the Peanut ELISA kit, respectively. Reproducibility relative standard deviations of peanut standard protein in four food extracts were 23.5-44.4%, 9.6-28.4% for the Peanut kit and the Peanut ELISA kit, respectively. The detection limits of both ELISA methods were 2-2.5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of peanut protein levels in extracts of biscuit, sauce, chocolate and butter.

Determination of enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase by LC/MS.

A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed. EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants. The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system. EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein). Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp. strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P. In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein. The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment. This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods.

Cleanup of food samples with pre-packed multi-layer silica gel column for the analysis of PCDDs, PCDFs and dioxin-like PCBs.

The cleanup procedure for the determination of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (PCBs) in food samples using a disposable pre-packed multi-layer silica gel column (multi-layer dioxin tube; D-tube) was evaluated. The blank test showed the need for conditioning of the column with n-hexane. To compare the method with the D-tube and the conventional method for the analyses of actual food samples, seven food samples (spinach, komatsuna, rice, salmon, beef, egg and butter) were extracted by shaking with acetone-n-hexane or n-hexane after alkaline treatment, and then the extracts were cleaned up by use of the D-tube or the prepared conventional column, followed by several column chromatographic steps. Both cleanup procedures gave similar values at each isomeric concentration level and showed similar efficiency with favorable recoveries. The results suggest that the D-tube is applicable to cleanup for the analysis of PCDD/Fs and dioxin-like PCBs in foods.

Validation of the CALUX bioassay for the screening of PCDD/Fs and dioxin-like PCBs in retail fish.

The chemical-activated luciferase expression (CALUX) assay is a reporter gene assay that detects dioxin-like compounds based on their ability to activate the aryl hydrocarbon receptor (AhR) and thus expression of the reporter gene. In this paper, the CALUX assay was examined for its application in the screening of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) in retail fish. The sample extracts were cleaned up on a sulfuric acid-silica gel column followed by an activated carbon column, and the AhR activity of the separated PCDD/F and dioxin-like PCB fractions was determined using the assay. The quantitative limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was 0.98 pg ml(-1) (0.19 pg assay(-1) in the standard curve, corresponding to 0.16 pg g(-1) of CALUX-based toxic equivalency (2,3,7,8-TCDD equivalents) in the tested sample. Recovery tests in which dioxins were added to fish samples resulted in acceptable recoveries (77-117%). The CALUX assay performed well in the analysis of dioxins in fish samples and a comparative study revealed a strong correlation between the CALUX assay and high-resolution gas chromatography-high-resolution mass spectrometry analysis for the determination of PCDD/Fs (r = 0.89) and dioxin-like PCBs (r = 0.91) in retail fish (n = 22). These data revealed that the CALUX assay would be a useful screening method for PCDD/Fs and dioxin-like PCBs in retail fish.

Activation of the aryl hydrocarbon receptor by some vegetable constituents determined using in vitro reporter gene assay.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological action of many aromatic environmental pollutants. In this study, we investigated the activation of the AhR by some vegetable constituents using the AhR-based bioassay for dioxins, i.e., the chemical activated luciferase gene expression (CALUX) assay. Ninety-five vegetable constituents, including flavonoids, tannins, saponins, and terpenes, were tested in vitro. Among them, isoflavones such as daidzein, resveratrol having a stilbene structure, and some flavonoids such as naringenin, hesperetin, and baicalein showed AhR activation.

Evaluation of an aqueous KOH digestion followed by hexane extraction for analysis of PCDD/Fs and dioxin-like PCBs in retailed fish.

Aqueous KOH digestion followed by hexane extraction has been employed in the extraction of dioxins (polychlorinated dibenzo- p -dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls) for biological samples, but there are no reports on its evaluation. Therefore, we report here the evaluation of this extraction for the analysis of dioxins in retailed fish. The effect of the alkaline digestion on dioxins was evaluated by estimation of recoveries. The recoveries of dioxins after the alkaline digestion were good (79-106%) in various kinds of retailed fish except tuna. In tuna, loss of octachlorodibenzofuran (OCDF) was clearly observed, however, the loss was corrected by internal-standard quantification; (13)C12-labeled OCDF was added before the alkaline digestion. Comparative study showed that alkaline digestion followed by hexane extraction provides extraction efficiencies of dioxins equal to those of conventional Soxhlet extraction in fish. Additionally, in analysis of a certified reference fish sample with this extraction, the values obtained for certified isomers were almost equal to the certified values. Since the present method is very simple and inexpensive, it would be useful for analysis of dioxins in retailed fish.

[Inter-laboratory evaluation studies of notified ELISA methods for allergic substances (egg)].

Inter-laboratory evaluation studies were conducted for the notified ELISA methods for allergic substances (Egg). Standard extracts of egg spiked in extracts of sausage, sauce, cookie, bread and cereal at a level of 5-20 ng/mL as the sample solution were analyzed in replicate in 10 laboratories. Coefficients of variation (CVs) of all three ELISA methods using an Egg Protein ovalbumin ELISA Kit (ovalbumin kit), an Egg Protein ovomucoid ELISA Kit (ovomucoid kit) and a FASTKIT Egg ELISA kit (Egg ELISA kit) were mostly less than 10%. Mean recoveries of the standard extract of egg were over 40% in the three ELISA methods. Repeatability relative standard deviations of egg standard solution in five food extracts were in the ranges of 18.7-25.5%, 18.6-41.8%, 21.3-43.3% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. Reproducibility relative standard deviations of egg standard solution in five food extracts were 16.8-35.1%, 19.6-35.8%, 24.7-51.1% for the ovalbumin kit, the ovomucoid kit and the Egg ELISA kit, respectively. The detection limits of all the ELISA methods were 4-5 ng/mL in sample solutions. These results suggested that the notified ELISA methods are reliable and reproducible for the inspection of egg protein levels in extracts of sausage, sauce, cookie, bread and cereal.

[Simultaneous determination of dichlorvos and trichlorfon in agricultural products by GC-FPD].

We studied a gas chromatographic method for the determination of dichlorvos (DDVP) and trichlorfon (DEP) in agricultural products. DDVP and DEP were extracted from agricultural products with acetone and re-extracted with ethyl acetate instead of dichloromethane. DDVP and DEP were eluted in one fraction on a silica gel column using n-hexane-acetone (1:1). DEP is a thermally labile compound, so it was derived to a more thermally stable compound by acylation with N-methylbis(trifluoroacetamide) and pyridine in acetone at 60 degrees C for 2 hours. DDVP and the DEP-TFA derivative were determined simultaneously by GC-FPD. The recoveries of DDVP and DEP from agricultural products spiked at levels of 0.1 microgram/g were 72.6-117.7% and 86.2-106.6%, respectively. The detection limits were 0.03 microgram/g in powdered tea and < or = 0.01 microgram/g in other samples. An interlaboratory study by 6 laboratories was conducted to validate this proposed method for 6 crops. Repeatability ranged from 3.1 to 7.8% for DDVP and from 3.4 to 8.3% for DEP, and reproducibility, from 6.9 to 15.5% for DDVP, and from 7.9 to 21.8% for DEP. Precision values were well within statistically predicted levels.