Meju Samples Research Articles

Inhibitory Effects of Bacillus subtilis Isolated from Meju (Fermented Soybean Brick) on the Growth of Aspergillus parasiticus

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis.

Characterization of Nonaflatoxigenic Aspergillus flavus/oryzae Strains Isolated from Korean Traditional Soybean Meju

Filamentous fungi that could be classified into Aspergillus flavus/oryzae were isolated from traditionally fermented meju commercially available in Korea. The samples were analyzed for aflatoxin B1 and ochratoxin A contamination by HPLC; however, no toxin was detected. In addition, fungal and bacterial metagenomic sequencing were performed to analyze the microbial distribution in the samples. The results revealed that the distribution and abundance of fungi and bacteria differed considerably depending on the production regions and fermentation conditions of the meju samples. Through morphological analysis, ITS region sequencing, and assessment of the aflatoxin-producing ability, a total of 32 A. flavus/oryzae strains were identified. PCR analysis of six regions with a high mutation frequency in the aflatoxin gene cluster (AGC) revealed a total of six types of AGC breaking point patterns. The A. flavus/oryzae strains did not exhibit the high amylase activity detected in the commercial yellow koji strain (starter mold). However, their peptidase and lipase activities were generally higher than that of the koji isolates. We verified the safety of the traditionally fermented meju samples by analyzing the AGC breaking point pattern and the enzyme activities of A. flavus/oryzae strains isolated from the samples. The isolated strains could possibly be used as starter molds for soybean fermentation.

Comparative Evaluation of Quality and Metabolite Profiles in Meju Using Starter Cultures of Bacillus velezensis and Aspergillus oryzae.

The production of good Meju soybean paste primarily depends on the selection of raw materials and fermenting microorganisms, which together influence its characteristic metabolites, taste, and aroma. In this study, we analyzed the relationship between properties and metabolites in Meju samples fermented by Aspergillus oryzae alone or with Bacillus velezensis. We developed fast-stable processing techniques to obtain Meju from A. oryzae and B. velezensis using the inoculation method, thereby ensuring safety in the production of soybean paste. The amino-type nitrogen content increased from an initial 180–228 mg% to a final 226–776 mg% during fermentation and was higher in Meju inoculated separately with the fungi and bacteria (C group) than in Meju co-inoculated with both the starters concurrently (D group). The levels of metabolites such as glucose, myo-inositol, glycerol, and fatty acids (palmitic, stearic, oleic, and linoleic acids) in Meju fermented by A. oryzae with B. velezensis were higher than those in Meju fermented by A. oryzae alone. Fungal growth was affected by the inoculated bacteria, which often occurs during the fermentation of co-inoculated Meju.

Integrated Metabolomics and Volatolomics for Comparative Evaluation of Fermented Soy Products.

Though varying metabolomes are believed to influence distinctive characteristics of different soy foods, an in-depth, comprehensive analysis of both soluble and volatile metabolites is largely unreported. The metabolite profiles of different soy products, including cheonggukjang, meju, doenjang, and raw soybean, were characterized using LC-MS (liquid chromatography–mass spectrometry), GC-MS (gas chromatography–mass spectrometry), and headspace solid-phase microextraction (HS-SPME) GC-MS. Principal component analysis (PCA) showed that the datasets for the cheonggukjang, meju, and doenjang extracts were distinguished from the non-fermented soybean across PC1, while those for cheonggukjang and doenjang were separated across PC2. Volatile organic compound (VOC) profiles were clearly distinct among doenjang and soybean, cheonggukjang, and meju samples. Notably, the relative contents of the isoflavone glycosides and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) conjugated soyasaponins were higher in soybean and cheonggukjang, compared to doenjang, while the isoflavone aglycones, non-DDMP conjugated soyasaponins, and amino acids were significantly higher in doenjang. Most VOCs, including the sulfur containing compounds aldehydes, esters, and furans, were relatively abundant in doenjang. However, pyrazines, 3-methylbutanoic acid, maltol, and methoxyphenol were higher in cheonggukjang, which contributed to the characteristic aroma of soy foods. We believe that this study provides the fundamental insights on soy food metabolomes, which determine their nutritional, functional, organoleptic, and aroma characteristics.

High-throughput sequencing of the microbial community associated with the physicochemical properties of meju (dried fermented soybean) and doenjang (traditional Korean fermented soybean paste)

Doenjang, a traditional Korean soybean paste consisting of fermented meju has been consumed for centuries as a protein source. In this study, the physicochemical factors and microbial communities of samples prepared from four batches of meju and doenjang were investigated to identify the factors that affect the food quality. The water content and salt concentration were higher in doenjang than in meju, and the acidity and amino-type nitrogen varied depending on the batch. After fermentation of doenjang, the pH, salt concentration, and amino-type nitrogen increased in all batches. Enterococcus and Bacillus were present in all meju samples, whereas Enterococcus, Tetragenococcus, and Bacillus were present in all doenjang samples. The dominant fungi in meju and doenjang was Aspergillus. The abundance of certain fungi, such as Debaryomyces, Candida, Mucor, and Gibberella, varied batch to batch. The amino-type nitrogen, which determines the quality of doenjang, was correlated with the relative abundance of Aspergillus in doenjang prior to fermentation. In addition, these strains had a negative correlation with the salt concentration. This study has elucidated the composition of microbial communities in meju and doenjang, which may lead to improved quality in the production of fermented food.

Natural Occurrence of Aflatoxigenic <i>Aspergillus</i> Species and Aflatoxins in Traditional Korean Fermentation Starters, <i>Meju</i> and <i>Nuruk</i>

Meju and nuruk (respectively soybean and malt) are traditional Korean fermentation starters that are vulnerable to contamination by harmful microorganisms such as aflatoxigenic fungi and their associated aflatoxins (AFs). In this study, Aspergillus spp. were isolated and identified from a total of 57 meju and 18 nuruk samples collected from Korean markets. Their potential aflatoxigenicity was investigated by examining the presence of three aflatoxin biosynthetic genes (aflO, aflP, and aflR) using multiplex polymerase chain reaction (mPCR) assays. Thereafter, aflatoxin production of isolates and the natural occurrence of AFs in meju and nuruk samples were analyzed by high-performance liquid chromatography (HPLC). A total of 177 Aspergillus isolates were identified and 130 isolates were obtained from meju samples. Of these, 25 isolates (19.2%) contained all three aflatoxin biosynthetic genes, and five (20%) of these isolates produced aflatoxins. Forty-seven of the Aspergillus isolates were obtained from nuruk samples, five of which (10.6%) expressed all three AF biosynthetic genes; however, none of these strains produced AFs. HPLC analysis showed that 88% (51/58) of the meju samples and 39% (7/18) of nuruk samples were not contaminated with AFs (below limit of detection). Among the isolates isolated from meju and nuruk, there were aflatoxigenic strains containing all three aflatoxin biosynthetic genes or producing aflatoxin in medium, but the frequency of aflatoxin contamination was low in the meju and nuruk samples.

Natural occurrence of aflatoxins and ochratoxin A in meju and soybean paste produced in South Korea

In this study, we investigated the occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in meju and soybean paste produced in South Korea. Samples were collected from three regions divided on the basis of climate in South Korea. A total of 100 meju samples were analyzed over 3 years (2012–2015), and 45 soybean paste samples were analyzed in 2016. Mycotoxins were extracted with an immunoaffinity column method and quantified by high-performance liquid chromatography. AFs were detected in 10 of meju (10%) and 11 of soybean paste samples (24.4%) with concentrations of 0.2–48.3 μg/kg and 0.88–16.17 μg/kg, respectively. OTA was detected in 50 of meju (50%) and 22 of soybean paste samples (48.9%) with concentrations of 0.1–193.2 μg/kg and 0.88–26.29 μg/kg, respectively. Mycotoxin contamination in meju was more common in the central region than in the southern areas. Thus, more mycotoxins were produced in the central region owing to less fungal competition in meju during fermentation inside households. We also found that about 91% of AFs and 73% of OTA in meju were degraded after the production of soybean paste and soy sauce. Even after degradation of AFs and OTA, the levels of AFB1 and OTA were 0.5 µg/kg and 7.5 µg/kg in soy sauce and 11.9 µg/kg and 190.4 µg/kg in soybean paste, respectively. Thus, our results suggest the need for constant monitoring of meju and soybean paste for AFs and OTA.

Detection of biogenic amines and microbial safety assessment of novel Meju fermented with addition of Nelumbo nucifera, Ginkgo biloba, and Allium sativum

Meju, a cooked and fermented soy bean based food product, is used as a major ingredient in Korean traditional fermented foods such as Doenjang. We developed a novel type of Meju using single and combined extracts of Allium sativum (garlic clove), Nelumbo nucifera (lotus leaves), and Ginkgo biloba (ginkgo leaves) at 1% and 10% concentrations to improve the safety of Meju-based fermented products. Biogenic amines (BAs) in protein-rich fermented food products pose considerable toxical risks. The objective of this study was to investigate the effects of adding selected plant extracts in Meju samples during fermentation. Nine BAs, including tryptamine, 2-phenylethylamine, putrescine, cadaverine, agmatine, histamine, tyramine, spermidine and spermine, were isolated from Meju samples after sample derivatization with dansyl chloride and analyzed by high performance liquid chromatography. As a result, all tested Meju samples with added plant extracts showed total BAs levels in the range of 20.12 ± 2.03 to 118.42 ± 10.68 mg/100 g, which were below the safety limit set by various regulatory authorities (USFDA/KFDA/EFSA). However, among all tested Meju samples, LOM10 (Meju fermented with Nelumbo nucifera at 10% concentration) showed higher levels of BAs content than others either due to batch-to-batch variability or reduced beneficial microorganisms and/or due to increase in BA forming microorganisms. Also, none of the samples showed the aflatoxin level above the detection limit. Furthermore, all the tested Meju samples improved microbial safety as confirmed by the complete absence of Salmonella species and Staphylococcus aureus. However, some of the Meju samples showed the presence of coliforms (in range of 1.6 × 100–1.1 × 103 CFU/g), which is under regulatory limits. These results suggested that the use of plant extracts in Meju during fermentation have potential to improve microbial and toxicological safety of Meju products.

Development of novel Meju starter culture using plant extracts with reduced Bacillus cereus counts and enhanced functional properties

We developed a novel type of Meju starter culture using single and combined extracts of Allium sativum (garlic clove), Nelumbo nucifera (lotus leaves), and Ginkgo biloba (ginkgo leaves) to improve the quality and functionality of Meju-based fermented products. Meju samples fermented with plant extracts (10 mg/ml) showed phenolic contents of 11.4–31.6 mg/g (gallic acid equivalents). Samples of extracts (garlic clove, lotus leaves, ginkgo leaves and their combination) fermented with Meju strongly inhibited tyrosinase, α-glucosidase, and elastase activities by 36.43–64.34%, 45.08–48.02%, and 4.52–10.90%, respectively. Specifically, ginkgo leaves extract added to fermented Meju samples at different concentrations (1% and 10%) strongly inhibited tyrosinase, α-glucosidase, and elastase activities and exhibited a potent antibacterial effect against Bacillus cereus with a significant reduction in bacterial counts compared with the effects observed for garlic clove and lotus leaf added to Meju samples. Scanning electron microscopy revealed severe morphological alterations of the B. cereus cell wall in response to ginkgo leaf extracts. Gas chromatographic mass spectroscopic analysis of plant extract-supplemented Meju samples and control Meju samples identified 113 bioactive compounds representing 98.44–99.98% total extract. The proposed approach may be useful for the development of various fermented functional foods at traditional and commercial levels.

Evaluation of fungal microflora for aflatoxin producing possibility in novel quality Meju fermented with single and/or multiple additions of Nelumbo nucifera, Ginkgo biloba, and Allium sativum extracts

AbstractThis study was aimed to evaluate the fungal microflora in Meju samples fermented using single and/or multiple additions of ethanolic extracts from plants, including Nelumbo nucifera leaves, Ginkgo biloba leaves, and Allium sativum cloves, at different concentrations (1 and 10%) and mixtures of these extracts at a 1:1:1 ratio. Fungal species were grouped based on morphological characteristics, and selected fungal strains were identified based on DNA sequence analysis. Aspergillus species were the dominant microflora in Meju samples. A total of 10 fungal strains isolated from the tested Meju products resulted in the identification of five genera, including Agaricaceae, Aspergillus (synonym: Eurotium), Mucor, Penicillium, and Paecilomyces. Further, Meju samples fermented with selected individual and/or multiple ethanolic extracts resulted in the occurence of various fungal strains, including Aspergillus, Mucor, Paecilomyces, and Penicillium chrysogenum. Conversely, Meju samples fermented without ethanolic extracts as a control resulted in the isolation of Aspergillus ruber, which might be capable of producing aflatoxin B1 and ochratoxin A. The outcome of this study confirms that fermentation of Meju using plant extracts may significantly influence fungal microflora, leading to improved quality of Meju production, because of diminishing effects of the extracts against toxin producing fungal pathogens.Practical applicationsFor decades, fermented foods have been used traditionally as health beneficial products in Asian countries including Korea. Contamination of fermented food starter culture, such as soybean‐based Meju, by pathogenic fungi or toxin producing fungi, such as Aspergillus species, has been reported as a severe concern to consumers. Medicinal plants have shown remarkable efficacy to control a number of pathogenic microbes including fungi. In this study, Nelumbo nucifera leaves, Ginkgo biloba leaves, and Allium sativum cloves exhibited significant potential to diminish the growth of toxin producing fungi, suggesting potential application of medicinal plants for the improved quality of Meju production.

Aflatoxin B1 Detoxification by Aspergillus oryzae from Meju, a Traditional Korean Fermented Soybean Starter.

Aflatoxins are classified as Group 1 (carcinogenic to humans) by the International Agency for Research on Cancer. In this study, a total of 134 fungal strains were isolated from 65 meju samples, and two fungal isolates were selected as potential aflatoxin B1 (AFB1)-biodetoxification fungi. These fungi were identified as Aspergillus oryzae MAO103 and A. oryzae MAO104 by sequencing the beta-tubulin gene. The two A. oryzae strains were able to degrade more than 90% of AFB1 (initial concentration: 40 μg/l) in a culture broth in 14 days. The mutagenic effects of AFB1 treated with A. oryzae MAO103 and MAO104 significantly decreased to 5.7% and 6.4%, respectively, in the frame-shift mutation of Ames tests using Salmonella typhimurium TA98. The base-substituting mutagenicity of AFB1 was also decreased by the two fungi. Moreover, AFB1 production by Aspergillus flavus was significantly decreased by the two A. oryzae strains on soybean-based agar plates. Our data suggest that the two AFB1-detoxifying A. oryzae strains have potential application to control AFB1 in foods and feeds.

Biodegradation of Ochratoxin A by Aspergillus tubingensis Isolated from Meju.

Ochratoxin A (OTA), a mycotoxin, contaminates agricultural products and poses a serious threat to public health worldwide. Microbiological methods are known to be a promising approach for OTA biodegradation because physical and chemical methods have practical limitations. In the present study, a total of 130 fungal isolates obtained from 65 traditional Korean meju (a fermented starter for fermentation of soybeans) samples were examined for OTA-biodegradation activity using thin-layer chromatography. Two fungal isolates were selected for OTA-biodegradation activity and were identified as Aspergillus tubingensis M036 and M074 through sequence analysis of the beta-tubulin gene. After culturing both A. tubingensis isolates in Soytone-Czapek medium containing OTA (40 ng/ml), OTA-biodegradation activity was analyzed using high-performance liquid chromatography (HPLC). Both A. tubingensis strains degraded OTA by more than 95.0% after 14 days, and the HPLC analysis showed that the OTA biodegradation by the A. tubingensis strains led to the production of ochratoxin α, which is much less toxic than OTA. Moreover, crude enzymes from the cultures of A. tubingensis M036 and M074 led to OTA biodegradation of 97.5% and 91.3% at pH 5, and 80.3% and 75.3% at pH 7, respectively, in a buffer solution containing OTA (40 ng/ml) after 24 h. In addition, the OTA-biodegrading fungi did not exhibit OTA production activity. Our data suggest that A. tubingensis isolates and their enzymes have the potential for practical application to reduce levels of OTA in food and feed.

The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes

Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.

Analyses of bacterial communities in meju, a Korean traditional fermented soybean bricks, by cultivation-based and pyrosequencing methods

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before.

Metabolomic analysis of meju during fermentation by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS)

Changes in the water-soluble metabolites of meju during fermentation were analysed by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS), and the resultant data were statistically processed by partial least squares-discriminant analysis (PLS-DA). Various metabolites, including amino acids, small peptides, nucleosides, urea cycle intermediates, and organic acids, which are responsible for the unique taste and nutritional and functional quality of fermented soy foods, were clearly altered by increasing the fermentation period. Changes in these metabolites allowed discrimination among meju samples with different fermentation periods (0, 10, 20, 40, and 60d) on a PLS-DA score plot, and the fermentation was mainly processed between 10 and 40d of fermentation. Twenty-two metabolites (phenylalanine, glutamic acid, leucine, adenine, citrulline, arginine, glutamine, γ-aminobutyric acid, proline, acetylornithine, valine, pipecolic acid, methionine, citric acid, xanthine, tyrosine, isoleucine, Glu-Tyr, Ser-Pro, tryptophan, Glu-Phe, and Leu-Val-Pro-Pro) with high PLS-DA values of over 1.00 were determined as the major compounds contributing to the discrimination of meju samples. These metabolites, which were positively related to the sensory quality of meju, can be used as fermentation biomarkers for the production of meju and to construct the meju fermentation metabolic pathway. Therefore, our results indicate that monitoring the changes in metabolites during meju fermentation might be important for producing meju-related foods with good nutritional and sensory quality.

Effects of Alternatively Prepared Meju Methanolic Extracts on Dietary Lipid Digestion

We examined the effect of extracts of meju prepared with traditional and standardized methods on pancreatic lipase and the absorption of dietary lipid. Aqueous methanolic (80%, v/v) extracts of meju dose-dependently inhibited the activities of porcine pancreatic lipase. The plasma triglyceride levels in Imprinting Control Region mice after a single oral administration of lipid emulsion containing aqueous methanolic extracts from meju made by the standardized methods were lower than that of the group given a lipid emulsion containing the extracts of meju made by traditional methods. The inhibitory activity of the meju extract on dietary lipid digestion appears to be more closely associated with aglycone forms of phenolic compounds such as free isoflavones than with glycosides, since meju samples with higher total phenolic or free isoflavone content showed the stronger inhibition against pancreatic lipase. Furthermore, the data suggest that meju made using the standardized method, which contains higher levels of total isoflavones relative to traditionally prepared meju, could effectively suppress digestion of dietary lipids and therefore have the potential to help ameliorate hyperlipidemia and obesity.

Determination of microbial diversity in meju, fermented cooked soya beans, using nested PCR-denaturing gradient gel electrophoresis

To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Using conventional bacterial 16S rDNA, bacilli-specific 16S rDNA or fungi 18S rDNA-specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. A variety of bacterial and fungal micro-organisms were identified in meju samples, in addition to the micro-organisms already known to be present. This is the first report showing the differences and similarities in the populations of micro-organisms in meju samples using nested PCR-DGGE, a culture-independent method. The results may be applicable to the development of improved meju, in which the indigenous micro-organisms required for fermentation can be standardized.

Detection of Aflatoxin-Producing Molds in Korean Fermented Foods and Grains by Multiplex PCR

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.

Natural occurrence of aflatoxins in Korean meju

Aflatoxin B1 (AFB1) was found in 35 of 60 (58.3%) meju samples with an average concentration of 7.3ng/g by ELISA. Contamination of AFB1 was confirmed in 25 of 60 samples (41.6%) using HPLC, with an average concentration of 6.9ng/g. Mean recoveries from meju ranged from 107% to 170% for AFB1 using ELISA at a spiking range of 1 to 50ng/g. Over the same range, recoveries using HPLC were from 70% to 83%. The levels of AFB1 determined by ELISA and by HPLC demonstrated a close relationship between the two methods (r2 = 0.9324) employed in this study. In order to evaluate the potential health risks of AFB1 on Koreans consuming meju, we calculated the estimated probable daily intake (PDI) based on the average contamination levels and compared it with the estimated tolerable daily intake (TDI). The PDIs of AFB1 from kanjang and dwenjang were determined to be 0.04 and 0.21ng/kg bw/day, respectively, and were higher than TDIs.

A study on the isolation of aflatoxin-producing strains from agricultural products in Korea.

In order to isolate aflatoxin-producing strains from agricultural products in the Youngnam district of Korea, rice, barley, unhulled barley, corn, soybean, Meju (soybean paste), peanut, and soil samples were collected from markets or homes. From the 347 samples, 280 strains of Aspergillus spp. were isolated. As a result of screening by thin layer chromatography (TLC), 29 strains showed fluorescent spot and four strains had the same Rf values as those of standard aflatoxins. The percentage of contamination of aflatoxin-producing strains was 1.4%. The four strains were classified as the Aspergillus flavus group by the examination of characteristics and morphology.