Original Paper Evaluation of the Biogenic Amine-Production Ability

http://www.jsfst.or.jp

Original paper

Evaluation of the Biogenic Amine-production Ability of Lactic Acid Bacteria Isolated from Tofu-misozuke

1 1 2 1 1 1* Youhei Takebe , Misato Takizaki , Hiroko Tanaka , Hiroto Ohta , Takuro Niidome and Shigeru Morimura

1Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami Chuou-ku, Kumamoto City, Kumamoto 860-8555, Japan 2Applied Chemistry & Biochemistry, Kumamoto University, 2-39-1 Kurokami Chuou-ku, Kumamoto City, Kumamoto 860- 8555, Japan

Received November 19, 2015 ; Accepted June 6, 2016

Tofu-misozuke is a traditional fermented food in Japan, and contains biogenic amines that function as neurotransmitters, but are toxic when consumed at high levels. Analysis of the amine production of bacterial communities is important for controlling the amine content in fermented foods. We investigated the bacterial community and biogenic amine content of tofu-misozuke. The lactic acid bacteria (LAB) ratio in the community and tyramine content were highly related. LAB were isolated to identify the biogenic amine-producing species. Isolated strains from tofu-misozuke were identified, and their tyramine formation potential was evaluated by PCR detection of the tyrosine decarboxylase gene and by cultivation tests. We detected three tyramine-producing bacteria, Enterococcus faecium, Weissella viridescens, and Lactobacillus curvatus, in tofu-misozuke.

Keywords: tofu-misozuke, lactic acid bacteria, biogenic amines, tyrosine decarboxylase

Introduction Asian fermented foods (Murooka and Yamashita, 2008). It is Fermented soybean products, such as miso, soy sauce, natto, important to analyze the microbial communities in fermented foods tempeh, and fermented tofu, are common in Asian households to effectively regulate flavor and taste. (Chen et al., 2012). Tofu-misozuke is a traditional fermented However, the bacterial community in tofu-misozuke has not soybean product originating in Kumamoto, Japan. It is made by yet been characterized. soaking tofu with miso paste, and has a flavor and texture similar Biogenic amines (BAs) are low-molecular weight nitrogenous to cheese. Recently, a new type of tofu-misozuke was produced by compounds of biological importance in microorganisms, plants, soaking in liquid seasoning containing koji (Aspergillus oryzae) and animals (Bai et al., 2013). They are mainly formed by during the manufacturing process. The flavor and taste of this novel decarboxylation of amino acids via substrate-specific tofu-misozuke is different from that traditionally produced. Fungal decarboxylases derived from microbes in foods (Santos, 1996). proteases involved in protein degradation and peptide production The most well known BAs, such as serotonin, dopamine, generate the unique flavors and textures associated with tofu- noradrenaline, and histamine, are neurotransmitters and/or misozuke (Funaki et al., 1996, Funaki et al., 1997). In miso paste, allergens. Those less well known include tyramine, tryptamine, and a raw material of tofu-misozuke, lactic acid bacteria (LAB) and β-phenylethylamine, and are abundant in fermented foods. bacteria of the genus Bacillus have been observed (Kim et al., Furthermore, some fermented foods contain putrescine, which 2010). LAB are the most common bacteria isolated from traditional belongs to a group of ubiquitous polycationic amines (Shukla et

*To whom correspondence should be addressed. E-mail: [email protected] 674 Y. Takebe et al. al., 2011). Putrescine can be synthesized either directly from DNA Ligation Kit (Takara Bio, Kusatsu, Japan) according to the ornithine by ornithine decarboxylase (ODC) or indirectly from manufacturer’s protocol. Then, 10 µL of the ligation mixture was arginine via arginine decarboxylase (ADC) (Costantini, 2013, transformed to 100 µL of Escherichia coli DH5α (Takara Bio) as Pereira et al., 2009). ADC converts arginine to agmatine, then competent cells by standard methods. After colonies were formed agmatine deiminase (AgDI) biosynthetically converts agmatine to on an LB-ampicillin plate, white colonies were selected and putrescine via the AgDI pathway (Nakada and Itoh, 2003). plasmids were extracted using the Wizard SV Minipreps DNA Therefore, arginine contained in soybeans can be a precursor of Purification System (Promega, Madison, WI, USA). Extracted putrescine (Chen et al., 2012). The consumption of foods plasmids were digested by EcoRI and PstI (Takara Bio) to containing high amounts of BAs may have toxicological effects, as determine the size of the inserted DNA. After electrophoresis, observed in a number of food-related incidents. In particular, DNA bands were visualized by staining the gels in 5 mg/L ethidium tyramine consumption causes headaches, heart palpitations, hyper/ bromide solution for 20 min. FAS-III (Toyobo, Osaka, Japan) was hypotension, and several allergic disorders (Kim and Kim, 2014). used to view the DNA bands. A sequence analysis of the inserted Therefore, understanding the relationship between BA production DNA was completed by Takara Bio. Data were compared with the and the bacterial community in fermented foods is important to National Center for Biotechnology Information (NCBI) database improve the safety of the manufacturing process. using a Basic Local Alignment Search Tool (BLAST) search to The objective of this study was to identify the bacterial identify the clones. The ratio of LAB was determined by the community in tofu-misozuke and to evaluate the tyramine- number of LAB clones to that of total bacterial clones. producing ability of LAB isolated from tofu-misozuke. Isolation of LAB from tofu-misozuke MRS medium (peptone 10 g/L, meat extract 20 g/L, yeast extract 5 g/L, glucose 20 g/L,

Materials and Methods Tween80 1 g/L, K2HPO4 2 g/L, sodium acetate 5 g/L, diammonium

Samples Five tofu-misozuke samples (A to E) were purchased hydrogen citrate 2 g/L, MgSO4·7H2O 0.2 g/L, MnSO4·nH2O 0.05 g/ from companies in Kumamoto Prefecture, Japan. Samples A and E L), and LM17 medium (casein 5 g/L, peptone 5 g/L, meat extract were produced by the traditional handmade method of soaking in 5 g/L, yeast extract 2.5 g/L, ascorbic acid 0.5 g/L, MgSO4·7H2O miso paste, while the others were produced by soaking in liquid 0.25 g/L, disodium-β-glycerophosphate 10 g/L, lactose 0.25 g/L) seasoning. were used to cultivate and isolate LAB. Amounts of 1 g/L each of Chemicals Peptone and tryptic soy broth (TSB) were sodium azide and cycloheximide were added to MRS or LM17 purchased from Difco (Sparks, MD, USA). Meat extract was medium at a final concentration of 0.01 g/L each. Agar (15 g/L) purchased from Merck KGaA, Darmstadt, Germany. Yeast extract was added to prepare the solid medium. Tofu-misozuke was cut and casein were purchased from Nacalai Tesque, Kyoto, Japan. and inoculated into MRS or LM17 medium in a flask. The flask The other chemicals were purchased from Nacalai Tesque and was incubated statically at 30℃ for 2 days. The culture broth was Wako Pure Chemical Industries, Osaka, Japan. diluted and spread on the MRS plate. Colonies grown on the MRS Identification of the bacterial community of tofu-misozuke plate were observed based on color, size, and shape. Various Twenty-five grams of each sample was homogenized in 225 mL of colonies were picked and inoculated onto an MRS slant, and sterilized saline. The homogenized solution was centrifuged at incubated at 30℃ for 2 days. They were subsequently stored at 4℃. 8,000 × g at 4℃ for 15 min. The precipitate was suspended in Identification of isolated LAB based on partial 16S rRNA gene sterilized water and the suspension was centrifuged again under the sequences Isolated strains were analyzed taxonomically by partial same conditions. The precipitate was then suspended in 200 μL of 16S rRNA gene sequencing. Part of the 16S rRNA gene was sterilized water. DNA was extracted from 50 μL of each sample amplified by colony PCR with the 27F and 518R primer set. GoTaq solution using the Fast DNA SPIN Kit for Soil (MP Biomedicals, Master Mix (Promega) was used for colony PCR. The PCR Solon, OH, USA) according to the protocol. The extracted DNA reaction contained the picked colony as a template, 2 µL of forward was used as a template for PCR targeting the partial 16S rRNA and reverse primers (25 µM each), 25 µL of GoTaq green, and gene, using the primer set of 27F 20 µL of nuclease-free water. After preheating at 95℃ for 5 min, a (5’-AGAGTTTGATCCTGGCTCAG-3’) and 518R cycle of heating at 95℃ for 1 min, annealing at 50℃ for 1 min, (5’-GTATTACCGCGGCTGCTGG-3’). AmpliTaq Gold (Applied and extension at 72℃ for 2 min was repeated 25 times. A sequence Biosystems, Carlsbad, CA, USA) was used for PCR analysis. After analysis of the amplified DNA from isolates was completed by preheating at 95℃ for 5 min, a cycle of heating at 95℃ for 1 min, Takara Bio. Both PCR primers were used as the sequence primer annealing at 50℃ for 1 min, and extension at 72℃ for 2 min was for the sequence analysis. The obtained partial 16S rRNA gene repeated 25 times. PCR products were purified using the Ultra sequences were compared to those in the NCBI database using Clean PCR Clean-up Kit (MO-BIO, Carlsbad, CA, USA) according BLAST. to the manufacturer’s protocol. Purified PCR products were ligated Detection of tyrosine decarboxylase (tdc) gene GoTaq Master with the pT7Blue vector (Novagen, Darmstadt, Germany) using the Mix (Promega) was used. PCR was performed as described above, LAB Isolated from Tofu-misozuke 675 and included 5 min at 95℃ followed by 35 cycles of 30 s at 95℃, chromatography (HPLC). An HPLC system (Shimadzu, Kyoto, 30 s at 55℃, and 90 s at 72℃. The primer set of TD5 Japan) equipped with an SPD-M20A detector and LC solution (5’-CAAATGGAAGAAGAAGTWGG-3’) and TD2 software was employed. L-column ODS (4.6 mm I.D. × 150 mm; (5’-ACATAGTCAACCATRTTGAA-3’) was used for Chemicals Evaluation and Research Institute, Tokyo, Japan) was

Enterococcus and Weissella. TD2-Lac used with H2O (solvent A) and acetonitrile (solvent B) as the (5’-GTCCAATCGACCATATTGAA-3’), instead of TD2, was mobile phases at a total flow rate of 1 mL/min. The program was used for Lactobacillus. Five microliters of PCR products and 1 µL set for a linear gradient starting from 50% solvent B to reach 90% of loading buffer were mixed and analyzed by electrophoresis to after 30 min. A sample volume of 20 µL was injected, and the determine the size of the amplified fragments. sample was monitored at 254 nm. Determination of tyramine content in tofu-misozuke samples and the biogenic amine-producing ability of isolates An analysis Results and Discussion of biogenic amines in tofu-misozuke was performed following the Sample characteristics and tyramine content The procedure described by Byun and Mah (2012). Briefly, 20 mL of characteristics and tyramine content of tofu-misozuke samples are 0.4 M perchloric acid was added to 5 g of sample, and the mixture summarized in Table 1. The two types of tofu-misozuke differed was homogenized using a vortex mixer. The sample was kept in a with respect to taste and flavor. The flavor and taste of samples A cold chamber at 4℃ for 2 h, and centrifuged at 3000 × g at 4℃ for and E (samples soaked in miso paste) were similar to cheese, while 10 min. The supernatant was collected, and the residue was the samples of novel tofu-misozuke tasted like seasoning, such as extracted again with an equal volume of 0.4 M perchloric acid. soy sauce, or lightly of sake. Since this involves only soaking in Both supernatants were mixed, and the final volume was adjusted liquid seasoning, it is not thought to promote fermentation during to 50 mL with 0.4 M perchloric acid. The extract was filtered manufacturing. However, the addition of sake is known to through Whatman paper No. 1. prevent bacterial contamination during manufacturing. In Biogenic amines in the culture broth were determined addition, the tyramine content differed between the two types. We according to the procedure developed by Mah et al. (2003) and detected a high concentration of tyramine in samples A and E. modified by Burdychova and Komprada (2007). A loopful of the β-Phenylethylamine and putrescine were also detected in both isolated strain was inoculated into 5 mL of TSB with 0.25% (w/v) samples (as shown in Fig. 1a for sample E). Chromatograms of L-tyrosine disodium salt hydrate, L-phenylalanine, L-arginine, or standard BAs, including tyramine, β-phenylethylamine, histamine, L-ornithine hydrochloride (pH 5.8) with 0.0005% pyridoxal-HCl. cadaverine, tryptamine, and putrescine, are shown in Fig. 1b. The After incubation at 30℃ for 24 h, 100 µL of the culture broth was amine content of other fermented soybean foods showed a similar transferred to a new tube containing 5 mL of the same broth and pattern to that of tofu-misozuke (Byun and Mah, 2012, Yang et al., incubated at 30℃ for 24 h. After cultivation, the broth was taken 2014). It was suggested that tofu-misozuke would be a typical BA- up using a sterile syringe and filtered through a 0.2-µm membrane. containing fermented soybean food. Then, 9 mL of 0.4 M perchloric acid was added to 1 mL of the Bacterial diversity in tofu-misozuke We amplified the partial filtrate and mixed well using a vortex mixer. The mixture was kept 16S rRNA gene from the extracted DNA. The concentration of in a cold chamber at 4℃ for 2 h, and centrifuged at 3000 × g at 4℃ extracted DNA (ng/μL) from samples A, B, C, D, and E was 21.0, for 10 min. The extract was filtered through Whatman paper No. 1. 6.5, 6.2, 9.6, and 16.0, respectively. These concentrations may Standard solutions of biogenic amines were separately prepared at indicate the bacterial concentration in each sample. The amplified a concentration of 100 or 1000 mg/L to a final volume of 10 mL. PCR product was cloned into E. coli DH5α competent cells for the Determination of biogenic amines in extracts and standards construction of a bacterial clone library. An analysis of biogenic amines was carried out according to the The number of analyzed clones was 30, 74, 24, 24, and 48 for procedures described by Eerola et al. (1993) with minor samples A, B, C, D, and E, respectively. We determined the clones modifications. One milliliter of each solution was mixed with with the highest sequence similarity by BLAST searches, and the 200 µL of 2 M sodium hydroxide and 300 µL of saturated sodium bacterial distribution of each sample is shown in Fig. 2. The ratio bicarbonate. Then, 2 mL of dansyl chloride solution (10 mg/mL) dissolved in acetone was added to the mixture and incubated at Table 1. Characteristics of tofu-misozuke samples 40℃ for 45 min. Residual dansyl chloride was removed by adding 100 µL of 25% ammonium hydroxide. After 30 min of incubation at room temperature, the volume of the amine solution was adjusted with acetonitrile to 5 mL. Finally, the mixture was centrifuged at 3000 × g for 5 min, and the supernatant was filtered through a 0.20-µm syringe filter. The filtered supernatant was kept _ at 25℃ until it was assayed by high-performance liquid N.D., Not detected 676 Y. Takebe et al.

Fig. 3. The linear fitting between tyramine content and LAB ratio in tofu-misozuke samples.

Gardini, 2003). Therefore, it was suggested that W. viridescens might be involved in BA production. The ratios of LAB to all bacteria detected in the manufactured type (samples B, C, and D) were very low: 5%, 0%, and 0%, respectively. Staphylococcus was Fig. 1. Typical HPLC chromatograms of biogenic amines in sample E (a) and standard solution (b) (1, tryptamine; 2, β-phenylethylamine; 3, dominant in sample B. Staphylococcus spp. are often found in putrescine; 4, cadaverine; 5, histamine; 6, tyramine). fermented foods such as soy sauce (Wei et al., 2013). B. amyloliquefaciens and B. subtilis were the dominant species in of LAB in samples A and E (soaked in miso paste) were as high as samples C and D, respectively. These Bacillus spp. have been 80% and 92%, respectively. In the case of sample A, Weissella found in miso (Byun and Mah, 2012) and douchi, a fermented viridescens (36%), Lactobacillus sakei (28%), and L. curvatus soybean curd (Chen et al., 2012). However, Bacillus spp. are likely (16%) were the dominant species with highly similar sequences. not involved in the BAs produced in tofu-misozuke. Bacillus megaterium (16%) and Deinococcus geothermalis (4%) Figure 3 shows the correlation between the ratio of LAB and were also detected. In sample E, L. homohiochii (50%), tyramine content in tofu-misozuke samples. This result is consistent W. viridescens (27%), L. sakei (8%), and L. curvatus (8%) with the generation of tyramine by LAB. It was suggested that were the dominant LAB. W. viridescens was the major bacterial LAB might be involved in BA production in tofu-misozuke as with species identified in samples A and E. W. viridescens was reported fermented protein foods. to be isolated from fermented dry sausage (Papamanoli et al., Identification of isolated LAB In the previous section, LAB 2003), and dry sausage showed high BA content (Suzzi and constituted a large proportion of the bacteria detected in

Fig. 2. Comparison of bacterial diversity among 5 tofu-misozuke samples LAB Isolated from Tofu-misozuke 677

Table 2. Identification and detection of the tyramine-producing ability of isolates

samples A and E. Therefore, we attempted to isolate LAB from samples A and E. Based on the results of partial 16S rRNA gene sequencing, we identified the isolated strains as 7 strains belonging to 3 genera (Table 2). Other than Enterococcus faecium, almost all of the strains detected in samples A and E (shown in Fig. 2) were isolated using MRS medium. Enterococcus was not detected in Fig. 2. However, Enterococcus was isolated using LM17 medium for lactic streptococci. The number of Enterococcus in the samples was likely very small, but we isolated E. faecium most frequently when using LM17 medium. We isolated Lactobacillus and Weissella using MRS medium. We Fig. 4. Typical HPLC chromatogram of the putrescine production test from AM7 (W. hellenica) detected two W. viridescens isolates from sample A; however, the accession numbers of their closest matches were different. We isolated W. hellenica and Weissella sp. from sample A, and W. Determination of non-tyramine BA-producing ability of LAB viridescens, L. homohiochii, L. curvatus, and L. sakei from sample To examine biogenic amines other than tyramine, we performed E. cultivation tests using the isolated strains. In the analysis of Determination of tyramine-producing ability of isolated LAB samples A and E, we detected phenylethylamine, putrescine, and The tyramine-producing ability of the isolated LAB is summarized tyramine (see sample E in Fig. 1a). We tested for phenylethylamine in Table 2. The tyrosine decarboxylase coding gene (tdc) was and putrescine production using phenylalanine and arginine or detected by PCR, and tyramine-producing ability was examined by ornithine as precursors of each amine. Phenylethylamine was cultivation tests and HPLC analysis. E. faecium was positive in produced by the addition of phenylalanine only by E. faecium. both tests. Enterococcus has been isolated from cheese and Thus, it might be produced via tyrosine decarboxylase, as reported produces tyramine (Burdychova and Komprada, 2007). Isolates previously (Bargossi et al., 2015). Only isolate AM7 (close to W. from tofu-misozuke closest to Enterococcus also produced hellenica) generated putrescine from agmatine, but not from tyramine. On the other hand, isolates close to W. viridescens were ornithine, with the supplemented medium (Fig. 4). Therefore, divided into two groups, namely, tyramine-producing and non- isolate AM7 appeared to produce putrescine via the AgDI pathway. producing bacteria. Thus, tyramine-producing ability is not species- The peak detected at a retention time of 21.5 min in Fig. 4 was specific, but strain-specific. Isolates close to W. hellenica and confirmed as residual phenylalanine that was added to the test Weissella sp. were negative in both tests. L. curvatus and L. medium. homohiochii have been shown to produce tyramine (Pereira et al., BA production was related to the ratio of LAB in the final 2001). In this study, the isolate identified as L. curvatus tested products. However, the BA producing ability of isolated LAB was positive using both PCR and HPLC. In contrast, the isolate close to dependent on the strain. Therefore, the content of BAs could be L. homohiochii was negative using both methods. In tofu-misozuke reduced by using non-producing LAB as the starter strain for the samples A and E, isolated bacteria that were genetically close to E. production of tofu-misozuke. faecium, W. viridescens and L. curvatus showed tyramine- producing ability. Acknowledgements We thank Mr. Toshiro Toyama at the 678 Y. Takebe et al. main Toyama store for his kind advice regarding tofu-misozuke. bacterial and fungal communities in Japanese- and Chinese-fermented soybean pastes using nested PCR-DGGE. Curr. Microbiol., 60, 315-320. References Mah, J. H., Ahn, J. B., Park, J. H., Sung, H. C., and Hwang, H. J. (2003). Bai, X., Byun, B. Y., and Mah, J. H. (2013). Formation and destruction of Characterization of biogenic amine-producing microorganisms isolated biogenic amines in Chunjang (a black soybean paste) and Jajang (a from myeolchi-jeot, Korean salted and fermented anchovy. J. Microbial. black soybean sauce). Food Chemistry, 141, 1026-1031. Biotechnol., 13, 692-699. Bargossi, E., Tabanelli, G., Montanari, C., Lanciotti, R., Gatto, V., Gardini, Nakada, Y. and Itoh, Y. (2003). Identification of the putrescine biosynthetic F., and Torriani, S. (2015). Tyrosine decarboxylase activity of genes in Pseudomonas aeruginosa and characterization of agmatine enterococci grown in media with different nutritional potential: tyramine deiminase and N-carbamoylputrescine amidohydrolase of the arginine and 2-phenylethylamine accumulation and tyrDC gene expression. decarboxylase pathway. Microbiology, 149, 707-714. Frontiers in Microbiology, 6, Article 259. Papamanoli, E., Tzanetakis, N., Tzanetaki, E.L., and Kotzekidou, P. (2003). Burdychova, R. and Komprada, T. (2007). Biogenic amine-forming Characterization of lactic acid bacteria isolated from a Greek dry- microbial communities in cheese. FEMS. Microbiol. Lett., 276, 149-155. fermented sausage in respect of their technological and probiotic Byun, B. Y. and Mah, J. H. (2012). Occurrence of biogenic amines in Miso, properties. Meat Science, 65, 859-867. Japanese traditional fermented soybean paste. J. Food Sci., 77, 216-223. Pereira, C. I., Crespo, M. T. B., and Romao, M. V. S. (2001). Evidence for Chen, K. I., Erh, M. H., Su, N. W., Liu, W. H, Chou, C. C., and Cheng, K. proteolytic activity and biogenic amines production in Lactobacillus C. (2012). Soyfoods and soybean products: from traditional use to curvatus and L. homohiochii. Int. J. Food Microbiol, 68, 211-216. modern applications. Appl. Microbiol. Biotechnol. 96, 9-22. Pereira, C. I., Romao, M.V.S., Lolkema, J.S., and Crespo, M. T. B. (2009). Chen, T., Jiang, S., Xiong, S., Wang, M., Zhu, D., and Wei, H. (2012). Weissella halotolerans W22 combines arginine deiminase and ornithine Application of denaturing gradient gel electrophoresis to microbial decarboxylation pathways and converts arginine to putrescine. J. Appl. diversity analysis in Chinese Douchi. J. Sci. Food. Agric, 92, 2171-2176. Microbiol., 107, 1894-1902. Costantini, A., Pietroniro, R., Doria, F., Pessione, E., and Moruno, E. G. Santos, M. H. S. (1996). Biogenic amines: their importance in foods. Int. J. (2013). Putrescine production from different amino acid precursors by Food Microbiol., 29, 213-231. lactic acid bacteria from wine and cider. Int. J. Food Microbiol, 165, 11- Shukla, S., Kim, J. K., and Kim, M. (2011). Occurrence of biogenic amines 17. in soybean food products. Soybean and Health, 9, 181-206. Eerola, S., Hinkknen, R., Lindfors, E., and Hirvi, T. (1993). Liquid Suzzi, G. and Gardini, F. (2003). Biogenic amines in dry fermented chromatographic determination of biogenic amines in dry sausages. J. sausages: a review. Int. J. Food Microbiol., 88, 41-54. Ass. Off. Anal. Chem. Int., 76, 575-577. Wei, C. L., Chao, S. H., Tsai, W. B., Lee, P. S., Tsau, N. H., Chen, J. S., Funaki, J., Yano, M., and Hayabuchi, H. (1996). Proteolysis of Tofu- Lai, W. L., Tu, J. C. Y., and Tsai, Y. C. (2013). Analysis of bacterial misozuke during ripening. Nippon Shokuhin Kagaku Kaishi., 43, 546- diversity during the fermentation of inyu, a high temperature fermented 551 (in Japanese). soy sauce, using nested PCR-denaturing gradient gel electrophoresis and Funaki, J., Yano, M., Misaki, T., Abe, K., and Arai, S. (1997). Purification the plate count method. Food Microbiology, 33, 252-261. and characterization of a neutral protease that contributes to the unique Yang, J., Ding, X., Qin, Y., and Zeng, Y. (2014). Safety assessment of the flavor and texture of tofu-misozuke.J. Food Biochem., 21, 191-202. biogenic amines in fermented soya beans and fermented bean curd. J. Kim, T. W., Lee, J. H., Park, M. H., and Kim, H. Y. (2010). Analysis of Agric. Food Chem, 62, 7947-7954.