DOI QR코드

DOI QR Code

Isolation and Identification of Fungi from a Meju Contaminated with Aflatoxins

  • Jung, Yu Jung (Department of Food Science and Technology, Kongju National University) ;
  • Chung, Soo Hyun (Department of Food and Nutrition, College of Health Science, Korea University) ;
  • Lee, Hyo Ku (Department of Food Science and Technology, Kongju National University) ;
  • Chun, Hyang Sook (Food Safety Research Division, Korea Food Research Institute) ;
  • Hong, Seung Beom (Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA)
  • Received : 2012.07.24
  • Accepted : 2012.09.28
  • Published : 2012.12.28

Abstract

A home-made meju sample contaminated naturally with aflatoxins was used for isolation of fungal strains. Overall, 230 fungal isolates were obtained on dichloran rosebengal chloramphenicol (DRBC) and dichloran 18% glycerol (DG18) agar plates. Morphological characteristics and molecular analysis of a partial ${\beta}$-tubulin gene and the internal transcribed spacer (ITS) of rDNA were used for the identification of the isolates. The fungal isolates were divided into 7 genera: Aspergillus, Eurotium, Penicillium, Eupenicillium, Mucor, Lichtheimia, and Curvularia. Three strains from 56 isolates of the A. oryzae/flavus group were found to be aflatoxigenic A. flavus, by the presence of the aflatoxin biosynthesis genes and confirmatory aflatoxin production by high-performance liquid chromatography (HPLC). The predominant isolate from DRBC plates was A. oryzae (42 strains, 36.2%), whereas that from DG18 was A. candidus (61 strains, 53.5%). Out of the 230 isolates, the most common species was A. candidus (34.3%) followed by A. oryzae (22.2%), Mucor circinelloides (13.0%), P. polonicum (10.0%), A. tubingensis (4.8%), and L. ramosa (3.5%). A. flavus and E. chevalieri presented occurrence levels of 2.2%, respectively. The remaining isolates of A. unguis, P. oxalicum, Eupenicillium cinnamopurpureum, A. acidus, E. rubrum, P. chrysogenum, M. racemosus, and C. inaequalis had lower occurrence levels of < 2.0%.

Keywords

References

  1. Barbesgaard, P., H. P. Heldt-Hansen, and B. Diderichsen. 1992. On the safety of Aspergillus oryzae: A review. Appl. Microbiol. Biotechnol. 36: 569-572.
  2. Chang, P. K. and K. C. Ehrlich. 2010. What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae? Int. J. Food Microbiol. 15: 189-199.
  3. Chang, P. K., B. W. Horn, and J. W. Dorner. 2005. Sequence break points in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genet. Biol. 42: 914-923. https://doi.org/10.1016/j.fgb.2005.07.004
  4. Chelkowski, J. 1991. Mycological quality of mixed feeds and ingredients, pp. 217-227. In J. Chelkowski (ed.). Cereal Grain, Mycotoxins, Fungi and Quality in Drying and Storage. Elsevier, Amsterdam, London, New York.
  5. Cho, D. H. and W. J. Lee. 1970. Microbiological studies of Korean native soy-sauce fermentation; A study on the microflora of fermented Korean maeju loaves. J. Kor. Agric. Chem. Soc. 13.
  6. Criseo, G., C. Racco, and O. Romeo. 2008. High genetic variability in non-aflatoxigenic A. flavus strains by using quadruplex PCR-based assay. Int. J. Food Microbiol. 125: 341-343. https://doi.org/10.1016/j.ijfoodmicro.2008.04.020
  7. Degola, F., E. Berni, C. Dall'Asta, E. Spotti, R. Marchelli, I. Ferrero, and F. M. Restivo. 2006. A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus. J. Appl. Microbiol. 103: 409-417.
  8. Fraga, M. E., F. Curvello, M. J. Gatti, L. R. Cavaglieri, A. M. Dalcero, and C. A. da Rocha Rosa. 2007. Potential aflatoxin and ochratoxin A production by Aspergillus species in poultry feed processing. Vet. Res. Commun. 31: 343-353.
  9. He, C. H., Y. H. Fan, G. F. Liu, and H. B. Zhang. 2008. Isolation and identification of a strain of Aspergillus tubingensis with deoxynivalenol biotransformation capability. Int. J. Mol. Sci. 9: 2366-2375. https://doi.org/10.3390/ijms9122366
  10. Hillis, D. M. and M. T. Dixon. 1991. Ribosomal DNA: Molecular evolution and phylogenetic inference. Q. Rev. Biol. 66: 411-453. https://doi.org/10.1086/417338
  11. Hocking, A. D. and J. I. Pitt. 1980. Dichloran-glycerol medium for enumeration of xerophilic fungi from low moisture foods. Appl. Environ. Microbiol. 39: 488-492.
  12. Horton, T. R. and T. D. Bruns. 2001. The molecular revolution in ectomycorrhizal ecology: Peeking into the black box. Mol. Ecol. 10: 1855-1871. https://doi.org/10.1046/j.0962-1083.2001.01333.x
  13. Hussein, S. H. and J. M. Brasel. 2001. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 167: 101-134. https://doi.org/10.1016/S0300-483X(01)00471-1
  14. Jumpponen, A. 2009. Analysis of rhizosphere fungal communities using rRNA and rDNA, pp. 29-40. In A. Varma and A. C. Kharkwal (eds.). Symbiotic Fungi, Soil Biology, 18th. Ed. Springer-Verlag, Berlin.
  15. Kim, D. H., S. H. Kim, Y. K. Kim, S. O. Kim, S. J. Kim, and S. B. Hong. 2009. Reidentification of Aspergillus spp. isolated from clinical specimens of patients suspected as pulmonary aspergillosis in Korea. Kor. J. Med. Mycol. 14: 133-144.
  16. Kim, D. M., S. H. Chung, and H. S. Chun. 2011. Multiplex PCR assay for the detection of aflatoxigenic and nonaflatoxigenic fungi in meju, a Korean fermented soybean food starter. Food Microbiol. 28: 1402-1408. https://doi.org/10.1016/j.fm.2011.06.017
  17. Kim, J. Y., S. H. Yeo, S. Y. Baek, and H. S. Choi. 2011 Molecular and morphological identification of fungal species isolated from bealmijang meju. J. Microbiol. Biotechnol. 21: 1270-1279. https://doi.org/10.4014/jmb.1105.05013
  18. Kiyota, T., R. Hamada, K. Sakamoto, K. Iwashita, O. Yamada, and S. Mikami. 2011. Aflatoxin non-productivity of Aspergillus oryzae caused by loss of function in the aflJ gene product. J. Biosci. Bioeng. 111: 512-517. https://doi.org/10.1016/j.jbiosc.2010.12.022
  19. Kwon, D. J. 2002. Comparison of characteristics of koji manufactured with Bacillus subtilis B-4 and Aspergillus oryzae F-5. Kor. J. Food Sci. Technol. 34: 873-878.
  20. Lee, C. H. and S. S. Lee. 2002. Cereal fermentation by fungi. Appl. Mycol. Biotechnol. 2: 151-170.
  21. Lee, S. S., K. H. Park, K. J. Choi, and S. A. Won. 1993. Identification and isolation of Zygomycetous fungi found on maeju, a raw material of Korean traditional soysauces. Kor. J. Mycol. 21: 172-187.
  22. Lee, S. S., K. H. Park, K. J. Choi, and S. A. Won. 1993. A study on Hyphomycetous fungi found on maejus, a raw material of Korean traditional soysauce. Kor. J. Mycol. 21: 242-272.
  23. Lee, S. W., S. K. Park, and H. C. Kim. 2001. Characteristics of red mold isolated from traditional meju. Kor. J. Post-harvest Sci. Technol. 8: 199-205.
  24. Niessen, L. 2008. PCR-based diagnosis and quantification of mycotoxin-producing fungi. Adv. Food Nutr. Res. 54: 81-138.
  25. Park, J. H., S. J. Kang, S. S. Oh, and D. H. Chung. 2001. The screening of aflatoxin producing fungi from commercial meju and soybean paste in western Gyeongnam by immunoassay. J. Food Hyg. Safety 16: 274-279.
  26. Park, K.-Y., K.-B. Lee, and L. B. Bullerman. 1988. Aflatoxin production by Aspergillus parasiticus and its stability during the manufacture of Korean soy paste (doenjang) and soy sauce (kanjang) by traditional method. J. Food Prot. 51: 938-945.
  27. Peterson, S. W. 2008. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia 100: 205-226. https://doi.org/10.3852/mycologia.100.2.205
  28. Pitt, J. I. and A. D. Hocking. 2009. Fungi and Food Spoilage, 3rd Ed. Springer, New York.
  29. Rank, C., M. L. Klejnstrup, L. M. Petersen, S. Kildgaard, J. C. Frisvad, C. H. Gotfredsen, and T. O. Larsen. 2012. Comparative chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites 2: 39-56. https://doi.org/10.3390/metabo2010039
  30. Reddy, B. N. and C. R. Raghavender. 2007. Outbreaks of aflatoxicoses in India. Afr. J. Food Agric. Nutr. Devel. 7: 1-15.
  31. Samson, R. A., E. S. Hoekstra, and J. C. Frisvad. 2004. Introduction to Food and Airborne Fungi, 7th Ed. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
  32. Samson, R. A., K. A. Seifert, A. F. A. Kuijpers, J. A. M. P. Houbraken, and J. C. Frisvad. 2004. Phylogenetic analysis of Penicillium subgenus Penicillium using partial ${\beta}$-tubulin sequences. Stud. Mycol. 49: 175-200.
  33. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. https://doi.org/10.1093/molbev/msm092
  34. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL X Windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882. https://doi.org/10.1093/nar/25.24.4876
  35. Tominaga, M., Y. H. Lee, R. Hayashi, O. Suzuki, K. Tamada, K. Skamoto, K. Gotoh, and O. Akita. 2006. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl. Environ. Microbiol. 72: 484-490. https://doi.org/10.1128/AEM.72.1.484-490.2006
  36. Tsai, G. J. and S. C. Yu. 1997. An enzyme-linked immunosorbent assay for the detection of Aspergillus parasiticus and Aspergillus flavus. J. Food Prot. 60: 978-984.
  37. Wei, D. L. and S. C. Jong. 1986. Production of aflatoxins by strains of the Aspergillus flavus group maintained in ATCC. Mycopathologia 93: 19-24. https://doi.org/10.1007/BF00437010
  38. White, T. J., T. Bruns, S. Lee, and J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, pp. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (eds.). PCR Protocols: A Guide to Methods and Applications. Academic Press Inc., NY.
  39. Yoo, J. Y. and H. G. Kim. 1998. Characteristics of traditional mejus of nation-wide collection. J. Kor. Soc. Food Sci. Nutr. 27: 259-267.

Cited by

  1. Effect of incubation temperature on variations in bacterial communities grown in fermenting meju and the nutritional quality of soy sauce vol.23, pp.6, 2012, https://doi.org/10.1007/s10068-014-0262-6
  2. Non-Aflatoxigenicity of Commercial Aspergillus oryzae Strains Due to Genetic Defects Compared to Aflatoxigenic Aspergillus flavus vol.24, pp.8, 2012, https://doi.org/10.4014/jmb.1311.11011
  3. An Evaluation of Aflatoxin and Cyclopiazonic Acid Production in Aspergillus oryzae vol.77, pp.6, 2012, https://doi.org/10.4315/0362-028x.jfp-13-448
  4. Mycoflora and Enzymatic Characterization of Fungal Isolates in Commercial Meju, Starter for a Korean Traditional Fermented Soybean Product vol.42, pp.3, 2012, https://doi.org/10.5941/myco.2014.42.3.291
  5. Safety Evaluation of Filamentous Fungi Isolated from Industrial Doenjang Koji vol.24, pp.10, 2012, https://doi.org/10.4014/jmb.1403.03007
  6. 순창군 장류로부터 분리된 황국균의 동정 및 특성 vol.42, pp.4, 2012, https://doi.org/10.4489/kjm.2014.42.4.282
  7. Making soy sauce from defatted soybean meal without the mejus process by submerged cultivation using thermophilic bacteria vol.52, pp.8, 2012, https://doi.org/10.1007/s13197-014-1536-y
  8. Making soy sauce using direct fermentation of defatted soybean meal without the meju (soybean Koji) preparation process vol.25, pp.3, 2012, https://doi.org/10.1007/s10068-016-0132-5
  9. Antifungal Activity of Protamine Salmine Hydrochloride and ε‐Poly‐l‐Lysine in Actual Food Systems, Rice‐ or Wheat‐Based Confectioneries vol.40, pp.6, 2012, https://doi.org/10.1111/jfpp.12700
  10. Biodegradation of Ochratoxin A by Aspergillus tubingensis Isolated from Meju vol.26, pp.10, 2016, https://doi.org/10.4014/jmb.1606.06016
  11. 국내에서 분리된 황국균을 활용한 된장 제조 및 특성 분석 vol.44, pp.1, 2016, https://doi.org/10.4014/mbl.1511.11012
  12. Aflatoxin B1 Detoxification by Aspergillus oryzae from Meju, a Traditional Korean Fermented Soybean Starter vol.27, pp.1, 2017, https://doi.org/10.4014/jmb.1607.07064
  13. A molasses habitat-derived fungus Aspergillus tubingensis XG21 with high β-fructofuranosidase activity and its potential use for fructooligosaccharides production vol.7, pp.1, 2012, https://doi.org/10.1186/s13568-017-0428-8
  14. Natural Occurrence of Aflatoxigenic Aspergillus Species and Aflatoxins in Traditional Korean Fermentation Starters, Meju and Nuruk vol.35, pp.5, 2012, https://doi.org/10.13103/jfhs.2020.35.5.438
  15. Critical thresholds of 1-Octen-3-ol shape inter-species Aspergillus interactions modulating the growth and secondary metabolism vol.10, pp.None, 2012, https://doi.org/10.1038/s41598-020-68096-x
  16. Contamination characteristics and risk assessment of aflatoxins in homemade soybean paste, a traditional fermented soybean food, in South Korea vol.424, pp.no.pc, 2012, https://doi.org/10.1016/j.jhazmat.2021.127576