Mycobiology

The Korean Society of Mycology (한국균학회)
권호 표지
DOI QR코드

DOI QR Code

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

  • Sang-Cheol Jun (Department of Pharmaceutical Engineering, Woosuk University) ;
  • Yu-Kyung Kim (Department of Pharmaceutical Engineering, Woosuk University) ;
  • Kap-Hoon Han (Department of Pharmaceutical Engineering, Woosuk University)
  • Received : 2022.08.11
  • Accepted : 2022.12.02
  • Published : 2022.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

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.

Keywords

Acknowledgement

This work was supported by a grant (21162MFDS028) from the Ministry of Food and Drug Safety in 2022, Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A3B06035312) and Woosuk University.

References

  1. Hedayati MT, Pasqualotto AC, Warn PA, et al. Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology. 2007;153(6):1677-1692. https://doi.org/10.1099/mic.0.2007/007641-0
  2. Latg e JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev. 1999;12(2):310-350. https://doi.org/10.1128/CMR.12.2.310
  3. Apostolopoulou A, Esquer Garrigos Z, Vijayvargiya P, et al. Invasive pulmonary aspergillosis in patients with SARS-CoV-2 infection: a systematic review of the literature. Diagnostics. 2020;10(10):807.
  4. Lai CC, Yu WL. COVID-19 associated with pulmonary aspergillosis: a literature review. J Microbiol Immunol Infect. 2021;54(1):46-53. https://doi.org/10.1016/j.jmii.2020.09.004
  5. Keller NP. Fungal secondary metabolism: regulation, function and drug discovery. Nat Rev Microbiol. 2019;17(3):167-180.
  6. Pusztahelyi T, Holb IJ, P ocsi I. Secondary metabolites in fungus-plant interactions. Front Plant Sci. 2015;6:573.
  7. Spiteller P. Chemical ecology of fungi. Nat Prod Rep. 2015;32(7):971-993.
  8. Macheleidt J, Mattern DJ, Fischer J, et al. Regulation and role of fungal secondary metabolites. Annu Rev Genet. 2016;50:371-392. https://doi.org/10.1146/annurev-genet-120215-035203
  9. Park HS, Jun SC, Han KH, et al. Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol. 2017;100:161-202. https://doi.org/10.1016/bs.aambs.2017.03.001
  10. Toghueo RMK, Boyom FF. Endophytic Penicillium species and their agricultural, biotechnological, and pharmaceutical applications. 3 Biotech. 2020;10(3):107.
  11. Bennett JW, Klich M. Mycotoxins. Clin Microbiol Rev. 2003;16(3):497-516. https://doi.org/10.1128/CMR.16.3.497-516.2003
  12. Bhatnagar D, Cary JW, Ehrlich K, et al. Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development. Mycopathologia. 2006;162(3):155-166. https://doi.org/10.1007/s11046-006-0050-9
  13. Linz JE, Wee J, Roze LV. Aspergillus parasiticus SU-1 genome sequence, predicted chromosome structure, and comparative gene expression under aflatoxin-inducing conditions: evidence that differential expression contributes to species phenotype. Eukaryot Cell. 2014;13(8):1113-1123. https://doi.org/10.1128/EC.00108-14
  14. Kjaerbolling I, Vesth T, Frisvad JC, et al. A comparative genomics study of 23 Aspergillus species from section Flavi. Nat Commun. 2020;11(1):1106.
  15. Frisvad JC, Hubka V, Ezekiel CN, et al. Taxonomy of Aspergillus section Flavi and their production of aflatoxins, ochratoxins and other mycotoxins. Stud Mycol. 2019;93:1-63.
  16. Kusumoto K, Nogata Y, Ohta H. Directed deletions in the aflatoxin biosynthesis gene homolog cluster of Aspergillus oryzae. Curr Genet. 2000;37(2):104-111. https://doi.org/10.1007/s002940050016
  17. Lee CZ, Liou GY, Yuan GF. Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology. 2006;152(1):161-170. https://doi.org/10.1099/mic.0.27618-0
  18. Tominaga M, Lee YH, Hayashi R, et al. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl Environ Microbiol. 2006;72(1):484-490.
  19. Ehrlich KC, Chang PK, Yu J, et al. Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation. Appl Environ Microbiol. 2004;70(11):6518-6524. https://doi.org/10.1128/AEM.70.11.6518-6524.2004
  20. Horn BW, Greene RL, Sobolev VS, et al. Association of morphology and mycotoxin production with vegetative compatibility groups in Aspergillus flavus, A. parasiticus, and A. tamarii. Mycologia. 1996;88(4):574-587. https://doi.org/10.1080/00275514.1996.12026688
  21. Horn BW, Dorner JW. Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States. Appl Environ Microbiol. 1999;65(4):1444-1449.
  22. Chang PK, Ehrlich KC. What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae? Int J Food Microbiol. 2010;138(3):189-199. https://doi.org/10.1016/j.ijfoodmicro.2010.01.033
  23. Jung JY, Lee SH, Jeon CO. Microbial community dynamics during fermentation of doenjang-meju, traditional korean fermented soybean. Int J Food Microbiol. 2014;185:112-120. https://doi.org/10.1016/j.ijfoodmicro.2014.06.003
  24. Hong SB, Kim DH, Samson RA. Aspergillus associated with meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. Mycobiology. 2015;43(3):218-224. https://doi.org/10.5941/MYCO.2015.43.3.218
  25. Jung YJ, Chung SH, Lee HK, et al. Isolation and identification of fungi from a meju contaminated with aflatoxins. J Microbiol Biotechnol. 2012;22(12):1740-1748. https://doi.org/10.4014/jmb.1207.07048
  26. Woo SY, Ryu SY, Tian F, et al. Simultaneous determination of twenty mycotoxins in the korean soybean paste doenjang by LC-MS/MS with immunoaffinity cleanup. Toxins. 2019;11(10):594.
  27. Green MR, Sambrook J. Precipitation of DNA with ethanol. Cold Spring Harb Protoc. 2016;2016(12):pdb.prot093377.
  28. Orgiazzi A, Lumini E, Nilsson RH, et al. Unravelling soil fungal communities from different Mediterranean land-use backgrounds. PLoS One. 2012;7(4):e34847.
  29. Wu X, Zhang H, Chen J, et al. Comparison of the fecal microbiota of dholes high-throughput illumina sequencing of the V3-V4 region of the 16S rRNA gene. Appl Microbiol Biotechnol. 2016;100(8):3577-3586. https://doi.org/10.1007/s00253-015-7257-y
  30. Chang PK, Horn BW, Dorner JW. Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates. Fungal Genet Biol. 2005;42(11):914-923. https://doi.org/10.1016/j.fgb.2005.07.004
  31. Ceska M. Enzymatic catalysis in solidified media. Eur J Biochem. 1971;22(2):186-192. https://doi.org/10.1111/j.1432-1033.1971.tb01531.x
  32. Hankin L, Anagnostakis SL. The use of solid media for detection of enzyme production by fungi. Mycologia. 1975;67(3):597-607. https://doi.org/10.1080/00275514.1975.12019782
  33. Mourey A, Kilbertus G. Simple media containing stabilized tributyrin for demonstrating lipolytic bacteria in foods and soils. J Appl Bacteriol. 1976;40(1):47-51. https://doi.org/10.1111/j.1365-2672.1976.tb00589.x
  34. Segers FJ, Meijer M, Houbraken J, et al. Xerotolerant Cladosporium sphaerospermum are predominant on indoor surfaces compared to other Cladosporium species. PLoS One. 2015;10(12):e0145415.
  35. Segers FJ, van Laarhoven KA, Huinink HP, et al. The indoor fungus Cladosporium halotolerans survives humidity dynamics markedly better than Aspergillus niger and Penicillium rubens despite less growth at lowered steady-state water activity. Appl Environ Microbiol. 2016;82(17):5089-5098. https://doi.org/10.1128/AEM.00510-16
  36. Jung WY, Jung JY, Lee HJ, et al. Functional characterization of bacterial communities responsible for fermentation of doenjang: a traditional Korean fermented soybean paste. Front Microbiol. 2016;7:827.
  37. Oh DG, Jang YK, Woo JE, et al. Metabolomics reveals the effect of garlic on antioxidant- and protease-activities during Cheonggukjang (fermented soybean paste) fermentation. Food Res Int. 2016;82:86-94. https://doi.org/10.1016/j.foodres.2016.01.019
  38. Lee DE, Shin GR, Lee S, et al. Metabolomics reveal that amino acids are the main contributors to antioxidant activity in wheat and rice gochujangs (Korean fermented red pepper paste). Food Res Int. 2016;87:10-17. https://doi.org/10.1016/j.foodres.2016.06.015
  39. Lee SY, Kim HY, Lee S, et al. Mass spectrometry-based metabolite profiling and bacterial diversity characterization of Korean traditional meju during fermentation. J Microbiol Biotechnol. 2012;22(11):1523-1531. https://doi.org/10.4014/jmb.1207.07003
  40. Alshannaq AF, Gibbons JG, Lee MK, et al. Controlling aflatoxin contamination and propagation of Aspergillus flavus by a soy-fermenting Aspergillus oryzae strain. Sci Rep. 2018;8(1):16871.