Mycobiology

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

DOI QR Code

Taxonomic Characterization, Evaluation of Toxigenicity, and Saccharification Capability of Aspergillus Section Flavi Isolates from Korean Traditional Wheat-Based Fermentation Starter Nuruk

  • Bal, Jyotiranjan (Department of Molecular Biology, Institute for Molecular Biology and Genetics, Chonbuk National University) ;
  • Yun, Suk-Hyun (Department of Molecular Biology, Institute for Molecular Biology and Genetics, Chonbuk National University) ;
  • Chun, Jeesun (Department of Molecular Biology, Institute for Molecular Biology and Genetics, Chonbuk National University) ;
  • Kim, Beom-Tae (Department of Bioactive Material Sciences, Institute for Molecular Biology and Genetics, Chonbuk National University) ;
  • Kim, Dae-Hyuk (Department of Molecular Biology, Institute for Molecular Biology and Genetics, Chonbuk National University)
  • Received : 2016.09.09
  • Accepted : 2016.09.21
  • Published : 2016.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillus oryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through ${\alpha}-amylase$ and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in ${\alpha}-amylase$ and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that ${\alpha}-amylase$ activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.

Keywords

References

  1. Park JW, Lee KH, Lee CY. Identification of filamentous molds isolated from Korean traditional nuruk and their amylolytic activities. Korean J Appl Microbiol Biotechnol 1995; 23:737-46.
  2. Machida M, Yamada O, Gomi K. Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res 2008;15:173-83. https://doi.org/10.1093/dnares/dsn020
  3. Rokas A. The effect of domestication on the fungal proteome. Trends Genet 2009;25:60-3. https://doi.org/10.1016/j.tig.2008.11.003
  4. Liang Y, Pan L, Lin Y. Analysis of extracelular proteins of Aspergillus oryzae grown on soy sauce koji. Biosci Biotechnol Biochem 2009;73:192-5. https://doi.org/10.1271/bbb.80500
  5. Yu TS, Kim J, Kim HS, Hyun JS, Ha HP, Park MG. Bibliographical study on microorganisms of traditional Korean nuruk (since 1945). J Korean Soc Food Sci Nutr 1998;27:789-99.
  6. Kim HS, Hyun JS, Kim J, Ha HP, Yu TS. Enzymological characteristics and identification of useful fungi isolated from traditional Korean nuruk. Korean J Appl Microbiol Biotechnol 1998;26:456-64.
  7. Wang L, Yokoyama K, Miyaji M, Nishimura K. Mitochondrial cytochrome b gene analysis of Aspergillus fumigatus and related species. J Clin Microbiol 2000;38:1352-8.
  8. Henry T, Iwen PC, Hinrichs SH. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J Clin Microbiol 2000;38:1510-5.
  9. Kumeda Y, Asao T. Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus section Flavi strains. Appl Environ Microbiol 2001;67:4084-90. https://doi.org/10.1128/AEM.67.9.4084-4090.2001
  10. Carbone I, Kohn LM. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999;91:553-6. https://doi.org/10.2307/3761358
  11. Tominaga M, Lee YH, Hayashi R, Suzuki Y, Yamada O, Sakamoto K, Gotoh K, Akita O. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl Environ Microbiol 2006;72:484-90. https://doi.org/10.1128/AEM.72.1.484-490.2006
  12. Ehrlich KC, Chang PK, Yu J, Cotty PJ. Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation. Appl Environ Microbiol 2004;70:6518-24. https://doi.org/10.1128/AEM.70.11.6518-6524.2004
  13. Bal J, Yun SH, Song HY, Yeo SH, Kim JH, Kim JM, Kim DH. Mycoflora dynamics analysis of Korean traditional wheatbased nuruk. J Microbiol 2014;52:1025-9. https://doi.org/10.1007/s12275-014-4620-0
  14. Serra R, Peterson SW. Penicillium astrolabium and Penicillium neocrassum, two new species isolated from grapes and their phylogenetic placement in the P. olsonii and P. brevicompactum clade. Mycologia 2007;99:78-87. https://doi.org/10.1080/15572536.2007.11832602
  15. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-20. https://doi.org/10.1007/BF01731581
  16. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4. https://doi.org/10.1093/molbev/msw054
  17. Bernfeld P. Amylase ${\alpha}$ and ${\beta}$. In: Colowick SP, Kaplan NO, editors. Methods in enzymology. New York: Academic Press Inc.; 1955. p. 149-58.
  18. Yang S, Choi SJ, Kwak J, Kim K, Seo M, Moon TW, Lee YW. Aspergillus oryzae strains isolated from traditional Korean Nuruk: fermentation properties and influence on rice wine quality. Food Sci Biotechnol 2013;22:425-32. https://doi.org/10.1007/s10068-013-0097-6
  19. Anson ML. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 1938;22:79-89. https://doi.org/10.1085/jgp.22.1.79
  20. Yang S, Lee J, Kwak J, Kim K, Seo M, Lee YW. Fungi associated with the traditional starter cultures used for rice wine in Korea. J Korean Soc Appl Biol Chem 2011;54:933-43. https://doi.org/10.1007/BF03253183
  21. Bal J, Yun SH, Yeo SH, Kim JM, Kim DH. Metagenomic analysis of fungal diversity in Korean traditional wheat-based fermentation starter nuruk. Food Microbiol 2016;60:73-83. https://doi.org/10.1016/j.fm.2016.07.002
  22. Varga J, Frisvad JC, Kocsube S, Brankovics B, Toth B, Szigeti G, Samson RA. New and revisited species in Aspergillus section Nigri. Stud Mycol 2011;69:1-17. https://doi.org/10.3114/sim.2011.69.01
  23. 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;43:218-24. https://doi.org/10.5941/MYCO.2015.43.3.218
  24. Rodrigues P, Santos C, Venancio A, Lima N. Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDITOF ICMS, and molecular approaches. J Appl Microbiol 2011;111:877-92. https://doi.org/10.1111/j.1365-2672.2011.05116.x
  25. Hong SB, Lee M, Kim DH, Chung SH, Shin HD, Samson RA. The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes. J Microbiol 2013;51:766-7 https://doi.org/10.1007/s12275-013-3128-3