Isolation and Identification of Bacillus sp. with High Protease and Amylase Activity from Sunchang Traditional Kochujang

  • Jung, Sung-Tae (Harim Ltd.) ;
  • Kim, Min-Hwa (Faculty of Biotechnology (Food Science & Technology Major), Chonbuk National University) ;
  • Shin, Dong-Hwa (Faculty of Biotechnology (Food Science & Technology Major), Chonbuk National University) ;
  • Kim, Yong-Suk (Research Center for Industrial Development of BioFood Materials, Chonbuk National University)
  • Published : 2008.06.30

Abstract

To improve the quality of traditional kochujang, strains with high protease and amylase activity were isolated and identified from Sunchang traditional kochujang. Twenty-three strains strongly producing protease and 16 strains strongly producing $\alpha$- and $\beta$-amylase were isolated by using 1% isolated soy protein agar medium and 2% starch agar medium, respectively. Protease activities of the IA7, I5, and IA2 strain were 22.5, 21.2, and 20.6 unit/mL, respectively, and were higher than those of the other strains. Stains with high $\alpha$-amylase activity included K9 (967.8 unit/mL), K14 (828.3 unit/mL), K13 (662.5 unit/mL), K8 (601.5 unit/mL), and K11 (405.9 unit/mL). The $\beta$-amylase activity of the K11 strain was the highest, 34.3 unit/mL, among the isolated strains. Based on morphological, physiological properties, and API 50CHB-kit test for assimilation of 49 carbohydrates, 8 strains selected according to protease, $\alpha$-amylase, and $\beta$-amylase activities were tentatively identified as Bacillus megaterium (IA2), Bacillus subtilis (IA7, 15), Bacillus amyloliquefaciens (K8, K9, K11, and K13), and Bacillus stearothermophillus (K14). The IA7, 15, and K11 strains were finally identified as B. subtilis (99% ID) based on 16S rDNA sequencing.

References

  1. Ahn IS, Do MS, Kim SO, Jung HS, Kim YI, Kim HJ, Park KY. Antiobesity effect of kochujang (Korean fermented red pepper paste) extract in 3T3-L1 adipocytes. J. Med. Food 9: 15-21 (2006) https://doi.org/10.1089/jmf.2006.9.15
  2. Oh JY, Kim YS, Shin DH. Changes in microorganisms and enzyme activities of low-salted kochujang added with horseradish powder during fermentation. Korean J. Food Sci. Technol. 37: 463-467 (2005)
  3. Yun GH, Lee ET, Kim SD. Purification and characterization of a fibrinolytic enzyme produced from Bacillus amyloliquefaciens K42 isolated from Korean soy sauce. Korean J. Microbiol. Biotechnol. 31: 284-291 (2003)
  4. Lee SH, Lee MS. Utilization the tofu-residue for production of the bacteriocin. J. Food Hyg. Saf. 15: 271-276 (2000)
  5. Paik HD, Kim IG, Lee JH, Lee JH, Park KY, Ji GE, Jin TE, Rhim SL. Heterologous expression of $\alpha$-amylase gene of Bifidobacterium adolescentis Int57 in Bacillus polyfermenticus SCD. Food Sci. Biotechnol. 16: 655-658 (2007)
  6. Thompson JD, Higgins DG, Gibson TJ, Clustal W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties, and weight matrix choice. Nucleic Acids Res. 22: 4673-4680 (1994) https://doi.org/10.1093/nar/22.22.4673
  7. Oh HI, Shon SH, Kim JM. Changes in microflora and enzyme activities of kochujang prepared with Aspergillus oryzae, Bacillus licheniformis, and Saccharomyces rouxii during fermentation. Korean J. Food Sci. Technol. 32: 410-416 (2000)
  8. Moon TW, Kim ZU. Some chemical physical characteristics and acceptability of kochujang from various starch sources. J. Korean Agr. Chem. Soc. 31: 387-393 (1998)
  9. Kim YS. Studies on the changes in physicochemical characteristics and volatile flavor compounds of traditional kochujang during fermentation. PhD thesis, Sejong University, Seoul, Korea (1993)
  10. Kim YS, Kwon DJ, Koo MS, Oh HI, Kang TS. Changes in microflora and enzyme activities of traditional kochujang during fermentation. Korean J. Food Sci. Technol. 25: 502-509 (1993)
  11. Jung YC, Choi WJ, Oh NS, Han MS. Distribution and physiological characteristics of yeasts in traditional and commercial kochujang. Korean J. Food Sci. Technol. 28: 253-259 (1996)
  12. Lee JS, Choi YJ, Kwon SJ, Yoo JY, Chung DH. Screening and characterization of osmotolerant and gas-producing yeasts from traditional doenjang and kochujang. Food Sci. Biotechnol. 5: 54-58 (1996)
  13. Rhee SH, Kong KR, Jung KO, Park KY. Decreasing effect of kochujang on body weight and lipid levels of adipose tissues and serum in rats fed a high-fat diet. J. Korean Soc. Food Sci. Nutr. 32: 882-886 (2003) https://doi.org/10.3746/jkfn.2003.32.6.882
  14. Lee TS. Studies on the brewing of kochujang (red pepper paste) by the addition of yeast. J. Korean Agr. Chem. Soc. 22: 65-90 (1979)
  15. Lee KH, Lee MS, Park SO. Studies on the microflora and enzymes influencing on Korea native kochujang (red pepper soybean paste) aging. J. Korean Agr. Chem. Soc. 19: 82-92 (1976)
  16. Shin DH, Ahn EY, Kim YS, Oh JY. Changes in the microflora and enzyme activities of kochujang prepared with different koji during fermentation. Korean J. Food Sci. Technol. 33: 94-99 (2001)
  17. Hwang CS, Kim HH, Oh BC, Kim YS, Shin DH. Identification and characteristics of microorganism isolated from spoiled red bean paste. Food Sci. Biotechnol. 13: 758-761 (2004)
  18. Oh BH, Kim YS, Jeong PH, Shin DH. Quality characteristics of kochujang meju prepared with Aspergillus species and Bacillus subtilis. Food Sci. Biotechnol. 15: 549-554 (2006)
  19. Yoon JH, Lee ST, Park YH. Inter- and intraspecific phylogenetic analysis of genus nocardiodies and related taxa based on 16S rDNA sequences. Int. J. Syst. Bacteriol. 48: 187-194 (1996) https://doi.org/10.1099/00207713-48-1-187
  20. Oh JY, Kim YS, Shin DH. Changes in microorganisms, enzyme activities, and gas formation by the addition of mustard powder on kochujang with different salt concentration. Food Sci. Biotechnol. 15: 298-302 (2006)
  21. Choo JJ. Anti-obesity effects of kochujang in rats fed on high-fat diet. J. Korean Soc. Food Nutr. 33: 787-793 (2000)
  22. Sneath PHA, Mair NS, Sharpe ME, Holt JG. Bergey's Manual of Systematic Bacteriology. Vol. 2. Williams & Wilkins, Baltimore, MD, USA (1994)
  23. Kim MS, Kim IW, Oh JA, Shin DH. Effect of different koji and irradiation on the quality of traditional kochujang. Korean J. Food Sci. Technol. 31: 196-295 (1999)
  24. Kim YS, Ahn YS, Jeong DY, Shin DH. Isolation and identification of Bacillus cereus from fermented red pepper-soybean paste (kochujang), and its heat resistance characteristics. Food Sci. Biotechnol. 17: 123-129 (2008)
  25. Shin DH, Kim DH, Choi U, Lim MS, Ahn EY. Changes in microflora and enzymes activity of traditional kochujang prepared with various raw materials. Korean J. Food Sci. Technol. 29: 901- 906 (1997)
  26. Kim DH, Choi HJ. Physicochemical properties of kochujang prepared by Bacillus sp. koji. Korean J. Food Sci. Technol. 35: 1174-1181 (2003)
  27. Kim HJ, Lee JJ, Cheigh SY. Amylase, protease, peroxidase, and ascorbic acid oxidase activity of kimchi ingredients. Korean J. Food Sci. Technol. 30: 1333-1338 (1998)
  28. Choi JY, Lee TS, Noh BS. Quality characteristics of the kochujang prepared with mixture of meju and koji during fermentation. Korean J. Food Sci. Technol. 32: 125-131 (2000)
  29. Lee JM, Jang JH, Oh NS, Han MS. Bacterial distribution of kochujang. Korean J. Food Sci. Technol. 28: 260-266 (1996)
  30. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428 (1959) https://doi.org/10.1021/ac60147a030
  31. Kim YC, Yoo BS. Rheological properties of traditional kochujang. Korean J. Food. Sci. Technol. 32: 1313-1318 (2000)