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High-yield fermentative preparation of tetramethylpyrazine by Bacillus sp. using an endogenous precursor approach

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A spore-forming Bacillus sp. was isolated from a high-temperature Daqu, a starter culture of Chinese Maotai-flavor liquor, using an endogenous precursor screening strategy. The Bacillus sp. was capable of producing a high level of 2,3,5,6-tetramethylpyrazine (TTMP) via a precursor of 3-hydroxy-2-butanone (HB). The strain was characterized as Bacillus subtilis based on morphological, physiological, and biochemical properties as well as on partial 16S rRNA gene sequences. Different carbon and nitrogen sources as well as fermentation conditions were investigated. Optimization tests showed that oxygen supply and fermentation temperature were the most important parameters determining the production process. The production of >4.08 g/l TTMP was achieved together with a high level of endogenous precursor HB accumulation (>20 g/l) in both flask and fermentor cultures when the optimized medium and cultivation conditions were applied. Our data demonstrates the effectiveness of the endogenous precursor strategy for screening microorganisms that produce flavor compounds with structure-related precursors. The high yield of TTMP and the inexpensiveness of the agro-industrial product used as the substrate (soybean meal) indicate the potential of this process for industrial application.

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References

  1. Adachi T, Kamiya H, Kosuge T (1964) Studies on the metabolic products of Bacillus subtilis. IV. determination and mechanism of formation of tetramethylpyrazine. Yakugaku Zasshi 84:545–548

    CAS  PubMed  Google Scholar 

  2. Amranihemaimi M, Cerny C, Fay LB (1995) Mechanisms of formation of alkylpyrazines in the Maillard reaction. J Agric Food Chem 43:2818–2822

    Article  CAS  Google Scholar 

  3. Besson I, Creuly C, Gros JB, Larroche C (1997) Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans. Appl Microbiol Biotechnol 47:489–495

    Article  CAS  Google Scholar 

  4. Chen X, Chen S, Sun M, Yu Z (2005) Medium optimization by response surface methodology for poly-γ-glutamic acid production using dairy manure as the basis of a solid substrate. Appl Microbiol Biotechnol 69:390–396

    Article  Google Scholar 

  5. Demain AL, Jackson M, Trenner NR (1967) Thiamine-dependent accumulation of tetramethylpyrazine accompanying a mutation in isoleucine-valine pathway. J Bacteriol 94:323–326

    Article  CAS  PubMed  Google Scholar 

  6. Fan WL, Xu Y, Zhang YH (2007) Characterization of pyrazines in some Chinese liquors and their approximate concentrations. J Agric Food Chem 55:9956–9962

    Article  CAS  PubMed  Google Scholar 

  7. Grundy FJ, Turinsky AJ, Henkin TM (1994) Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA. J Bacteriol 176:4527–4533

    CAS  PubMed  Google Scholar 

  8. Ho JW, Jie M (2007) Pharmacological activity of cardiovascular agents from herbal medicine. Cardiovasc Hematol Agents Med Chem 5:273–277

    Article  CAS  PubMed  Google Scholar 

  9. Huang M, Oppermann-Sanio FB, Steinbuchel A (1999) Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway. J Bacteriol 181:3837–3841

    CAS  PubMed  Google Scholar 

  10. Huang TC, Fu HY, Ho CT (1996) Mechanistic studies of tetramethylpyrazine formation under weak acidic conditions and high hydrostatic pressure. J Agric Food Chem 44:240–246

    Article  CAS  Google Scholar 

  11. Jalbout AF, Shipar MAH (2007) Formation of pyrazines in hydroxyacetaldehyde and glycine nonenzymatic browning Maillard reaction: a computational study. Food Chem 103:1208–1216

    Article  CAS  Google Scholar 

  12. Johansen L, Bryn K, Stormer FC (1975) Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes. J Bacteriol 123:1124–1130

    CAS  PubMed  Google Scholar 

  13. Kim KS, Lee HJ, Shon DH, Chung DK (1994) Optimum conditions for the production of tetramethylpyrazine flavor compound by aerobic fed-batch culture of Lactococcus lactis subsp. lactis biovar. diacetylactis FC1. J Microbiol Biotechnol 4:327–332

    CAS  Google Scholar 

  14. Kosuge T, Kamiya H, Adachi T (1962) Isolation of tetramethylpyrazine from culture of Bacillus natto, and biosynthetic pathways of tetramethylpyrazine. Nature 195:1103

    Article  CAS  PubMed  Google Scholar 

  15. Larroche C, Besson I, Gros JB (1999) High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans. Process Biochem 34:667–674

    Article  CAS  Google Scholar 

  16. Li HB, Chen F (2004) Preparative isolation and purification of chuanxiongzine from the medicinal plant Ligusticum chuanxiong by high-speed counter-current chromatography. J Chromatogr A 1047:249–253

    CAS  PubMed  Google Scholar 

  17. Liu B, Dean JR, Price R (1999) Extraction of tetramethylpyrazine from Ligusticum chuanxiong Hort. using phytosol solvent and supercritical fluid. Zhongguo Yiyao Gongye Zazhi 30:196–198

    Google Scholar 

  18. Liu XH, Li J, Li QX, Ai YX, Zhang L (2008) Protective effects of ligustrazine on cisplatin-induced oxidative stress, apoptosis and nephrotoxicity in rats. Environ Toxicol Pharmacol 26:49–55

    Article  CAS  Google Scholar 

  19. Masuda H, Mihara S (1988) Olfactive properties of alkylpyrazines and 3-substituted 2-alkylpyrazines. J Agric Food Chem 36:584–587

    Article  CAS  Google Scholar 

  20. Meara RAQO (1931) A simple delicate and rapid method of detecting the formation of acetylmethylcarbinol by bacteria fermenting carbohydrate. J Pathol Bacteriol 34:401–406

    Article  Google Scholar 

  21. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  22. Moes J, Griot M, Keller J, Heinzle E, Dunn IJ, Bourne JR (1985) A microbial culture with oxygen-sensitive product distribution as a potential tool for characterizing bioreactor oxygen-transport. Biotechnol Bioeng 27:482–489

    Article  CAS  PubMed  Google Scholar 

  23. Nakashimada Y, Kanai K, Nishio N (1998) Optimization of dilution rate, pH and oxygen supply on optical purity of 2, 3-butanediol produced by Paenibacillus polymyxa in chemostat culture. Biotechnol Lett 20:1133–1138

    Article  CAS  Google Scholar 

  24. Owens JD, Allagheny N, Kipping G, Ames JM (1997) Formation of volatile compounds during Bacillus subtilis fermentation of soya beans. J Sci Food Agric 74:132–140

    Article  CAS  Google Scholar 

  25. Rizzi GP (1988) Formation of pyrazines from acyloin precursors under mild conditions. J Agric Food Chem 36:349–352

    Article  CAS  Google Scholar 

  26. Schrader J (2007) Microbial flavour production. In: Berger RG (ed) Flavours and fragrances: chemistry, bioprocessing and sustainability. Springer, Berlin, pp 507–574

    Google Scholar 

  27. Sikkema J, Debont JAM, Poolman B (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222

    CAS  PubMed  Google Scholar 

  28. Xiao ZJ, Xie NZ, Liu PH, Hua DL, Xu P (2006) Tetramethylpyrazine production from glucose by a newly isolated Bacillus mutant. Appl Microbiol Biotechnol 73:512–518

    Article  CAS  PubMed  Google Scholar 

  29. Xiao ZJ, Xu P (2007) Acetoin metabolism in bacteria. Crit Rev Microbiol 33:127–140

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Financial support from the Ministry of Science and Technology, P. R. China under National Key Technology R&D Program (2007 BAK36B02 and 2008 BAI63B06), the Ministry of Education, P. R. China under the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (No. IRT0532), the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-6), the Natural Science Foundation of China (NSFC) (No. 30470046, 20872050), and the State Key Laboratory of Food Science and Technology, Jiangnan University (No. SKLF-MB-200801) is gratefully acknowledged.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, 214122, Wuxi, Jiangsu, China

    Bing-Feng Zhu, Yan Xu & Wen-Lai Fan

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  1. Bing-Feng Zhu
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  2. Yan Xu
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  3. Wen-Lai Fan
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Correspondence to Yan Xu.

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Zhu, BF., Xu, Y. & Fan, WL. High-yield fermentative preparation of tetramethylpyrazine by Bacillus sp. using an endogenous precursor approach. J Ind Microbiol Biotechnol 37, 179–186 (2010). https://doi.org/10.1007/s10295-009-0661-5

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  • DOI: https://doi.org/10.1007/s10295-009-0661-5

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