Annals of Microbiology

, Volume 68, Issue 10, pp 655–665 | Cite as

Coproduction of menaquinone-7 and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy

  • Han Wang
  • Xiaojuan Sun
  • Li Wang
  • Hefang Wu
  • Genhai Zhao
  • Hui Liu
  • Peng WangEmail author
  • Zhiming ZhengEmail author
Original Article


Numerous physiological functions of menaquinone-7 (MK-7) act to reduce vascular calcification, suggesting that MK-7 may be a potential therapy for Alzheimer’s and Parkinson’s disease, and in this study, we attempted to increase the concentration of MK-7 synthesized by Bacillus subtilis natto, a standard nattokinase (NK) producing strain. Different Bacillus subtilis isolates demonstrated positive correlations between MK-7 and NK concentrations. Response surface methodology (RSM) was employed to optimize a culture medium for the simultaneous production of these molecules; the optimized medium contained the following components (%, w/v): soybean curd residue, 12.2; soya peptone, 5.7; lactose, 2.6; and K2HPO4, 0.6. The fermentation process was subsequently optimized based on online feedback control of fermentation process parameters. The dissolved oxygen (DO) concentration played an important role in the production of MK-7 and NK. With increased DO concentrations, the cell growth rate and NK activity increased. In contrast, at low DO concentrations, the concentration of MK-7 rapidly increased during the late fermentation stage. Thus, in this study, the production of MK-7 and NK by Bacillus subtilis was accomplished using soybean curd residue through medium optimization and DO control. This novel coproduction strategy was developed by controlling the aeration rate during the fermentation process. The concentrations of MK-7 and NK achieved in this study reached 91.25 mg/L and 2675.73 U/mL, respectively.


Bacillus subtilis Menaquinone-7 Nattokinase Optimization of fermentation Process control Response surface 



This research was funded by The National High Technology Research and Development Program of China (2014AA021704) and by the Major Projects of Science and Technology in Anhui Province “Development and Demonstration of Vitamin K2 Functional Food” grant (grant no. 17030801036).

Compliance with ethical standards

Conflicts of interest

We declare that we have no conflict of interest. We also thank Dr. Shen, Zhiyong for his suggestions in the writing of this manuscript.

Research involving human participants and/or animals

This study does not involve human or animal experiments.

Informed consent

This study does not involve human or animal experiments.

Supplementary material

13213_2018_1372_MOESM1_ESM.docx (27 kb)
ESM 1 (DOCX 27 kb)


  1. Anson ML (1938) The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J Gen Physiol 22:79–89. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Berenjian A et al (2011) Efficient media for high menaquinone-7 production: response surface methodology approach. New Biotechnol 28:665–672. CrossRefGoogle Scholar
  3. Berenjian A, Mahanama R, Talbot A, Regtop H, Kavanagh J, Dehghani F (2014) Designing of an intensification process for biosynthesis and recovery of Menaquinone-7. Appl Biochem Biotechnol 172:1347–1357. CrossRefPubMedGoogle Scholar
  4. Bhatt PC, Pathak S, Kumar V, Panda BP (2018) Attenuation of neurobehavioral and neurochemical abnormalities in animal model of cognitive deficits of Alzheimer's disease by fermented soybean nanonutraceutical. Inflammopharmacology 26:105–118. CrossRefPubMedGoogle Scholar
  5. Cai D, Zhu C, Chen S (2017) Microbial production of nattokinase: current progress, challenge and prospect. World J Microbiol Biotechnol 33.
  6. Chen H, Xiao X, Wang J, Wu L, Zheng Z, Yu Z (2008) Antagonistic effects of volatiles generated by Bacillus subtilis on spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnol Lett 30:919–923. CrossRefPubMedGoogle Scholar
  7. Cho Y-H et al (2010) Production of nattokinase by batch and fed-batch culture of Bacillus subtilis. New Biotechnol 27:341–346. CrossRefGoogle Scholar
  8. Dabbagh F et al (2014) Nattokinase: production and application. Appl Microbiol Biotechnol 98:9199–9206. CrossRefPubMedGoogle Scholar
  9. Dajanta K, Chukeatirote E, Apichartsrangkoon A (2012) Improvement of thua nao production using protein-rich soybean and Bacillus subtilis TN51 starter culture. Ann Microbiol 62:785–795. CrossRefGoogle Scholar
  10. Dedavid e Silva LA, Macedo AJ, Termignoni C (2014) Production of keratinase by Bacillus subtilis S14. Ann Microbiol 64:1725–1733. CrossRefGoogle Scholar
  11. Deepak V, Kalishwaralal K, Ramkumarpandian S, Babu SV, Senthilkumar SR, Sangiliyandi G (2008) Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. Bioresour Technol 99:8170–8174. CrossRefPubMedGoogle Scholar
  12. Garg R, Thorat BN (2014) Nattokinase purification by three phase partitioning and impact of t-butanol on freeze drying. Sep Purif Technol 131:19–26. CrossRefGoogle Scholar
  13. Gong G, Zheng Z, Chen H, Yuan C, Wang P, Yao L, Yu Z (2009) Enhanced Production of Surfactin by Bacillus subtilis E8 Mutant Obtained by Ion Beam Implantation Food Technology and Biotechnology 47:27–31Google Scholar
  14. Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:4501–4523. CrossRefPubMedGoogle Scholar
  15. Hu X-C, Liu W-M, Luo M-M, Ren L-J, Ji X-J, Huang H (2017) Enhancing Menaquinone-7 production by Bacillus natto R127 through the nutritional factors and surfactant. Appl Biochem Biotechnol. CrossRefGoogle Scholar
  16. Kirrolia A, Bishnoi NR, Singh R (2014) Response surface methodology as a decision-making tool for optimization of culture conditions of green microalgae chlorella spp. for biodiesel production. Ann Microbiol 64:1133–1147. CrossRefGoogle Scholar
  17. Kumar R, Mahajan S, Kumar A, Singh D (2011) Identification of variables and value optimization for optimum lipase production by Bacillus pumilus RK31 using statistical methodology. New Biotechnol 28:65–71. CrossRefGoogle Scholar
  18. Kwon E-Y, Kim KM, Kim MK, Lee IY, Kim BS (2011) Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis. Bioprocess Biosyst Eng 34:789–793. CrossRefPubMedGoogle Scholar
  19. Li C, Xia JY, Chu J, Wang YH, Zhuang YP, Zhang SL (2013a) CFD analysis of the turbulent flow in baffled shake flasks. Biochem Eng J 70:140–150. CrossRefGoogle Scholar
  20. Li S, Zhu D, Li K, Yang Y, Lei Z, Zhang Z (2013b) Soybean curd residue: composition, utilization, and related limiting factors. ISRN Ind Eng 2013:1–8. CrossRefGoogle Scholar
  21. Liu D et al (2015) Simultaneous production of butanol and acetoin by metabolically engineered clostridium acetobutylicum. Metab Eng 27:107–114. CrossRefPubMedGoogle Scholar
  22. Mandinia E, Demirci A, Berenjian A (2017) Production and application of menaquinone-7 (vitamin K2): a new perspective. World J Microbiol Biotechnol 33:7. CrossRefGoogle Scholar
  23. Meriem G, Mahmoud K (2017) Optimization of chitinase production by a new Streptomyces griseorubens C9 isolate using response surface methodology. Ann Microbiol 67:175–183. CrossRefGoogle Scholar
  24. Narasimhan MK, Chandrasekaran M, Rajesh M (2015) Fibrinolytic enzyme production by newly isolated Bacillus cereus SRM-001 with enhanced in-vitro blood clot lysis potential. J Gen Appl Microbiol 61:157–164. CrossRefPubMedGoogle Scholar
  25. Ni H, Guo PC, Jiang WL, Fan XM, Luo XY, Li HH (2016) Expression of nattokinase in Escherichia coli and renaturation of its inclusion body. J Biotechnol 231:65–71. CrossRefPubMedGoogle Scholar
  26. Nikiforova OA, Klykov S, Volski A, Dicks LMT, Chikindas ML (2016) Subtilosin a production by Bacillus subtilis KATMIRA1933 and colony morphology are influenced by the growth medium. Ann Microbiol 66:661–671. CrossRefGoogle Scholar
  27. Reddy LV, Kim Y-M, Yun J-S, Ryu H-W, Wee Y-J (2016) L-lactic acid production by combined utilization of agricultural bioresources as renewable and economical substrates through batch and repeated-batch fermentation of Enterococcus faecalis RKY1. Bioresour Technol 209:187–194. CrossRefPubMedGoogle Scholar
  28. Sato T, Yamada Y, Ohtani Y, Mitsui N, Murasawa H, Araki S (2001) Production of menaquinone (vitamin K-2)-7 by Bacillus subtilis. J Biosci Bioeng 91:16–20. CrossRefPubMedGoogle Scholar
  29. Sekar BS, Seol E, Park S (2017) Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd). Biotechnol Biofuels 10.
  30. Sharma V, Meganathan R, Hudspeth MES (1993) Menaquinone (vitamin-K2) biosynthesis - cloning, nucleotide-sequence, and expression of the menc gene from escherichia-coli. J Bacteriol 175:4917–4921CrossRefGoogle Scholar
  31. Shimatani A, Iwata T, Tsutsui M (2006) Method for separating vitamin K2 from bacillus bacterium culture. USGoogle Scholar
  32. Singh V, Haque S, Niwas R, Srivastava A, Pasupuleti M, Tripathi CK (2016) Strategies for fermentation medium optimization: an in-depth review. Front Microbiol 7:2087. CrossRefPubMedGoogle Scholar
  33. Song J et al (2014) Enhanced production of vitamin K-2 from Bacillus subtilis (natto) by mutation and optimization of the fermentation medium. Braz Arch Biol Technol 57:606–612. CrossRefGoogle Scholar
  34. Thadathil N, Kuttappan AKP, Vallabaipatel E, Kandasamy M, Velappan SP (2014) Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products. Ann Microbiol 64:671–681. CrossRefGoogle Scholar
  35. Urano T et al (2001) The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1. J Biol Chem 276:24690–24696. CrossRefPubMedGoogle Scholar
  36. Vos M, Verstreken P, Klein C (2015) Stimulation of electron transport as potential novel therapy in Parkinson's disease with mitochondrial dysfunction. Biochem Soc Trans 43:275–279. CrossRefPubMedGoogle Scholar
  37. Vossen LM et al (2015) Menaquinone-7 supplementation to reduce vascular calcification in patients with coronary artery disease: rationale and study protocol (VitaK-CAC trial). Nutrients 7:8905–8915. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Wang H, Xia J, Zheng Z, Zhuang YP, Yi X, Zhang D, Wang P (2018) Hydrodynamic investigation of a novel shear-generating device for the measurement of anchorage-dependent cell adhesion intensity. Bioprocess Biosyst Eng. CrossRefGoogle Scholar
  39. Wang S-L, Chen H-J, Liang T-W, Lin Y-D (2009) A novel nattokinase produced by Pseudomonas sp TKU015 using shrimp shells as substrate. Process Biochem 44:70–76. CrossRefGoogle Scholar
  40. Wei HF, Zhao GH, Liu H, Wang H, Ni WF, Wang P, Zheng ZM (2018) A simple and efficient method for the extraction and separation of menaquinone homologs from wet biomass of Flavobacterium. Bioprocess Biosyst Eng 41:107–113. CrossRefPubMedGoogle Scholar
  41. Xie MH, Xia JY, Zhou Z, Chu J, Zhuang YP, Zhang SL (2014) Flow pattern, mixing, gas hold-up and mass transfer coefficient of triple-impeller configurations in stirred tank bioreactors. Ind Eng Chem Res 53:5941–5953. CrossRefGoogle Scholar
  42. Xin B, Tao F, Wang Y, Liu HY, Ma CQ, Xu P (2017) Coordination of metabolic pathways: enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis. Metab Eng 41:102–114. CrossRefPubMedGoogle Scholar
  43. Xu Z et al (2015) Economic process to co-produce poly(epsilon-L-lysine) and poly(L-diaminopropionic acid) by a pH and dissolved oxygen control strategy. Bioresour Technol 187:70–76. CrossRefPubMedGoogle Scholar
  44. Zheng Z, Li W, Huang X, Qin W (2017) Effect of trace elements and optimization of their composition for the nitrification of a heterotrophic nitrifying bacterium, Acinetobacter harbinensis HITLi7(T), at low temperature. Ann Microbiol 67:715–725. CrossRefGoogle Scholar
  45. Zhu M et al. (2017) Vitamin K2 analog menaquinone-7 shows osteoblastic bone formation activity in vitro Biomedical Research-India 28:1364–1369Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Authors and Affiliations

  1. 1.Key Laboratory of High Magnetic Field And Ion Beam Physical Biology, Hefei Institutes of Physical ScienceChinese Academy of SciencesHefeiPeople’s Republic of China
  2. 2.University of Science and Technology of ChinaHefeiPeople’s Republic of China

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