Journal of Food Science and Technology

, Volume 52, Issue 8, pp 5212–5219 | Cite as

Development of menaquinone-7 enriched nutraceutical: inside into medium engineering and process modeling

  • Rishipal Singh
  • Alka Puri
  • Bibhu Prasad Panda
Original Article


Menaquinone 7 (MK-7) is nutritionally important metabolite found by fermentation mainly using B. subtilis species. In this study, soybean medium was modified to improve the MK-7 production using Bacillus subtilis NCIM 2708 under solid state fermentation. The objective of this study was to produce large amount of MK-7 within a short period of time. Nine nutritional components viz. glycerol, mannitol, dextrose, sucrose, yeast extract, malt extract, K2HPO4, MgSO4.7H2O and CaCl2 were investigated to obtain the maximum MK-7 concentration. The highest MK-7 concentration 39.039 μg/g was obtained after 24 h of fermentation in the following optimised medium components: soybean 20 g, glycerol 40 ml/kg, mannitol 60 g/kg, yeast extract 4 g/kg, malt extract 8 g/kg and calcium chloride 4 g/kg. The maximum production of MK-7 56.757 μg/g was predicted by point prediction tool of Design Expert 7.1 software (Statease Inc. USA). This data shows 68.78 % validity of the predicted model.


Soybean Solid state fermentation Menaquinone-7 Medium engineering 



The authors gratefully Professor S. K. Lal, IARI, PUSA, New Delhi for providing soybeans variety. All the authors acknowledge the Department of Science and Technology (DST), Govt. of India for providing fellowship for scholar under INSPIRE Programme.


  1. Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Kavanagh J, Dehghani F (2011a) The effect of amino-acids and glycerol addition on MK-7 production. Proc World Congr Eng Comput Sci 11:19–21Google Scholar
  2. Berenjian A, Mahanama R, Talbot A, Biffin R, Regtop H, Valtchev P, Kavanagh J, Dehghani F (2011b) Efficient media for high menaquinone-7 production: response surface methodology approach. New Biotechnol 28(6):665–672CrossRefGoogle Scholar
  3. Beulens JWJ, Bots ML, Atsma F, Bartelink MLEL, Prokop M, Geleijnse JM, Witteman JCM, Grobbee DE, Van Der Schouw YT (2009) High dietary menaquinone intake is associated with reduced coronary calcification. Atherosclerosis 203(2):489–493CrossRefGoogle Scholar
  4. Fujita Y, Iki M, Tamaki J, Kouda K, Youra A, Kadowaki E, Sato Y, Moon JS, Tomiaka N, Okamoto N, Kurumatani N (2011) Association between vitamin K intake from fermented soybeans, natto, and bone mineral density in elderly Japanese men: the Fujiwara-kyo Osteoporosis Risk in Men (FORMEN) study. Osteoporosis Int 23(2):705–714CrossRefGoogle Scholar
  5. Gast GCM, De Roos NM, Sluijs I, Bots ML, Beulens JWJ, Geleijnse JM, Witteman JC, Grobbee DE, Peeters PHM, Van Der Schouw YT (2009) A high menaquinone intake reduces the incidence of coronary heart disease. Nutr Metab Cardiovas 19(7):504–510CrossRefGoogle Scholar
  6. Hill KF, Mueller JP, Taber HW (1990) The Bacillus subtilis men CD promoter is responsive to extracellular pH. Arch Microbiol 153:355–359CrossRefGoogle Scholar
  7. Hu H, Yao S, Mei L, Zhu Z, Hur B (2000) Partial purification of nattokinase from Bacillus subtilis by expanded bed adsorption. Biotechnol Lett 22(17):1383–1387CrossRefGoogle Scholar
  8. Kamao M, Suhara Y, Tsugawa N, Uwano M, Yamaguchi N, Uenishi K, Ishida H, Saski S, Okano T (2007) Vitamin K content of foods and dietary vitamin K intake in japanese young women. J Nutr Sci Vitaminol 53(6):464–470CrossRefGoogle Scholar
  9. Kennedy M, Krouse D (1999) Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biot 23(6):456–475CrossRefGoogle Scholar
  10. Mahanama R, Berenjian A, Valtchev P, Talbot A, Biffin R, Regtop H, Dehghani F, Kavanagh JM (2011) Enhanced production of menaquinone 7 via solid substrate fermentation from Bacillus subtilis. Int J Food Eng 7(5): DOI:10.2202/1556-3758.2314Google Scholar
  11. Mahanama R, Boyce D, Berenjian A, Dehghani F, Kavanagh J (2012a) The effect of enzymatic pre- treatment on solid state fermentation of vitamin K2. J Am Sci 8(1):648–654Google Scholar
  12. Mahanama R, Berenjian A, Dehghani F, Kavanagh J (2012b) Modelling the effect of bed height and particle size for vitamin K2 production in a static bed fermenter. Eng Lett 20(1):16–17Google Scholar
  13. Rowland B, Taber H (1996) Duplicate isochorismate synthase genes of Bacillus subtilis: regulation and involvement in the biosyntheses of menaquinone and 2,3-dihydroxybenzoate. J Bacteriol 178(3):854–861Google Scholar
  14. Rowland B, Hill K, Miller P, Driscoll J, Taber H (1995) Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes. Gene 167(1–2):105–109CrossRefGoogle Scholar
  15. Sakano T, Notsumoto N, Nagaoka N (1988) Measurement of K vitamins in food by high performance liquid chromatography with fluorometric detection. Vitam (Jpn) 62(8):393–398Google Scholar
  16. Sato T, Yamada Y, Ohtani Y, Mitsui N, Murasawa H, Araki S (2001a) Efficient production of menaquinone (Vitamin K2) by minadione-resistant mutant of Bacillus subtilis. J Ind Microbiol Biot 26(3):115–120CrossRefGoogle Scholar
  17. Sato T, Yamadaa Y, Ohtania Y, Mitsuib N, Murasawab H, Araki S (2001b) Production of Menaquinone (vitamin K2)-7 by Bacillus subtilis. J Biosci Bioeng 91(1):16–20CrossRefGoogle Scholar
  18. Schurgers LJ, Teunissen KJF, Hamulyák K, Knapen MHJ, Vik H, Vermeer C (2007) Vitamin K-containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7. Blood 109(8):3279–3283CrossRefGoogle Scholar
  19. Seto H, Jinnai Y, Hiratsuka T, Fukawa M, Furhata K, Itoh N, Dairi T (2008) Studies on a new biosynthetic pathway for menaquinone. J Am Chem Soc 130(17):5614–5615CrossRefGoogle Scholar
  20. Shearer MJ (1990) Vitamin K and vitamin K-dependent proteins. Brit J Haematol 75(2):156–162CrossRefGoogle Scholar
  21. Sonenshein AL, Hoch JA, Losick R (eds) (2002) Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington DCGoogle Scholar
  22. Theuwissen E, Smit E, Vermeer C (2012) The role of vitamin K in soft-tissue calcification. Adv Nutr 3:166–173CrossRefGoogle Scholar
  23. Truong JT, Booth SL (2011) Emerging issues in vitamin K research. J Evid Based Complement Altern Med 16(2):73–79CrossRefGoogle Scholar
  24. Tsukamoto Y, Kasai M, Kakuda H (2001) Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance. Biosci Biotechnol Biochem 65(9):2007–2015CrossRefGoogle Scholar
  25. Vermeer C, Schurgers LJ (2000) A comprehensive review of vitamin K and vitamin K antagonists. Hematol Oncol Clin North Am 14(2):339–353CrossRefGoogle Scholar
  26. Wu WJ, Ahn BY (2011) Improved menaquinone (vitamin K2) production in cheonggukjang by optimization of the fermentation conditions. Food Sci Biotechnol 20(6):1585–1591CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2014

Authors and Affiliations

  • Rishipal Singh
    • 1
  • Alka Puri
    • 1
  • Bibhu Prasad Panda
    • 1
  1. 1.Microbial and Pharmaceutical Biotechnology Laboratory, Centre for Advanced Research in Pharmaceutical Science, Faculty of PharmacyJamia HamdardNew DelhiIndia

Personalised recommendations