Biotechnology and Bioprocess Engineering

, Volume 12, Issue 5, pp 548–555 | Cite as

Optimization of medium for the production of a novel aquaculture probiotic,Micrococcus MCCB 104 using central composite design

  • R. Preetha
  • N. S. Jayaprakash
  • Rosamma Philip
  • I. S. Bright Singh


A marine isolate ofMicrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic toVibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen,Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L). ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).


probiotic antagonism Micrococcus response surface methodology central composite design aquaculture 


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  1. 1.
    Karunasagar, I., R. Pai, G. R. Malahti, and I. Karunasagar (1994) Mass mortality ofPenaeus monodon larvae due to antibiotic-resistantVibrio harveyi infection.Aquaculture 128: 203–209.CrossRefGoogle Scholar
  2. 2.
    Abraham, T. J., and R. Manlcy (1995) Luminous and non-luminousVibrio harveyi associated with shell disease in culturedPenaeus indicus.J. Aqua. Trop. 10: 273–276.Google Scholar
  3. 3.
    Singh, I. S. B., P. Lakshmanaperumalsamy, and D. Chandramohan (1998) Bacterial flora of pond rearedPenaeus indicus (Milne Edwards).J. Aqua. Trop. 13: 133–142.Google Scholar
  4. 4.
    Abraham, T. J., R. Manley, R. Papanippan, and K. Dhevendran (1997) Pathogenicity and antibiotic sensitivity of luminousVibrio harveyi isolated from diseased penaeid shrimp.J. Aqua. Trop. 12: 1–8.Google Scholar
  5. 5.
    Tendencia, E. A. and L. D. de la Pena (2001) Antibiotic resistance of bacteria from shrimp ponds.Aquaculture 195: 193–204.CrossRefGoogle Scholar
  6. 6.
    Verschuere, L., G. Rombaut, P. Sorgeloos, and W. Verstraete (2000) Probiotic bacteria as biological control agents in aquaculture.Microbiol. Mol. Biol. Rev. 64: 655–671.CrossRefGoogle Scholar
  7. 7.
    Irianto, A., and B. Austin (2002) Probiotics in aquaculture.J. Fish Dis. 25: 633–642.CrossRefGoogle Scholar
  8. 8.
    Jayaprakash, N. S., S. S. Pai, A. Anas, R. Preetha, R. Philip, and I. S. B. Singh (2005) A marineMicrococcus MCCB 104 antagonistic to vibrios in prawn larval rearing systems.Dis. Aquat Organ. 68: 39–45.CrossRefGoogle Scholar
  9. 9.
    Leal-Sanchez, M. V., R. Jimenez-Diaz, A. Maldonado-Barragan, A. Garrido-Fernandez, and J. L. Ruiz-Barba (2002) Optimization of bacteriocin production by batch fermentation ofLactobacillus plantarum LPCO10.Appl. Environ Microbiol. 68: 4465–4471.CrossRefGoogle Scholar
  10. 10.
    Beg, Q. K., V. Sahai, and R. Gupta (2003) Statistical media optimization and alkaline protease production fromBacillus mojavensis in a bioreactor.Process Biochem. 39: 203–209.CrossRefGoogle Scholar
  11. 11.
    Adinarayana, K., P. Ellaiah, B. Srinivasulu, R. Bhavani Devi, and G. Adinarayana (2003) Response surface methodological approach to optimize the nutritional parameters for neomycin production byStreptomyces marinensis under solid-state fermentation.Process Biochem. 38: 1565–1572.CrossRefGoogle Scholar
  12. 12.
    Wang, Y.-X. and Z.-X. Lu (2004) Statistical optimization of media for extracellular polysaccharide byPholiota squarrosa (Pers.ex Fr.) Quel. AS 5.245 under submerged cultivation.Biochem. Eng. J. 20: 39–47.CrossRefGoogle Scholar
  13. 13.
    Xiong, C., C. Shouwen, S. Ming, and Y. Ziniu (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.CrossRefGoogle Scholar
  14. 14.
    Sambrook, J., and D. W. Russell (2001)Molecular Cloning: A Laboratory Manual 3ed ed., pp. A2–2, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, USA.Google Scholar
  15. 15.
    Oliver, J. D. (1982) Taxonomic scheme for the identification of marine bacteria.Deep Sea Res. 29: 795–798.CrossRefGoogle Scholar
  16. 16.
    Guerra, N. P., and L. Pastrana (2002) Nisin and pediocin production on mussel-processing waste supplemented with glucose and five nitrogen sources.Lett Appl. Microbiol. 34: 114–118.CrossRefGoogle Scholar
  17. 17.
    Pridmore, D., N. Rekhif, A. C. Pittet, B. Suri, and B. Mollet (1996) Variacin, a new lanthionine-containing bacteriocin produced byMicrococcus varians: comparison to the lacticin 481 ofLactococcus lactis.Appl. Environ. Microbiol. 62: 1799–1802.Google Scholar
  18. 18.
    Kim, M.-H., Y.-J. Kong, H. Baek, and H.-H. Hyun (2005) Purification and characterization of micrococcocin GO5, a bacteriocin produced byMicrococcus sp. Go5 isolated from kimchi.J. Food Prot. 68: 157–163.Google Scholar
  19. 19.
    Kim, M.-H., Y.-J. Kong, H. Back, and H.-H. Hyun (2006) Optimization of culture conditions and medium composition for the production of micrococcin GO5 byMicrococcus sp. GO5.J. Biotechnol. 121: 54–61.CrossRefGoogle Scholar
  20. 20.
    Gupta, N., V. Sahai, and R. Gupta (2007) Alkaline lipase from a novel strainBarkholderia multivorans: Statistical medium optimization and production in a bioreactor.Process Biochem. 42: 518–526.CrossRefGoogle Scholar
  21. 21.
    Huang, L., Z. Lu, Y. Yuan, F. Lu, and X. Bie (2006) Optimization of a protective medium for enhancing the viability of freeze-driedLactobacillus delbrueckii subsp.bulgaricus based on response surface methodology.J. Ind. Microbiol. Biotechnol. 33: 55–61.CrossRefGoogle Scholar
  22. 22.
    De Vuyst, L., and E. J. Vandamme (1992) Influence of the carbon source on nisin production inLactococcus lactis subsp.lactis batch fermentation.J. Gen. Microbiol. 138: 571–578.Google Scholar
  23. 23.
    De Vuyst, L., and E. J. Vandamme (1993) Influence of the phosphorus and nitrogen source on nisin production inLactococcus lactis subsp.lactis batch fermentations using a complex medium.Appl. Microbiol. Biotechnol. 40: 17–22.CrossRefGoogle Scholar
  24. 24.
    Liong, M. T., and N. P. Shah (2005) Optimization of cholesterol removal by probiotics in the presence of prebiotics by using a response surface method.Appl. Environ. Microbiol. 71: 1745–1753.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 2007

Authors and Affiliations

  • R. Preetha
    • 1
  • N. S. Jayaprakash
    • 1
  • Rosamma Philip
    • 2
  • I. S. Bright Singh
    • 1
  1. 1.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia
  2. 2.Department of Marine Biology, Microbiology and Biochemistry, School of Ocean Science and TechnologyCochin University of Science and TechnologyKochiIndia

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