OJVM  Vol.2 No.3 , September 2012
Production and Biochemestry-Molecular Analysis of Microbial Community Fermenting Whey as a Potential Probiotic for Use Animals
Abstract: The aims of this work were: To achieve a simple and low cost propagation of potential probiotic agents using plain whey as a culture medium, study the diversity of the members of the bacterial community (MC) in plain whey and to evaluate the probiotic capacity of this MC. After a systematic selection of agents according to their growing capacity in whey, the constituted MC was considered as a unit. Biochemical characterization of the lactic acid bacteria were performed using the API system. Molecular characterization of the lactic acid bacteria was realized using AFLPTM DNA-fingerprinting, partial 16S rDNA sequence analysis and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The physiological characterization of yeast was determined with the automated microplate method Allev/Biolomics and using yeast characterization system based on standard taxonomic criteria. The identification molecular was realized by PCR-fingerprinting. The resistance of MC to pH and bile salts were evaluated. The MC was composed of agents from different separated Dominium like Bacteria (Lactobacillum) and Eukaria (yeast). They are multispecies and also multistrain assuring high biodiversity. The MC grew at low pH and different concentrations bile salts.
Cite this paper: E. N. Esteban, M. Indart, S. Cerone, G. de Yaniz, A. G. Inza, H. Landi, S. Mogni, M. Juliarena and L. Igarza, "Production and Biochemestry-Molecular Analysis of Microbial Community Fermenting Whey as a Potential Probiotic for Use Animals," Open Journal of Veterinary Medicine, Vol. 2 No. 3, 2012, pp. 104-112. doi: 10.4236/ojvm.2012.23018.

[1]   M.R. Etzel, “Manufacture and use of dairy protein fractions”, Journal of Nutrition, vol. 134, No. 6, 2004, pp. 996S-1002S. Review.

[2]   A.A Koutinas, I. Athanasiadis, A. Bekatorou, M. Iconomopoulou and G. Blekas, “Kefir Yeast Technology: Scale-Up in SCP Production Using Milk Whey”, Biotechnology and Bioenginering, vol. 89, No.7, 2005, pp. 788-796. doi:10.1002/bit.20394

[3]   V.H. Holsinger, L.P. Posati and E.D. deVilbiss, “Whey Beverages: A Review”, Journal of Dairy Science, vol. 57, No 8, 1974, pp. 849-859. doi:10.3168/jds.S0022-0302(74)84976-3

[4]   M. Chartrain, L. Bhatnagar and J.G. Zeikus, “Microbial Ecophysiology of Whey Biomethanation: Comparison of Carbon Transformation Parameters, Species Composition, and Starter Culture Performance in Continuous Culture”, Applied Environmental Microbiology, vol. 53, No 5, 1987, pp. 1147-1156.

[5]   R. Fuller, “Probiotics in man and animals, A review”. Journal of Applied Microbiology, Vol. 66, No 5, 1989, pp 365-378. doi:10.1111/j.1365-2672.1989.tb05105.x

[6]   X. Chen, J. Xu, J. Shuai, J. Chen, Z. Zhang and W. Fang, “The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157: H7 and Salmonella typhimurium”, International Journal of Food Microbiology, vol. 115, No 3, 2007, pp. 307-312. doi:10.1016/j.ijfoodmicro.2006.11.007

[7]   R. D. Rolfe, “The role of probiotic cultures in the control of gastrointestinal health”. The Journal of Nutrition, vol. 130 (Suppl. 2S), 2000, pp. 396S-402S.

[8]   J.C. de Man, M. Rogosa and M.E. Sharpe, “A medium for the cultivation of lactobacilli. Journal of Appied Bacteriology, vol. 23, No.1, 1960, pp. 130-135. doi:10.1111/j.1365-2672.1960.tb00188.x

[9]   P. Raibaud, M. Caulet, J.V. Galpín and G. Mocquot, “Studies on the bacterial flora of the alimentary tract of pigs, II Streptococci: selective enumeration of the dominant groups”, Journal of Applied Microbiology, Vol. 24, No. 3, 1961, pp. 285-306. doi:10.1111/j.1365-2672.1961.tb00262.x

[10]   P. Vos, R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Hornes, A. Friters, J. Pot, J. Paleman, M. Kuiper and M. Zabeau, “AFLP: a new technique for DNA fingerprinting”, Nucleic Acids Research, vol. 2, No.21, 1995, pp.4407-4414. doi:10.1093/nar/23.21.4407

[11]   S. Gevers, G. Huys and J. Swings, “Applicability of rep-PCR fingerprinting for differentiation of Lactobacillus species”, FEMS Microbiology Letters, vol. 205, No 1, 2001, pp. 31-36. doi:10.1111/j.1574-6968.2001.tb10921.x

[12]   K. Van Hoorde, T. Verstraete, P. Vandamme and G. Huys, ”Diversity of lactic acid bacteria in two Flemish artisan raw milk Gouda-type cheeses”, Food Microbiology, vol. 25, No.7, 2008, pp. 929-935. doi:10.1016/

[13]   V. Robert, P. Evrard and G.L. Hennebert, “BCCM-TM/Allev 2.00 an automated system for the identification of yeast, Mycotaxon, vol. 64, 1997, pp.455-463.

[14]   N.J.W. Kreger-van Rij, “Classification of yeast”, In: Rose, A.H. and. Harrisonn J.S. Eds, The Yeast, Academic Press Inc., London, UK, 1987, vol.1, Biology of Yeast, 2nd ed. pp. 5-61.

[15]   J.P. Van der Walt and D. Yarrow, “Methods for de isolation, maintenance, classification and identification of yeast”, In: The yeast, a taxonomic study, Kreger-van Rij N.J.W. Ed, Elsevier science publishers B. V., Amsterdam, 1984, pp. 45-104.

[16]   C.P. Kurtzman and C.J. Robnett, “Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences”, Antonie Van Leeuwenhoek, Vol. 73, No. 4, 1998, pp.331-371. doi:10.1023/A:1001761008817

[17]   J.A. Barnett, R.W. Payne and D. Yarrow, “Yeasts: Characteristics and Identification”, Third Edition. Cambridge University Press, Cambridge, UK. 2000.

[18]   T. J. White, T. Bruns, S. Lee and J. W. Taylor, “Conserved primer sequences for PCR amplification and sequencing from nuclear ribosomal RNA”, 1990.

[19]   T. A. Hall, “BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT”, Nucleic Acids Symposium Series, vol. 41, No. 41, 1999, pp. 95-98.

[20]   M. Groenewald, H.M. Daniel, V. Robert, G.A. Poot and M.Th Smith,” Polyphasic re-examination of Debaryomyces hansenii strains and reinstatement of D. hansenii, D. fabryi and D. subglobosus”. Persoonia-Molecular Phylogeny and Evolution of Fungi, Vol. 21, No. 11, 2008, pp. 17-27. doi:10.3767/003158508X336576

[21]   G. Vassart, M. Georges, R. Monsieur, H. Brogas, A.S. Lequarre and D. Christophe, “ A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA”, Science, Vol. 235, No. 4789,1987, pp. 683-684. doi:10.1126/science.2880398

[22]   G.Kociubinski, P. Pérez and G. de Antoni, “ Screening of bile resistance and bile of precipitation in lactic acid bacteria and bifidobacteria”, Journal Food Protection, vol. 62, No.8, 1999, pp. 905-912.

[23]   E.R. Famworth, “Kefir, a complex probiotic”, Food Science and Technology Bulletin: Functional Foods, vol. 2, No.1, 2005, pp.1-17. doi:10.1616/1476-2137.13938

[24]   K.B Amor., E.E. Vaughan and W.M. de Vos, “Advanced Molecular Tools for the Identification of Lactic Acid Bacteria”, The Journal Nutrition, Vol. 137, No.3, 2007, pp. 741S-747S.

[25]   C. Vael and K. Desager, “The importance of development of the intestinal microbiota in infancy”, Current Opinion of Pediatrics, vol. 21, No.6, 2009, pp. 794-800. doi:10.1097/MOP.0b013e328332351b

[26]   M. Campieri and P. Gionchetti, “Probiotics in inflammatory bowel disease: new insight to pathogenesis or a possible therapeutic alternative?”, Gastroenterology, vol. 116, No. 5, 1999, pp. 1246– 1249. doi:10.1016/S0016-5085(99)70029-6

[27]   I. Salinas, L. Abelli, F. Bertoni, S. Picchietti, A. Roque, D. Furones, A. Cuesta, J. Meseguer and M.A. Esteban, “Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.)”, Fish & Shellfish Inmunology, vol. 25, No. 1-2, 2008, pp.114-123. doi:10.1016/j.fsi.2008.03.011

[28]   M. Collado, J. Meriluoto and S. Salminen, “Measurement of aggregation properties between probiotics and pathogens: in vitro evaluation of different methods”, Journal of Microbiological Methods, vol. 71, No. 1, 2007, pp. 71-74. doi:10.1016/j.mimet.2007.07.005

[29]   H.M. Timmerman, L.E. Niers, B.U. Ridwan, C.J. Koning, L. Mulder, L.M. Akkermans, F.M. Rombouts and G.T Rijkers, “Design of a multispecies probiotic mixture to prevent infectious complications in critically ill patients”, Clinical Nutrition, Vol. 26, No. 4, 2007, pp. 450-459. doi:10.1016/j.clnu.2007.04.008

[30]   T. Hosoi, A. Ametani, K. Kiuchi and S. Kaminogawa, “Improved growth and viability of lactobacilii in the presence of Bacillus subtilis, catalase, or subtilisin”, Canadian Journal of Microbiology, vol. 46, No. 10, 2000, pp. 892-897.

[31]   A.C. Ouwehand, E. Isolauri, P.V. Kirjavainen, S.T? Ikk? and S.J. Salminen, “The mucus binding of Bifidobacterium lactis Bb 12 is enhanced in the presence of Lactobacillus GG and Lact. Delbrüekii subsp. Bulgaricus”, Letters in Applied Microbiology, vol. 30, No. 1, 2000, pp.10-13. doi:10.1046/j.1472-765x.2000.00590.x

[32]   H.M. Timmerman, C. J. Koning, L. Mulder, F.M. Romboust and A.C. Beynen, “Monostrain, multistrain and multispecies probiotics: A comparison of functionality and efficacy”, International Journal of Food Microbiology, vol. 96, No. 219, 2004, pp.219-233. doi:10.1016/j.ijfoodmicro.2004.05.012

[33]   I. Salinas, A. Cuesta, M.A. Esteban and J. Meseguer, “Dietary administration of Lactobacillus delbrüeckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses”, Fish & Shellfish Inmunology, vol. 19, No. 1, 2005, pp. 67-77. doi:10.1016/j.fsi.2004.11.007

[34]   E. Renner, “Cultured dairy products in human nutrition”, Bulletin of the International Dairy Federation, vol. 225, 1991, pp 2-24.

[35]   E. L. Morrell, D. P. Moore, A. C. Odeón, M. A. Poso, E. Odriozola, G. Cantón, F. Paolicchi, F. Malena, M. R. Leunda, C. Marsella and C. M. Campero, “Retrospective study of bovine neonatal mortality: cases reported from INTA Balcarce, Argentina,” Revista Argentina de Microbiología, vol. 40, No. 3, 2008, pp. 151-157.

[36]   L. S. Frizzo, E. Bertozzi, L. P. Soto, M. V. Zbrun, G. J. Sequeira, R. Dalla Santina, R. Rodriguez Armestro, “The effect of supplementation with three lactic acid bacteria from bovine origin on growth performance and health status of group calves”, Journal of Animal and Veterinary Advances, vol. 7, No. 4, 2008, pp. 400-408.

[37]   M. R. Mokhber-Dezfouli, P. Tajik, M. Bolourchi, H. Mahmoudzadeh, “Effects of probiotics supplementation in daily milk intake of newborn calves on body weight gain, body height, diarrhea occurrence and health condition,” Journal of Biological Science, vol. 10, No. 18, 2007, pp. 3136-3140.

[38]   H.M. Timmerman, L. Mulder, H. Everts, D.C. van Espen, E. van der Wal, G. Klaassen, S.M. Rouwers, R. Hartemink, F.M. Rombousts and A.C. Beynen, “Health and growth of veal calves fed milk replacers with or without probiotics”, Journal of Dairy Science, vol. 88, No.6, 2005, pp.2154-2165. doi:10.3168/jds.S0022-0302(05)72891-5

[39]   Q. S and H.S. Gill, “Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 against Escherichia coli O157:H7 infection in mice”, FEMS Immonulogy and Medical Microbiology, vol. 34, No. 1, 2002, pp. 59-64.

[40]   E. Isolauri, Y. Sütas, P. Kankaanp??, H. Arvilommi and S. Salminen, “Probiotics: effects on immunity”, American Journal of Clinical Nutrition, vol. 73, No. 2, 2001, pp. 444S-450S.