Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus Ka ´ tia Gianni de Carvalho Lima * , Monika Francisca Kruger, Jorge Behrens, Maria Teresa Destro, Mariza Landgraf, Bernadette Dora Gombossy de Melo Franco Universidade de Sa ˜o Paulo, Faculdade de Cie ˆncias Farmace ˆuticas, Departamento de Alimentos e Nutriça ˜o Experimental, Av. Prof. Lineu Prestes 580, 05508-900 Sa ˜o Paulo, SP, Brazil article info Article history: Received 31 March 2008 Received in revised form 27 August 2008 Accepted 27 August 2008 Keywords: Lactic acid bacteria Probiotic Culture media Selectivity Enumeration abstract The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: non- selective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L. acidophilus La05, L. casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37  C or 42  C, anaerobiosis; L. acidophilus: MRS agar with bile salts, 37  C or 42  C, aero- biosis; L. casei: MRS agar with lithium chloride and sodium propionate, 37  C or 42  C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37  C or 42  C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC–MRS, 42  C, anaerobiosis, L. acidophilus: LC medium, 42  C, aerobiosis or anaerobiosis and L. casei: LP–MRS, 42  C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P  0.05). Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. 1. Introduction Probiotic bacteria have many beneficial effects on human health, and these effects are dependent on the number of viable microbial cells that reach the human gut (Leahy, Higgins, Fitzgerald, & Sin- deren, 2005). Thus, reliable determination of viability of bacteria in probiotic foods is important (Lahtinen, Gueimonde, Ouwehand, Reinikainenm, & Salminen, 2006; Saarela, Mogesen, Fonden, Matto, & Mattila-Sandholm, 2000). Traditionally, lactic acid bacteria are counted in MRS agar (De Man, Rogosa, Sharpe Agar), but this medium is not selective for enumeration of probiotic microorgan- isms when non-probiotic starter cultures are present. Effective isolation depends on medium formulation, fermentation condi- tions, time, temperature and atmosphere of incubation as well as on the type of food product under analysis (Aasen, Moretro, Katla, Axelsson, & Storro, 2000; Liew, Ariff, Raha, & Ho, 2005; Payne, Morris, & Beers, 1999; Roy, 2001; Talwalkar & Kailasapathy, 2004; Vinderola & Reinheimer, 1999, 2000). Besides MRS agar, several other culture media have been proposed for the enumeration of probiotic and starter cultures, which include the selective Lactobacillus casei agar (LC agar) described by Shah and Ravula (2000), MRS agar supplemented with bile salts, sodium propionate, lithium chloride, cystein, gentamycin or dicloxacillin (Ingham,1999; Lapierre, Undeland, & Cox, 1992; Lim, Huh, & Baek, 1995; Vinderola & Reinheimer, 2000) and MRS agar in which glucose was replaced by other carbohydrates, such as trehalose and maltose (Hull & Roberts, 1984). Bile salts are used to inhibit growth of non-intestinal microorganisms and sodium propionate and lithium chloride have been shown to be inhibitory for Lactobacillus acidophilus, Lactobacillus delbrueckii and Strepto- coccus thermophilus (Dave & Shah, 1996; Vinderola & Reinheimer, 2000). Supplementation with cystein was recommended by Shah (2000), because this substance reduces the redox potential, creating favorable conditions for the growth of microaerophilic microor- ganisms. Antibiotics, specially gentamycin and dicloxacillin, were used by Ingham (1999) and Lim et al. (1995), to inhibit certain species of lactic acid bacteria, without inhibiting the bifidobacteria. The aim of this study was to compare the growth capability of probiotic (L. acidophilus, L. casei and Bifidobacterium animalis) and * Corresponding author. Tel.: þ55 11 30912191; fax: þ55 11 38154410. E-mail address: gianni@usp.br (K.G.deC. Lima). Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt 0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.08.011 LWT - Food Science and Technology 42 (2009) 491–495