Enumeration of the contaminating bacterial microbiota in unfermented pasteurized milks enriched with probiotic bacteria

Abstract

Pasteurized and unfermented milks supplemented with probiotic bacteria are appearing on the market. It then becomes a challenge to ascertain the undesirable contamination microbiota in the presence of a largely superior population of probiotic bacteria. A method to enumerate the contaminating microbial microbiota in such probiotic-enriched milks was developed. The probiotic cultures, Lactobacillus rhamnosus Lb-Immuni-T™ and Bifidobacterium animalis subsp. lactis BB-12®, were added to a pasteurized unfermented milk to reach a minimum of 1 billion CFU per 250 mL portion, as ascertained by plating on de Man – Rogosa – Sharpe (MRS) agar in anaerobic conditions. No growth of B. animalis subsp. lactis BB-12 was noted on plate count agar (PCA) or Petrifilm™ plates, and the presence of this culture did not affect standard plate counts (SPC) of contaminating bacteria. However, L. rhamnosus formed colonies on PCA and Petrifilm™ plates. Attempts were thus made to inhibit the growth of the probiotic lactobacilli in PCA. The addition of 2% sodium phosphate (SP) or 5% glycerophosphate (GP) inhibited the growth of the lactobacilli in broths, but pin-point colonies of L. rhamnosus Lb-Immuni-T nevertheless appeared on PCA supplemented with phosphates. SPC could be obtained on PCA + 2% SP by only counting the large colonies, but this resulted in a significant (4.4 fold) underestimation of SPC values. On Petrifilm™ AC, at dilutions 0 to 2, all colonies were considered as being contaminants, while at dilutions 3 and 4, only large colonies were counted for SPC determinations. There was a direct correlation (R2 = 0.99) between SPC values with Petrifilm™ in uninoculated milks and those obtained on probiotic-enriched milks. The high correlation obtained over the 102 to 106 CFU/mL range of SPC values show that this Petrifilm™ method is appropriate to evaluate the microbiological quality of pasteurized milks enriched with L. rhamnosus Lb-Immuni-T and B. animalis subsp. lactis BB-12.