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

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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.

Des laits pasteurisés non fermentés supplémentés avec des bactéries probiotiques apparaissent sur le marché. Un défi devient alors de dénombrer le microbiote contaminant indésirable en présence d’une quantité largement supérieure de bactéries probiotiques. Une méthode d'énumérer le microbiote contaminant dans un tel un lait a été développée. Les cultures probiotiques Lactobacillus rhamnosus Lb-Immuni-T™ et Bifidobacterium animalis subsp. lactis BB-12®, ont été ajoutées à un lait pasteurisé non fermenté, pour atteindre un minimum de 1 milliard de CFU par portion de 250 mL, tel que révélé par dénombrement sur gélose MRS en anaérobiose. Aucune croissance de B. animalis subsp. lactis BB-12 n'a été notée sur la gélose « plate count agar » (PCA) ou sur Petrifilm™. Par contre, L. rhamnosus a formé des colonies sur PCA et Petrifilm™. Les tentatives ont donc été faites pour prévenir la croissance du lactobacille probiotique dans PCA. L’ajout de 2 % phosphate de sodium (PS) ou de 5 % glycerophosphate a prévenu la croissance du lactobacille dans des bouillons, mais de petites colonies de L. rhamnosus Lb-Immuni-T ont néanmoins apparu sur PCA supplémenté en PS. La population bactérienne contaminante pourrait être obtenue sur PCA + 2 % SP en comptant seulement les grandes colonies, mais ceci sous-estime la flore contaminante par un facteur de 4. Sur Petrifilm ™, aux dilutions 0 à 2, toutes les colonies étaient issues de contaminants tandis qu'aux dilutions 3 et 4 seules les grosses colonies furent comptées à cette fin. En utilisant cette approche, il y avait une corrélation directe (R2 = 0,99) entre les comptes totaux dans les laits non inoculés et les valeurs de bactéries contaminantes obtenues sur les produits enrichis en probiotiques. La haute corrélation obtenue sur l’échelle 102 à 106 CFU·mL-1 montre que cette méthode Petrifilm™ est appropriée pour évaluer la qualité microbiologique de laits enrichis avec L. rhamnosus Lb-Immuni-T et B. animalis subsp. lactis BB-12.

Document Type: Research Article

Publication date: April 1, 2009

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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