A modified reinforced clostridial medium for the isolation and enumeration of Lactobacillus delbrueckii ssp. bulgaricus in a mixed culture

Abstract

In this study, we modified reinforced clostridial medium (RCM) to selectively enumerate and isolate Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and important starter culture in the dairy industry. The disparity in the reported carbohydrate fermentation pattern of L. delbrueckii ssp. bulgaricus was used to develop a growth medium not only selective for L. delbrueckii ssp. bulgaricus but significantly inhibitory to the growth of other lactic acid bacteria. A recently modified RCM (mRCM) was optimized for this study by the addition of 0.5% fructose, 0.5% dextrose, 1% maltose, and 0.25% sodium pyruvate while replacing lactose as a carbohydrate source. The cell recovery and bacterial counts of L. delbrueckii ssp. bulgaricus in tested products (pure L. delbrueckii ssp. bulgaricus strains, starter culture, probiotic supplements, and yogurt) using our mRCM with sodium pyruvate (mRCM-PYR) were significantly higher than in the recently modified RCM and the common de Man, Rogosa, and Sharpe (MRS) culture medium. The growth of other lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri) and Bifidobacteria was retarded in this modified medium compared with their growth in MRS and mRCM. This result is a significant improvement in the enumeration and differentiation of L. delbrueckii ssp. bulgaricus in mRCM-PYR compared with the results in MRS and mRCM where the high background growth of similar species interferes with the accuracy of bacterial population counts. Our results thus suggest that mRCM-PYR could be recommended as a reliable alternative growth medium for the selective enumeration and isolation of L. delbrueckii ssp. bulgaricus in a mixed culture.