Novel microaerobic agar plate method delivers highly selective and accurate enumeration of probiotic lactobacilli in freeze-dried blends containing bifidobacteria

Abstract

The enumeration of viable bacteria is an essential metric in the dietary supplement and food industry to ensure quality of probiotic products. However, selective enumeration of lactobacilli in probiotic freeze-dried blends containing bifidobacteria is difficult to achieve with current Lactobacillus-specific agars (i.e., Rogosa and LAMVAB). Using a panel of Bifidobacterium and Lactobacillus commercial probiotic species, we found that Rogosa agar failed to inhibit all bifidobacteria while LAMVAB agar suppressed several lactobacilli. This prompted us to develop an alternative method of selection, hereby referred to as Lactobacillus Micro-Aerobic (LMA) method, which promotes growth under controlled microaerobic conditions (6–12% O2, 5–8% CO2) to leverage the different oxygen sensitivities of lactobacilli and bifidobacteria. Validation using pure cultures and multi-strain blends of 4 Bifidobacterium and 10 Lactobacillus species showed that LMA effectively suppressed all bifidobacteria and accurately enumerated all lactobacilli when compared to control methods. These results demonstrate the superior efficacy of modulating the redox environment to select for Lactobacillus within a Bifidobacterium-rich background, as opposed to applying acid and antibiotic pressures.