Rapid Quantification of Live/Dead Lactic Acid Bacteria in Probiotic Products

Positive probiotic effects have been demonstrated in specific population groups, especially in patients with diseases such as necrotizing enterocolitis, antibiotic-associated diarrhea, and acute infectious diarrhea. Lactic acid bacteria (LAB), the most common microbes employed as probiotics, are widely used in probiotic products like yogurts and fermented milk beverages to endow them with beneficial effects on intestinal health. To exert beneficial health effects, probiotic bacteria need to be viable and available at a high cell count (at least 10^6 cfu/ml). Probiotic products sold with claimed health benefits should meet this minimum viable count of bacteria. Thus, rapid and accurate quantification of viable LAB is of great importance for the quality assurance of probiotic products. The most frequently used method in the dairy industry to assess bacterial viability is plate counting, which is labor-intensive, time-consuming, and fails to detect bacteria that do not form colonies but are nevertheless metabolically active.

The Flow NanoAnalyzer was employed for the rapid and accurate detection of LAB and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red fluorescent nucleic acid stain, Propidium Iodide (PI), which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the Flow NanoAnalyzer. The method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands.

Figure 1. The Flow NanoAnalyzer analysis of UHT milk spiked with live LAB with SYTO 9 staining.

Figure 2. The Flow NanoAnalyzer analysis of UHT milk spiked with live/dead LAB with SYTO 9 and PI staining.

The Flow NanoAnalyzer has many potential applications in monitoring the fermentation process, assessing the quality, and studying the effect of prolonged storage of probiotic products.

Methods Appl. Fluoresc., 2017, 5(2), 024002.