Enumeration of Heat-Treated Probiotics in Functional Beverages

Area of Science:

• Microbiology
• Food Science
• Probiotics

Background:

• Thermal processing of functional beverages can induce a viable but non-culturable (VBNC) state in probiotics.
• Conventional colony-forming unit (CFU) assays often underestimate the actual viable probiotic populations due to the VBNC state.

Purpose of the Study:

• To develop and validate an anaerobic, catalase-assisted resuscitation method.
• To restore the culturability of heat-stressed Lacticaseibacillus rhamnosus GG.
• To ensure accurate enumeration of viable probiotics in heat-treated beverages.

Main Methods:

• Heat-stressed L. rhamnosus GG in dairy beverages were incubated anaerobically with catalase.
• Resuscitation was performed in Rogosa/MRS broth at 37°C for 16 hours.
• Viable populations were quantified using CFU counts and benchmarked against flow cytometry (AFU).

Main Results:

• Direct plating yielded no CFU, while flow cytometry detected 6.34 ± 0.04 log10 AFU/g.
• The resuscitation method recovered CFU counts to 6.05 ± 0.05 log10/g (CFU/AFU ratio = 0.86 ± 0.03).
• Omitting catalase or anaerobiosis significantly reduced recovery (>1.9 log10 CFU/g).

Conclusions:

• The developed protocol provides a reproducible workflow for probiotic enumeration in heat-treated beverages.
• This catalase-assisted, anaerobic resuscitation method accurately quantifies viable probiotics, addressing underestimation issues.
• The method is compatible with AOAC/ISO standards and suitable for quality control laboratories.