Enumeration of Heat-Treated Probiotics in Functional Beverages

Thermal processing of functional beverages often drives probiotics into a viable but non-culturable (VBNC) state, causing severe underestimation by conventional colony-forming unit (CFU) assays.

To develop and assess an anaerobic, catalase-assisted resuscitation approach that restores culturability of heat-stressed Lacticaseibacillus rhamnosus GG and aligns CFU counts with actual viable populations.

Dairy beverages (95 °C, 30 s; 2 × 106 cells/mL, pH 3.9) containing encapsulated L. rhamnosus GG were incubated at 37 °C for 16 h in Rogosa/MRS broth supplemented with 50-100 U/mL catalase under anaerobic conditions prior to plating. Recovery was benchmarked against flow cytometry (active fluorescent units, AFU), while pH and glucose monitoring confirmed resuscitation rather than growth. Method performance was examined across three production batches (single-laboratory study) and further evaluated in a multi-laboratory setting.

Direct plating yielded no CFU, whereas flow cytometry detected 6.34 ± 0.04 log10 AFU/g. After resuscitation, CFU recovered to 6.05 ± 0.05 log10/g (CFU/AFU ratio = 0.86 ± 0.03; p = 0.12). Omitting catalase or anaerobiosis reduced recovery by > 1.9 log10 CFU/g. CFU restoration occurred without acidification or glucose depletion, confirming true resuscitation. Single-laboratory reproducibility showed CV < 2%; inter-laboratory reproducibility was <15% RSD. Application to heat-treated milk (pH 6.85) yielded 1.74 × 106 CFU/g (CV 15.9%, n = 4), supporting method suitability across other dairy beverages.

The catalase-assisted, anaerobic resuscitation developed protocol provides a reproducible workflow for probiotic enumeration in heat-treated beverages. The method is compatible with AOAC/ISO standards, addresses underestimation of counts, and supports adoption into quality control (QC) laboratories for accurate probiotic labelling.

Heat-stressed L. rhamnosus GG cells recovered culturability when incubated with catalase under anaerobic conditions, restoring CFU counts to AFU-equivalent levels and confirming true resuscitation rather than growth.