Classification, quantification, and thermotolerance assessment of lactic acid bacteria in yogurt using bacterial melting curve analysis

Introduction: The expanding market for probiotic fermented foods like yogurt necessitates advanced methodologies to validate product claims. While multiple analytical approaches exist for characterizing LAB, most of them are either costly or inefficient. Methods: Here, for the first time, we put forward a bacterial melting curve analysis (BMCA) method to classify lactic acid bacteria (LAB) rapidly. This approach employs SYTO 9 and a thermoregulated fluorometer to simultaneously monitor LAB viability and double-stranded DNA (dsDNA) content. Ramping the temperature from physiological (37°C) to denaturing (98°C) levels allowed the concurrent recording of bacterial inactivation kinetics and dsDNA dissociation profiles, yielding distinct species-specific signatures. Results: Validation using three reference strains demonstrated clear differentiation of LAB via multivariate dimensionality reduction of the melting curves. The method was successfully applied to classify and quantify LAB in yogurt without using antibodies. Furthermore, BMCA was utilized to assess the thermotolerance of LAB in different formulations, revealing enhanced thermal stability when the bacteria were encapsulated within calcium alginate gel particles. Conclusion: The BMCA enabled LAB identification and measurement in yogurt samples without requiring antibody-based techniques. Additionally, the BMCA method can also be employed to evaluate thermal stability of LAB in formulations.