Influence of Heat Shock Temperatures and Fast Freezing on Viability of Probiotic Sporeformers and the Issue of Spore Plate Count Versus True Numbers

Background and Objectives: The purpose of the present study was to investigate effects of various heat shock conditions and fast freezing and subsequent thawing on the viability and recovery of Bacillus coagulans and Bacillus subtilis as probiotic sporeformers, and also to compare spore plate and microscopic counts. Materials and Methods: After preparing the final suspensions of B. coagulans and Bacillus subtilis subsp. Natto spores, they were spread-plated before and after fast freezing treatment (-70°C for about 1 min). Heat shock treatments of the spores were carried out at 68oC for 15, 20, and 30 min as well as at 80oC for 10 and 15 min. Concentrations of the examined probiotic sporeformers were determined simultaneously by plate enumerations and microscopically determined counts. Student’s t-test and one-way analysis of variance (ANOVA) of SPSS were used for statistical analysis of the data. Analysis of DoE results was carried out using Minitab. Results: The results presented here show that the highest recovery rates for B. coagulans (14.75 log CFU/mL) and B. subtilis spores (14.80 log CFU/mL) were under a heat shock condition of 68°C for 20 min in nutrient agar (p<0.05). In addition, the survival rates of B. coagulans and B. subtilis spores under the fast freezing and subsequent thawing condition were about 90% and 88%, respectively. Plate counts differed significantly from counts determined microscopically, with differences of almost 0.5 and 0.8 log for B. coagulans and B. subtilis spores, respectively (p<0.05). In addition, DoE results of the study revealed that both factors of spore count method and only freezing factor in fast freezing treatment have a significant effect on concentrations of the spores examined (p<0.05). Conclusions: Heat shock conditions, freezing and subsequent thawing circumstances, and plate counts or enumerations determined microscopically have significant influences on the viability of probiotic sporeformers and should be considered in determining of their accurate concentrations.