Assessment of temperature and acid tolerance of Bacillus subtilis isolated from a Brazilian fruit juice-added soy beverage

Abstract

Bacillus subtilis is a spore-forming bacterium and an important food contaminant. The aim of this study was to analyze the ability of B. subtilis spores to survive under conditions of low pH and high temperature. The package was purchased at a local supermarket, in Uberaba, Minas Gerais. A sample was collected, diluted and plated on Brain-Heart-Infusion agar (BHI). After incubation, suspected colonies of B. subtilis were transferred to BHI agar. Cell morphology, the presence of spores and Gram stain were examined, and the isolate was identified by 16S rRNA gene sequencing . The microscope evaluation indicated the presence of spores. The thermal tolerance of the spores was evaluated by the addition of 3x109spores/mL in test tubes containing peptone water. Heat treatments were carried out at 80 and 90°C at different incubation times (0, 10, 20, 30, 40, 50 and 60 min). After heating, the tubes were cooled and the number of viable spores was determined in BHI Agar. For the analysis of spore survival, D and Z values were calculated. Tolerance to acid conditions was evaluated using BHI broth with different pH values. After incubation, the bacterial concentration was determined by determining viable cell count on BHI Agar medium. The vegetative cells were transferred to the BHI broth and the pH was adjusted to different values (3, 4 or 5). Sampling were taken 8, 12 and 24 h after incubation. The samples were serially diluted in peptone water and spread in BHI Agar to determine the viable cell count . The 16S rRNA gene sequencing indicated high similarity (99.99%) with B. subtilis. D values were 17.01 min at 80°C and 13.42 min at 90°C. The Z-value was 97.13°C. B. subtilis was not able to grow at pH 3 and pH 4, but its survival was confirmed after the growth of colonies on BHI agar. At pH 5, B. subtilis grew after 24 h and the final pH changed to 7. Our results suggest that the spores of B. subtilis isolated from fruit juice-added soy beverage are tolerant to low pH and high temperature.