Bacillus subtilis (B. subtilis) produces spores, and Rhamnose lactobacillus produces lactic acid. In this study, to obtain functional bacteria with lactic acid and lactobacilli and simultaneously producing spores and digestive enzymes, we produced the protoplast as the parent strain of B. subtilis and Rhamnose lactobacillus and analyzed the production rate of spores, the ability to resist high temperature and hereditary stability. The results showed that RH-3 was a perfect protoplast from which 48 strains were obtained through regeneration, with a sporogenic rate of 69.1% and a colony number of 2.7×105 CFU/mL after an 80 °C high-temperature treatment. After 7 generations of fermentation, the average pH value was 4.47. The average number of colonies in the culture medium was 18.4. The production of protoplasts lays a foundation for research in microecological preparation.
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