Formulation of stable Bacillus subtilis AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers

To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90°C for 1 h, showed that spores produced under a sublethal temperature of 57°C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37°C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15–25°C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 μm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.