Development of a probiotic for animals and aquaculture based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains

Abstract: This article aims to develop a probiotic for animals and aquaculture based on the Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains. The selection of a nutrient medium was conducted for cultivating the inoculum of these microorganisms. Several bacteria fermentations of the Bacillus genus were performed in biological reactors with a capacity of 15 and 250 l. A technology for obtaining a finished probiotic for animals and aquaculture was developed. The results indicate that L-broth is the most optimal nutrient medium for cultivating the studied strains. The cultivation of B. toyonensis B-13249 and B. pumilus B-13250 strains in fermenters revealed that sporulation begins after 4–8 hours of fermentation. In contrast to the vegetative medium, the fermentative medium helped the bacilli develop a higher optical density (the maximum value in the B. pumilus strain – 2.400±0.149), pH value (maximum value in the B. toyonensis strain – 8.483±0.609) and titer (at least 1010 CFU/g). After 20–24 hours of incubation, both strains of bacilli in the fermenter, almost completely pass into endospores, which serve as a signal for the start of biomass centrifugation. This was indicated by the following: from a 15 l fermenter – 83.3±6.1 g of concentrate, from a 250 l fermenter – 499.8±51.4 g. The number of bacilli in a concentrated state was at least 1·1011 CFU/g for both strains. Obtaining a finished preparation required mixing bacterial concentrates with maltodextrin to a titer of at least 1·1010 CFU/g. The number of bacteria in the preparation checked every month during the year, recorded no value less than 1·1010 CFU/g. Thus, L-broth is most favorable for growing the mother culture of the B. toyonensis B-13249 and B. pumilus B-13250 strains, and fermentative nutrient medium – for the cultivation in fermenters. The expiry date of the bacilli-based biological preparation is at least 12 months, during which the drug’s polycomponence, color and consistency are preserved, in addition to the bacteria titer (at least 1·1010 CFU/g) and their viability.