Growth dynamics, antagonistic activity and acid production of Lactiplantibacillus (=Lactobacillus) plantarum KT-L18/1 and Bacillus subtilis BPT-B1 inoculants in alfalfa silage

Abstract

Aim. To study growth dynamics, antagonistic activity and acid formation of two inoculant strains, Lactiplantibacillus (=Lactobacillus) plantarum КТ-L18/1 and Ваcillus subtilis ВРТ-В1, that were introduced into alfalfa silage, up to 30 days of fermentation. Methods. A streptomycin-resistant strain of both above-mentioned organisms, obtained in earlier research, was used to achieve the research aims. Alfalfa was artificially wilted and dried to a dry matter content of 39–40 %. Fermentation was conducted under anaerobic conditions in polyethylene bags. Inoculant was added at a concentration of 108 CFU/kg alfalfa. The resulting 12 × 3 = 36 bags were kept at room temperature in a dark place for 30 days. At each examination date one sample per treatment was used and examined after cultivation of the two inoculants on elective De Man-Rogosa-Sharpe (MRS) agar and meat infusion agar (MIA), containing streptomycin. The antibacterial (ABA) and antifungal activities (AFA) were studied by the agar diffusion method. Results. After 30 days of ensiling the alfalfa samples, L. plantarum КТ-L18/1str and В. subtilis ВРТ-В1str were still present at levels of 9.2 lg CFU/g and 6.9 lg CFU/g respectively. In the natural population of LAB, L. plantarum КТ-L18/1str had a dominant position. After 30 days of the alfalfa fermentation L. plantarum КТ-L18/1str and В. subtilis ВРТ-В1str maintained ABA and AFA to the PPB strains (S. aureus, P. aeruginosa, S. typhimurium) and fungal strains tested. However, the antagonistic activities of the two inoculant strains decreased during the fermentation, for L. plantarum КТ-L18/1str from 7–21 %, and for В. subtilis ВРТ-В1str from 16–21 %. Compared to the uninoculated control treatment, the inoculated silage treatments had a lower pH level (5.2–5.3), an increased number of lactic acid bacteria (LAB) (by 32 %), and clostridia were no longer detected. The inoculation impacted fungi down to a level of 102–103 CFU/g that can ensure the aerobic stability of the feed. Conclusions. The inoculant strains and L. plantarum КТ-L18/1str and Ваcillus subtilis ВРТ-В1str showed a high competitiveness with background microbiota in an ensiling experiment with alfalfa. The antagonistic activity of the two strains to PPB after fermentation was preserved at a high level, albeit with a decrease of 7–21 %. Ваcillus subtilis ВРТ-В1str kept its AFA against all three fungal strains tested during the 30 days of fermentation. For L. plantarum КТ-L18/1str this was true for a strain of the Penicillium chrysogenum only. The inoculation of and L. plantarum КТ-L18/1str and Ваcillus subtilis ВРТ-В1str caused a decrease in pH of 8–9 % at the end of the fermentation period, an accumulation of LAB with 32 %, and the inhibition of the growth of clostridia up to undetectability in the fermented alfalfa. For both inoculants, compared to uninoculated control variant, fungi decreased down to 102–103 CFU/g of the feed, which can ensure the aerobic stability of the feed.