Characterization of Bacillus spp. strains for use as probiotic additives in pig feed

Abstract

Bacillus spp. are commonly used as probiotic species in the feed industry, however, their benefits need to be confirmed. This study describes a high throughput screening combined with the detailed characterization of endospore-forming bacteria with the aim to identify new Bacillus spp. strains for use as probiotic additives in pig feed. A total of 245 bacterial isolates derived from African fermented food, feces and soil were identified by 16S rRNA gene sequencing and screened for antimicrobial activity and growth in the presence of antibiotics, bile salts and at pH 4.0. Thirty-three Bacillus spp. isolates with the best characteristics were identified by gyrB and rpoB gene sequencing as B. amyloliquefaciens subsp. plantarum, B. amyloliquefaciens subsp. amyloliquefaciens, B. subtilis subsp. subtilis, B. licheniformis, B. mojavensis, B. pumilus and B. megaterium. These isolates were further investigated for their activity against the pathogenic bacteria, antibiotic susceptibility, sporulation rates, biofilm formation and production of glycosyl hydrolytic enzymes. Additionally, ten selected isolates were assessed for heat resistance of spores and the effect on porcine epithelial cells IPEC-J2. Isolates of B. amyloliquefaciens, B. subtilis and B. mojavensis, showed the best overall characteristics and, therefore, potential for usage as probiotic additives in feed. A large number of taxonomically diverse strains made it possible to reveal species and subspecies-specific trends, contributing to our understanding of the probiotic potential of Bacillus species.

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

“…Additionally, previous studies suggested that cellulase could increase the amylase and protease activities and then promote the growth of fish [47], which is extremely important for stomachless fish. In this study, all the B. subtilis isolates had the ability to produce cellulase, which was in agreement with other studies [48,49]. Furthermore, among the isolates, GC-5 and GC-22 had stronger ability to degrade cellulose, which might be good candidates as probiotic additives in aquatic feed to improve food utilization efficiencies.…”

“…In this study, the sporulation rates of GC-5 and GC-21 were almost 100% and that of GC-6 was more than 90% after 72 h incubation, which was coordinated with a recent report [29]. However, the sporulation rate of GC-22 was much weaker, which might be correlated with the medium, aeration or temperature [32] or because that GC-22 has different mechanism in sporulation process.…”

“…The results elucidated that the spores were not sensitive to 80 C and 90 C, and survived even up to 100 C, although the survival rate decreased with the increase of the heating time. Furthermore, the spores showed strong resistances to low pH and high bile concentration, which were also reported by many previous studies [4,29,51]. Thus, the isolates got the accession to feed additives.…”

“…In addition, antibiotic susceptibility assay showed that the isolates were sensitive to gentamicin, kanamycin, tetracycline, erythromycin, vancomycin, and chloramphenicol, but resistant to streptomycin and clindamycin according to the cut-off values of EFSA (2012). The resistance of B. subtilis to streptomycin was also reported previously, which was considered to be owing to the natural resistance gene of the strains [29,39]. However, few report indicated that B. subtilis is resistant to clindamycin, suggesting that clindamycin resistance might be an intrinsic characteristic of fish-sourced B. subtilis species.…”

“…The criteria used to evaluate the B. subtilis strains included species identification, antibiotic sensitivity, ability to inhibit pathogens, cellulase production, resistances to high temperature and gastrointestinal conditions, according to a previous study [29].…”