Studies by Fengru Deng et al. [17] and Cui et al. [27] showed that some probiotics Bacillus commercialized possess toxin genes, antibiotic resistance and hemolytic activity, this indicates the need for new Bacillus. Several studies have indicated that tolerating acid, alkali, bile salts, pancreatin, and nonhemolytic is the foundational basis for screening novel probiotic species of Bacillus. Spore-forming probiotic Bacillus receives attention in the use of probiotics because of its unique stress tolerance. However, the issues with safety of probiotics were redetected in the currently marketed strains. The screening of excellent isolated was supplemented to the library of probiotics is a good choice.
The objective of our research was to screen excellent probiotic Bacillus with antibiotic susceptibility and antimicrobial effect against intestinal pathogenic bacteria, including S. aureus CMCC26003, S. typhimurium CVCC541, E. coli CVCC230 and L. monocytogenes ATCC19111. Multiple traditional fermented food and soil samples were collected from various provinces in China. A total of three novel probiotic Bacillus were screened, 37326 was identified as B. halotolerans, 38327 and 38328 was identified as L. macrolides. Currently, L. macrolides can be applied to Cyprinus carpio as probiotics [28], but B. halotolerans could be used as potential probiotics have not been reported.
In this study, we optimized the operating procedure in order to achieve fast and efficient screening probiotics Bacillus using hemolysis test. Out of the 506 heat-resistant strains, 9 strains do not have hemolytic activity, and they showed tolerance to acid, alkali, bile and pancreatin. The results demonstrate that 9 strains have potential as oral probiotics, thereby corroborating the findings of Prieto et al. [29]. Based on the above experimental results, 9 strains were selected to conduct bacteriostatic activity test, and they had growth-inhibiting abilities against three of the four pathogenic bacteria. Previous work has revealed that the antimicrobial effect of the Bacillus is associated with their species. The Bacillus observed in our research correlate with other studies [30, 31]. Moreover, the antibacterial potency of the Bacillus isolated from soil was found in their study [32]. Barbosa et al. [33] showed a wide-spectrum antibacterial effect of B. subtilis isolated in the broiler gastrointestinal tract on the growth of L. monocytogenes, S. aureus, Listeria innocua, Clostridium perfringens and Enterococcus faecalis. The resistance to L. monocytogenes was similar to our study, however, only one strain showed weak inhibitory effect against E. coli in their study. In contrast, all of our 9 strains have significantly inhibitory effect on E. coli. Moreover, Bacillus isolated from seafood showed antimicrobial effect against Edwardsiella spp. [34]. Furthermore, three Bacillus inhibit the growths of Streptococcus agalactiae, Vibrio harveyi, and Aeromonas hydrophila [35]. These results are partially similar to ours, as our Bacillus showed statistically significant antimicrobial activity of indicator bacteria. Then, the 9 strains with good antimicrobial properties were selected for antibiotic susceptibility assessment.
In the antibiotic susceptibility assessment, the isolates of L. macrolides 38327 and 38328, B. halotolerans 37326 showed susceptibilities to almost all antibiotics. Some studies have revealed that inhibition of the Bacillus strains to antibiotics depends on strains and species [36]. As reported by Zhao et al. [23], the resistance to oxacillin among dairy products Bacillus isolates was found. Resistance to imipenem was reported by Hsu et al. [37] and Fiedler et al. [38] for the B. cereus group. These findings differ from our results. Three Bacillus were susceptible to ampicillin, erythromycin, and gentamicin [39]. Patel et al. [40] reported that Bacillus isolates were highly susceptible to ciprofloxacin. Our data are similar to these studies. Researchers note that the resistance of the natural isolates to drugs is not the main risk factor for human or animal welfare [41].
According to their broad-spectrum antibiotic sensitivities, antibacterial activities and other properties, three strains were selected to evaluate safety. And none of the Bacillus strains showed hemolytic activity, thereby confirming their security [42]. The mice in the experimental groups exhibited normal growth compared to the control group, and there were no pathological changes in various organs. According to the oral toxicity study of rats by Spears et al. [25], the safety of B. subtilis was determined. Kim et al. [43] have verified the safety of Bacillus through observation of pulmonary conditions. These research methods are similar to ours, which fully indicated the safety of 38327, 38328 and 37326.
We discovered B. halotolerans 37326 as potential probiotics candidates and the library of probiotics be extended. This finding indicates that the B. halotolerans can be directionally separated in future experiments. The loss of mice weight is significantly inhibited in 38327 and 38328 groups compared to the SM group in the anti-Salmonella infection trial. It was reported that anti-Salmonella infection is caused by multiple mechanisms including coaggregation, signal interference, intestinal barrier and bacteriostatic activity [44]. In our results, all Bacillus displayed inhibitory effects against S. typhimurium CVCC541 in vitro, this suggests that the mechanism of the mice against Salmonella infections in vivo may be due to the bacteriostatic activity. But the weak antibacterial activity of three strains prompted us to intensively investigate the mechanism. B. subtilis supplementation accelerates the exclusion of Salmonella enterica from the liver in a dose-dependent manner [26]. Similarly, 38327, 38328 and 37326 decreases the number of Salmonella in the liver of mice. Xie et al. [45] found B. coagulans improve the levels of T-AOC and SOD, which could inhibit the harmful bacteria. We found that T-AOC, GSH-Px and SOD levels were increased by 38327, 38328 and 37326, the results of antioxidant capacity are in agreement with theirs. These results demonstrate that the novel strains 37326, 38327 and 38328 have a significant potential to be probiotics, different from the strains of commercialized Bacillus probiotic products in use may be of safety concerns [16].
37326 has probiotic effects in vitro, which can be stated according to our results. Nevertheless, 37326 has no obvious protective effect in the anti-Salmonella infection model. But the results are enough to suggest that it is expected to screen L. halotolerans with intestinal protective effect in new samples. In later, large-scale animal experiments should be conducted and different animal species should be used for a comprehensive evaluation of safety. Moreover, the mechanism of 38327 and 38328 anti-Salmonella infection is undefined, further analysis and validation are needed.