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. 2021 Aug 17:12:649207.
doi: 10.3389/fmicb.2021.649207. eCollection 2021.

Probiotic Properties of Bacillus proteolyticus Isolated From Tibetan Yaks, China

Zhibo Zeng  1 , Xiaoling He  1 , Feiran Li  1 , Yan Zhang  1 , Zonghao Huang  1 , Yaping Wang  1 , Kun Li  1   2 , Yuhua Bao  3 , Mudassar Iqbal  1   4 , Muhammad Fakhar-E-Alam Kulyar  1 , Jiakui Li  1   5
Affiliations

Probiotic Properties of Bacillus proteolyticus Isolated From Tibetan Yaks, China

Zhibo Zeng et al. Front Microbiol. .

Abstract

Yaks (Bos grunniens) live primarily in high-altitude hypoxic conditions and have a unique intestinal micro-ecosystem, remarkable adaptability, and strong climatic resistance. Accumulating evidence revealed the importance of probiotics in host metabolism, gut microbiota, growth performance, and health. The goal of this study was to screen out probiotics with excellent probiotic potential for clinical application. In this study, four strains of Bacillus, i.e., Bacillus proteolyticus (named Z1 and Z2), Bacillus amyloliquefaciens (named J), and Bacillus subtilis (named K), were isolated and identified. Afterward, their probiotic potential was evaluated. Antioxidant activity tests revealed that Z1 had the highest DPPH and hydroxyl radical scavenging activity, whereas Z2 had higher reducing power and inhibited lipid peroxidation. Additionally, the antibacterial testing revealed that all strains were antagonistic to three indicator pathogens, Escherichia coli C83902, Staphylococcus aureus BNCC186335, and Salmonella enteritidis NTNC13349. These isolates also had a higher hydrophobicity, autoaggregation, and acid and bile tolerance, all of which helped to survive and keep dangerous bacteria out of the host intestine. Importantly, all strains could be considered safe in terms of antibiotic susceptibility and lack of hemolysis. In conclusion, this is the first study to show that B. proteolyticus and B. amyloliquefaciens isolated from yaks have probiotic potential, providing a better foundation for future clinical use.

Keywords: Bacillus amyloliquefaciens; Bacillus proteolyticus; antioxidant capacity; probiotics; yaks.

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Conflict of interest statement

YB was employed by company Tibet Autonomous Region Biological Drug Manufacturing Plant. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) The diagram shows the results of Gram staining for four isolates: Z1 and Z2 (Bacillus proteolyticus), J (Bacillus amyloliquefaciens), and K (Bacillus subtilis). (B) Phylogenetic tree constructed by using a neighbor-joining method on the basis for 16S rRNA gene sequences. J (Bacillus amyloliquefaciens), K (B. subtilis), Z1 and Z2 (B. proteolyticus).
FIGURE 2
FIGURE 2
(A,B) The scavenging ability of BS and BFE on hydroxyl radicals and DPPH. (C,D) The results of lipid peroxidation reducing ability inhibitory activity and anti-lipid peroxidation ability of BS and BFE. BS, bacterial saline suspension; BFE bacteria-free extract. The data were expressed as the mean ± SD. *P < 0.05, **P < 0.01.
FIGURE 3
FIGURE 3
The results of autoaggregation and cell surface hydrophobicity experiment. (A) Hydrophobic ability. (B) Autoaggregation ability.
FIGURE 4
FIGURE 4
The results of the antibacterial experiment. (A) The result shows the antimicrobial capacity of Z1. (B) The result shows the antimicrobial capacity of Z2. (C) The result shows the antimicrobial capacity of J. (D) The result shows the antimicrobial capacity of K.
FIGURE 5
FIGURE 5
The survival rate of strains under different bile salt concentrations. (A) The result determines tolerance to bile salts of Z1. (B) The result determines tolerance to bile salts of Z2. (C) The result determines tolerance to bile salts of J. (D) The result determines tolerance to bile salts of K.
FIGURE 6
FIGURE 6
The survival rate of different strains under different acid conditions. (A) The result determines tolerance to acid conditions of Z1. (B) The result determines tolerance to acid conditions of Z2. (C) The result determines tolerance to acid conditions of J. (D) The result determines tolerance to acid conditions of K.
FIGURE 7
FIGURE 7
The tolerance ability of all strains to 1, 0.7, 0.4, and 0% concentration of H2O2 solution. (A) The result demonstrates the different concentrations of H2O2 tolerance of Z1. (B) The result demonstrates the different concentrations of H2O2 tolerance of Z2. (C) The result demonstrates the different concentrations of H2O2 tolerance of J. (D) The result demonstrates the different concentrations of H2O2 tolerance of K.
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