Screening and functional study of Bacillus from intestines of largemouth bass
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State Key Laboratory of Agricultural Microbiology/College of Fisheries, Huazhong Agricultural University, Wuhan 430070,China

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    Abstract:

    In order to screen Bacillus strains which are beneficial to largemouth bass, the Bacillus strains from the intestine of largemouth bass were isolated, their hemolysis, enzymatic activity, biofilm formation ability, sensitivity to various common antibiotics, tolerance to gastric and intestinal fluids, and in vitro antagonism against Nocardia were detected.These strains were added to the feed of largemouth bass and weigh gain and expression of inflammatory cytokine were detected.A total of 94 Bacillus strains were identified.Four Bacillus subtilis strains, K-3, K-12, K-13, and K-22, were selected based on their non-hemolytic and broad enzymatic activity.These four strains all formed strong biofilms and were sensitive to various common antibiotics.Strains K-3 and K-13 showed in vitro antagonism against Nocardia.The 2-hour survival rates of the four strains in artificial gastric and intestinal fluids were above 80%.After feeding for 42 days, the weight gain of largemouth bass of K-13 group was significantly higher than that of the control group, while protease and lipase activities in the intestines in K-12 and K-13 groups were significantly higher than that of the control group. In addition, compared with the control group, the expression levels of pro-inflammatory factors in the head kidney, spleen, and hind gut of the K-13 experimental group were down-regulated, while the expression levels of anti-inflammatory factor were up-regulated, indicating that K-13 has the potential to reduce intestinal inflammation in largemouth bass. Overall, the B. subtilis strain K-13 selected in this study has high safety, broad enzymatic activity, antagonism against Nocardia, and promotes the growth of largemouth bass, making it a potential probiotic additive for largemouth bass feed.

    Table 5 Survival of Bacillus in artificial gastrointestinal fluids
    Table 4 Susceptibility of Bacillus to antibiotics
    Fig.1 Haemolysis of Bacillus on Columbia CNA blood agar plate
    Fig.2 Colony morphological characteristics of strains (A), gram stain (B) and growth curve (C)
    Fig.3 Biofilm formation(A) and ormation capacity(B) of 4 strains of Bacillus
    Fig.4 Antagonistic effect of four strains of Bacillus on Nocardia
    Fig.5 Growth rate of body mass of largemouth bass after feeding Bacillus(n=30)
    Fig.6 Intestinal digestive enzymes activity in largemouth bass after feeding Bacillus (n=3)
    Fig.7 Levels of mRNA expression of cytokines in largemouth bass after feeding Bacillus
    Table 1 Primer sequences
    Table 3 Physiological and biochemical identification of Bacillus
    Table 2 Determination of enzyme-producing ability of strains
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朱静静,张永安,涂加钢. Screening and functional study of Bacillus from intestines of largemouth bass[J]. Jorunal of Huazhong Agricultural University,2024,43(5):213-223.

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History
  • Received:May 04,2023
  • Online: October 08,2024
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