Isolation and identification of β-mannanase-producing Bacillus velezensis HF-14109 from common carp (Cyprinus carpio L.) and analysis of the enzymatic characteristics of the enzyme and biological characteristics of the strain

Abstract:

[Objective] Probiotics producing β-mannanase were isolated from the intestine of Cyprinus carpio. [Methods] The strain with high β-mannanase production was screened based on the transparent hydrolysis circle on the screening plate and shake flask fermentation. Through morphological observation, physiological and biochemical tests, 16S rRNA gene sequencing, and comparative genomics-based analysis, the strain was identified. The enzyme activity was evaluated with the dinitrosalicylic acid (DNS) method. The tolerance to temperature, acid, and bile salt, and bacteriostasis (Oxford cup method) of the strain were tested, and then the biosafety was evaluated with the filter paper method and intraperitoneal injection method. [Results] A total of 62 strains producing β-mannanase were isolated from C. carpio, among which HF-14109 was prominent in the enzyme production and was identified as Bacillus velezensis. The optimal reaction temperature and pH for the enzyme were 45 and 6.0. It was stable at 20-80 and pH 4.0-9.0. Cu²⁺, Fe³⁺, Zn²⁺, and Ba²⁺ can activate the enzyme, while Mn²⁺ and Ca²⁺ inhibited the enzyme. It can degrade konjac powder, guar gum, and sophora bean gum. HF-14109 showed tolerance to acid, high temperature, and bile salt and suppressed Escherichia coli EC 05, Edwardsiella tarda ET 03, Staphylococcus aureus SA 16, Aeromonas hydrophila Ah 01, among other bacteria. No death or abnormality occurred to the C. carpio injected with 10⁷-10⁹ CFU/mL HF-14109 suspension. It demonstrated no resistance to gentamicin, kanamycin, clindamycin, chloramphenicol, erythromycin, streptomycin, tetracycline, vancomycin, and ampicillin. [Conclusion] HF-14109 with high production of β-mannanase was isolated from C. carpio and the enzyme demonstrated high activity, good enzymatic properties and probiotic properties, and safety. HF-14109 can be used to remove mannan in aquatic feed.