鲤源产β-甘露聚糖酶Bacillus velezensis HF-14109的分离鉴定及其酶学和生物学特性研究

Isolation and identification ofβ-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

【目的】以黄河鲤为材料,从其肠道内分离具有产β-甘露聚糖酶功能的益生菌。【方法】采用平板水解圈法初筛,摇瓶发酵法复筛获得产β-甘露聚糖酶的菌株,通过形态学观察、生理生化试验、16S rRNA基因序列和比较基因组分析对该菌株进行鉴定,并用DNS定糖法测定酶学活性,用耐高温、耐酸、耐胆盐和平板打孔扩散法对其益生特性进行研究,用滤纸片法、腹腔注射法等对其生物安全性进行评价。【结果】本研究通过刚果红染色从鲤肠道中分离筛选出产β-甘露聚糖酶的细菌62株,其中HF-14109菌株产酶能力最强。通过形态学观察、生理生化试验、16S rRNA基因序列和比较基因组分析对该菌株进行鉴定,确定该菌株为贝莱斯芽孢杆菌(Bacillus velezensis)。酶学性质研究发现,该酶最适反应温度为45℃、最适pH为6.0,在温度20–80℃、pH 4.0–9.0范围内都较为稳定;Cu^(2+)、Fe^(3+)、Zn^(2+)、Ba^(2+)对该酶具有激活作用,而Mn^(2+)、Ca^(2+)对该酶具有抑制作用;对魔芋粉、瓜尔胶和槐豆胶都有较好的降解能力。生物学特性研究表明,HF-14109对酸、高温、胆盐具有较好的耐性,对大肠杆菌(Escherichia coli)EC 05、迟缓爱德华氏菌(Edwardsiella tarda)ET 03、金黄色葡萄球菌(Staphylococcus aureus)SA 16、嗜水气单胞菌(Aeromonas hydrophila)Ah 01等均有抑制作用。安全性评价显示,用107、108、109浓度HF-14109菌悬液对黄河鲤腹腔注射后,未出现死亡及异常状况;该菌株对庆大霉素、卡那霉素、克林霉素、氯霉素、红霉素、链霉素、四环素、万古霉素、氨苄西林等均不具有耐药性。【结论】本研究从黄河鲤(Cyprinus carpio)肠道筛选到一株产β-甘露聚糖酶的贝莱斯芽孢杆菌HF-14109,具有较高的酶活性和良好的酶学性质、益生特性和安全性,可作为潜在消除水产饲料中甘露聚糖抗营养因子的功能益生菌。

[Objective]Probiotics producingβ-mannanase were isolated from the intestine of Cyprinus carpio.[Methods]The strain with highβ-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 producingβ-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.Cu^(2+),Fe^(3+),Zn^(2+),and Ba^(2+)can activate the enzyme,while Mn^(2+)and Ca^(2+)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 ofβ-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.