海洋源芽孢杆菌的分离鉴定及其消化酶代谢产物的测定

Isolation and Identification of Marine Bacillus and Determination of Its Digestive Enzyme Metabolites

【目的】从海洋生物和环境中分离鉴定出产消化酶代谢产物的芽孢杆菌,为微生态制剂的研制提供益生菌种。【方法】从雷州半岛近岸海域采集样品,用高温处理、富集培养和分离培养的方法获得分离菌株;通过菌落形态、细胞结构、染色特征、生化试验和16S rRNA基因测序对分离菌株进行鉴定;采用福林-酚比色法、淀粉-碘比色法和橄榄油乳化法测定分离菌株代谢产物中的蛋白酶、淀粉酶和脂肪酶活性。【结果】从22份样品中初步鉴定出疑似芽孢杆菌43株,筛选3种消化酶综合活性最强的8株,经生化试验和16S rRNA基因测序鉴定,有蜡样芽孢杆菌3株、暹罗芽孢杆菌3株、特基拉芽孢杆菌1株、解木糖赖氨酸芽孢杆菌1株。其中,解木糖赖氨酸芽孢杆菌BC1产脂肪酶活力最高,为52.13 U/mL;蜡样芽孢杆菌BC3产蛋白酶活力最高,为4.78 U/mL;暹罗芽孢杆菌BC6产淀粉酶活力最高,为2.35 U/mL。【结论】成功筛选出产消化酶代谢产物的海洋源芽孢杆菌。

【Objective】The study is aimed at isolating and identifying Bacillus which can produce digestive enzyme metabolites from marine organisms and environment,and providing probiotics for the development of its microecologics.【Method】Samples were collected from the coastal waters of Leizhou Peninsula,and the isolated strains were obtained by high temperature treatment,enrichment culture,isolation and cultivation method.The isolated strains were identified by their colony morphology,cell structure,staining characteristics,biochemical test and 16S rRNA gene sequencing.The determination of protease,amylase and lipase activity in the metabolites of the isolated strains was detected by Folin phenol colorimetry,starch iodine colorimetry and olive oil emulsification method,respectively.【Result】Forty three suspected Bacillus strains are identified from 22 samples.Eight strains of them with the strongest comprehensive activities of three digestive enzymes are screened out,and identified through biochemical test and 16S rRNA gene sequencing as three strains of Bacillus cereus,three strains of Bacillus siamensis,one strain of Bacillus tequilensis,and one strain of Bacillus xylanilyticus.Among them,Bacillus xylolylysine BC1 produces the highest lipase activity(52.13 U/mL),Bacillus cereus BC3 produces the highest protease activity(4.78 U/mL)and Bacillus Siamese BC6 produces the highest amylase activity(2.35 U/mL).【Conclusion】Marine Bacillus producing digestive enzyme metabolites are screened out successfully.