朱鹮肠道微生物多样性与产酶活性

Intestinal microbial diversity and enzyme activities of crested ibis

摘要:

【背景】朱鹮是我国国家一级保护动物，属于世界上最濒危的鸟类之一。对朱鹮肠道微生物的多样性和产胞外酶活性进行分析，可为朱鹮种群数量恢复提供思路。【目的】了解朱鹮肠道微生物的多样性，测定其产胞外酶活性。【方法】采用纯培养的方法获得朱鹮肠道微生物，通过革兰氏染色和生理生化鉴定，结合16S rRNA基因扩增和序列分析对细菌进行鉴定。使用水解圈法筛选产淀粉酶、蛋白酶、纤维素酶、脂肪酶的菌株。【结果】从人工喂养的朱鹮新鲜粪便中共分离到296株细菌，共计2个门11个属。变形菌门(Proteobacteria) 236株，占分离总数的79.73%，分别为：埃希氏菌属(Escherichia) 137株，占分离总数的46.28%；哈夫尼亚菌属(Hafnia) 39株，占分离总数的13.18%；变形菌属(Proteus) 28株，占分离总数的9.46%；柠檬酸杆菌属(Citrobacter) 23株，占分离总数的7.77%；气单胞菌属(Aeromonas) 6株，占分离总数的2.03%；肠杆菌属(Enterobacter) 1株，占分离总数的0.34%；志贺菌属(Shigella) 1株，占分离总数的0.34%；克雷伯菌属(Klebsiella) 1株，占分离总数的0.34%。厚壁菌门(Firmicutes) 60株，占分离总数的20.27%，分别为：肠球菌属(Enterococcus) 33株，占分离总数的11.15%；库特氏菌属(Kurthia) 14株，占分离总数的4.73%；芽孢杆菌属(Bacillus) 13株，占分离总数的4.39%。优势菌群为变形菌门(Proteobacteria)中的埃希氏菌属(Escherichia)，占细菌总数的46.28%。经过生理生化鉴定，每个菌株生理生化鉴定出的种属与各自的16S rRNA基因鉴定出的种属相一致。产酶活力分析结果显示有238株产蛋白酶、25株产脂肪酶、24株产淀粉酶、15株产纤维素酶，分别占分离总数的80.41%、8.45%、8.11%和5.07%。【结论】朱鹮肠道微生物分离出的细菌可分为2门11属，优势菌群为变形菌门(Proteobacteria)中的埃希氏菌属(Escherichia)，占细菌总数的46.28%；产酶活性分析显示，80.41%的菌株具有产蛋白酶能力。

关键词:朱鹮;肠道微生物;纯培养;16S rRNA基因;产胞外酶

Abstract:

[Background] The crested ibis (Nipponia nippon) is a national protected animal in China and one of the most endangered birds in the world. In this study, we analyzed the microbial diversity and extracellular enzyme activity in the gut of crested ibis, aiming to provide ideas for restoring the population of crested ibis. [Objective] To reveal the intestinal microbial diversity and extracellular enzyme activities of crested ibis.[Methods] The intestinal microorganisms of crested ibis were isolated by the pure culture method. The bacteria were identified by Gram-staining, physiological and biochemical tests, and 16S rRNA gene sequencing. The strains producing amylase, protease, cellulose, and lipase were screened out by the hydrolysis circle method. [Results] A total of 296 strains of bacteria were isolated from the fresh excreta of artificially breeding Crested Ibis, belonging to 11 genera of 2 phyla. The 236 isolates of Proteobacteria accounted for 79.73% of the total isolates, including 137 (46.28%) isolates of Escherichia, 39 (13.18%) isolates of Hafnia, 28 (9.46%) isolates of Proteus sp., 23 (7.77%) isolates of Citrobacter sp., 6 (2.03%) isolates of Aeromonas, 1 (0.34%) isolate of Enterobacter, 1 (0.34%) isolate of Shigella, and 1 (0.34%) isolate of Klebsiella. The rest isolates (60, 20.27%) belonged to Firmicutes, including 33 (11.15%) isolates of Enterococcus, 14 (4.73%) isolates of Kurthia, 13 (4.39%) isolates of Bacillus. The dominant genus was Escherichia of Proteobacteria, with the isolates accounting for 46.28% of the total. The physiological and biochemical properties of each strain were consistent with the result of identification based on 16S rRNA gene sequence. The enzyme production experiments showed that 238, 25, 24, and 15 strains produced protease, lipase, amylase, and cellulase, accounting for 80.41%, 8.45%, 8.11%, and 5.07%, respectively. [Conclusion] The bacteria isolated from the intestine of crested ibis belonged to 11 genera of 2 phyla, among which Escherichia of Proteobacteria was dominant with the isolates accounting for 46.28% of the total, and 80.41% of the strains had the ability of producing protease.

Key words:crested ibis;gut microorganisms;pure culture;16S rRNA gene;production of extracellular enzymes