德国小蠊肠道可培养非厌氧细菌的分离、鉴定与产消化酶活性分析

Isolation and identification of culturable aerobic bacteria from the intestines of Blattella germanica and the activity of digestive enzymes produced by these bacteria

目的分离德国小蠊肠道可培养细菌,并测定其产消化酶活性。方法采用传统分离培养的方法分离纯化德国小蠊肠道细菌,通过16S rRNA序列分析进行分子鉴定;利用平板透明圈法筛选产淀粉酶、纤维素酶、蛋白酶和脂肪酶的细菌,通过水解圈与菌落直径的比值,比较不同细菌的产消化酶活性。利用SPSS 20.0软件进行统计学分析,组间比较采用单因素方差分析,当方差齐时用S-N-K多重比较方法;方差不齐时用Dunnett’s T3进行显著性分析。结果在德国小蠊肠道中共分离到12属20种细菌,其中肠球菌属4种,芽孢杆菌属和赖氨酸芽孢杆菌属各3种;柠檬酸杆菌属2种;沙雷菌属、根瘤菌属、假单胞菌属、摩根菌属、细杆菌属、肠杆菌属、金黄杆菌属和Ventosimonas属各1种。在所有细菌中,可产蛋白酶活性的细菌有7种,产淀粉酶活性的细菌有3种,未筛选到产纤维素酶和脂肪酶活性的细菌。粘质沙雷菌和Chryseobacterium massiliae的产蛋白酶活性较强,氧化微杆菌、解淀粉芽孢杆菌、Bacillus paramycoides、特基拉芽孢杆菌和铜绿假单胞菌较弱,7种菌的产蛋白酶活性差异有统计学意义(F=8.859,P=0.000),而产淀粉酶细菌的产酶活性差异无统计学意义(F=2.920,P=0.130)。结论德国小蠊肠道中有产蛋白酶和淀粉酶的细菌,这些细菌可能协助德国小蠊消化和分解食物。

Objective To isolate culturable bacteria from the intestines of Blattella germanica, and to investigate the activity of digestive enzymes produced by these bacteria. Methods Traditional isolation and culture method was used for the isolation and purification of the bacteria from the intestines of B. germanica, and 16 S rRNA sequencing was used for identification. The flat transparent circle method was used to screen out the bacteria producing amylase, cellulase, protease,or lipase, and the ratio of transparent circle diameter(D) to bacterial colony diameter(d) was used to compare the activities of digestive enzymes produced by different bacteria. SPSS 20.0 software was used for statistical analysis. A one-way analysis of variance was used for comparison between groups;the S-N-K multiple comparison method was used for comparison of data with homogeneity of variance, and the Dunnett’s T3 method was used for the significance analysis of data with heterogeneity of variance. Results A total of 20 species of bacteria belonging to 12 genera were isolated from the intestines of B. germanica, among which there were 4 species in Enterococcus, 3 species in Bacillus, 3 species in Lysinibacillus, 2 species in Citrobacter, and 1 specie each in Serratia, Rhizobium, Pseudomonas, Morganella, Microbacterium,Enterobacterium, Chryseobacterium, and Ventosimonas. Among these bacteria, 7 produced protease and 3 produced amylase;no bacteria produced cellulase or lipase. The protease produced by S. marcescens and C. massiliae had relatively high activity, while that produced by M. oxydans, B. amyloliquefaciens, B. paramycoides, B. tequilensis, and P. aeruginosa had relatively low activity;there was a significant difference in the activity of produced protease between the 7 species of bacteria(F=8.859, P=0.000), while there was no significant difference in the activity of amylase produced by different bacteria(F=2.920, P=0.130). Conclusion Some bacteria in the intestines of B. germanica can produce protease and amylase, and such bacteria can assist food digestion in B. germanica.