实验室品系家蝇幼虫肠道可培养细菌的分离、鉴定与产消化酶活性分析

doi:10.12140/j.issn.1000-7423.2022.03.012

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (3): 355-361.doi: 10.12140/j.issn.1000-7423.2022.03.012

实验室品系家蝇幼虫肠道可培养细菌的分离、鉴定与产消化酶活性分析

张可心¹(), 刘文娟¹, 张心雨¹, 张倩¹, 张瑞玲¹, 张忠¹^,²^,^*()

1.山东第一医科大学基础医学院,泰安 271016
2.山东第一医科大学生命科学学院,泰安 271016

收稿日期:2021-08-24 修回日期:2021-10-17 出版日期:2022-06-30 发布日期:2022-07-06
通讯作者: 张忠
作者简介:张可心（1997-）,女,硕士研究生,从事病媒生物与虫媒病研究。E-mail： Zhangkx2020021@126.com
基金资助:
国家自然科学基金(81572028)

Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly

ZHANG Ke-xin¹(), LIU Wen-juan¹, ZHANG Xin-yu¹, ZHANG Qian¹, ZHANG Rui-ling¹, ZHANG Zhong¹^,²^,^*()

1. School of Basic Medical Science, Shandong First Medical University, Tai’an 271016, China
2. School of Life Science of Shandong First Medical University, Tai’an 271016, China

Received:2021-08-24 Revised:2021-10-17 Online:2022-06-30 Published:2022-07-06
Contact: ZHANG Zhong
Supported by:
National Natural Science Foundation of China(81572028)

摘要/Abstract

摘要：

目的分离家蝇幼虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对家蝇幼虫消化食物以及生长发育的影响。方法采用传统细菌分离培养法分离纯化家蝇幼虫肠道细菌,采用16S rRNA序列分析进行分子鉴定;通过筛选培养基筛选产蛋白酶、脂肪酶、淀粉酶和纤维素酶的细菌,测量水解圈与菌落直径的比值（D/d值）,比较不同细菌的产消化酶活性。数据用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。结果在家蝇幼虫肠道中,通过厌氧培养分离到6属9种兼性厌氧细菌,其中普罗威登斯菌属3种（普罗维登斯菌DSM 19967、雷氏普罗维登斯菌株、斯氏普罗威登斯菌）,肠球菌属2种（铅黄肠球菌、粪肠球菌）;假柠檬酸杆菌属（粪假柠檬酸杆菌）、摩根菌属（摩根摩根菌）、肠杆菌属（霍氏肠杆菌）、克雷伯氏菌属（肺炎克雷伯菌）各1种。通过需氧培养分离到8属10种细菌,其中肠杆菌属2种（霍氏肠杆菌、阴沟肠杆菌）,普罗威登斯菌属2种（斯氏普罗威登斯菌、居幼虫普罗威登斯菌）;克雷伯氏菌属（肺炎克雷伯菌）、假单胞菌属（铜绿假单胞菌）、不动杆菌属（别雷斯不动杆菌）、乳球菌属（乳酸乳球菌）、梭形杆菌属（纺锤形赖氨酸芽孢杆菌）和芽孢杆菌属（沙福芽孢杆菌）各1种。经厌氧培养分离到的兼性厌氧细菌,在厌氧培养条件下,产淀粉酶活性的细菌有9种,产纤维素酶活性的细菌有8种,产脂肪酶活性的细菌有1种,未筛选到产蛋白酶活性的细菌;在需氧培养条件下,产淀粉酶活性的细菌有9种,产纤维素酶活性的细菌有8种,未筛选到产蛋白酶活性和脂肪酶活性的细菌。厌氧培养条件下,9种兼性厌氧细菌的产淀粉酶活性无差异（F = 1.953,P > 0.05）;8种兼性厌氧菌的产纤维酶活性中,铅黄肠球菌的产酶活性最强,D/d值为1.36 ± 0.06（F = 3.367,P < 0.05）;仅雷氏普罗维登斯菌具有产脂肪酶活性,D/d值为2.28 ± 0.16。需氧状态下,9种兼性厌氧菌的产淀粉酶活性中,粪肠球菌的产淀粉酶活性最强,D/d值为1.42 ± 0.06（F = 3.881,P < 0.05）;8种兼性厌氧菌的产纤维酶活性中,铅黄肠球菌的产纤维酶活性最强,D/d值为1.29 ± 0.01（F = 6.633,P < 0.05）。经需氧培养分离到的需氧可培养细菌中,铜绿假单胞菌和沙福芽孢杆菌有产蛋白酶活性,D/d值分别为3.67 ± 0.25、3.58 ± 0.31,差异无统计学意义（F = 0.087,P > 0.05）;仅筛选到肺炎克雷伯菌有产淀粉酶活性,D/d值为4.83 ± 0.12;未筛选到产纤维素酶和脂肪酶活性的细菌。结论家蝇幼虫肠道细菌中可培养细菌组成相对单一,但多数厌氧细菌和需氧细菌有分泌消化酶的功能。

关键词: 家蝇幼虫, 肠道厌氧细菌, 肠道需氧细菌, 产消化酶活性

Abstract:

Objective To isolate cultivable bacteria from the intestine of housefly larvae, investigate the activity of digestive enzymes produced by these bacteria, and explore the effects of intestinal bacteria on the digestion of food and the growth and development of housefly larvae. Methods The bacteria in the intestine of housefly larvae were isolated and purified by traditional bacterial isolation and culture method, and 16S rRNA gene sequence was analyzed for molecular identification of identify species. Using the bacteria culture medium of different property to screen the bacteria producing amylase, cellulase, protease, or lipase, and the diameter ratio of hydrolytic circle (D) to bacterial colony (d) was measured 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. Results A total of 9 species of facultative anaerobic bacteria, which belongs to 6 genera, were isolated from the intestines of housefly larvae cultured in an anaerobic environment, among which there were 3 species in Providence (Providencia sneebia DSM 19967, P. rettgeri and P. stuartii), 2 species in Enterococcus (E. casseliflavus and E. faecalis), and 1 specie each in Pseudocitrobacter (P. faecalis), Morganella (M. morganii), Enterobacter (E. hormaechei) and Klebsiella (K. pneumoniae). A total of 10 species of bacteria belonging to 8 genera were isolated from the intestines of housefly larvae cultured in an aerobic environment, among which there were 2 species in Enterobacter (E. hormaechei and E. cloacae), 2 species in Providence (P. stuartii and P. vermicola), and 1 specie each in Klebsiella (K. pneumoniae), Pseudomonas (P. aeruginosa), Acinetobacter (A. bereziniae), Lactococcus (L. lactis), Lysinibacillus (L. fusiformis) and Bacillus (B. safensis). Among these facultative anaerobes were isolated from the intestines of housefly larvae cultured in an anaerobic environment, 9 produced amylase, and 8 produced cellulase and 1 produced lipase; no bacteria produced protease under anaerobic conditions. Among these facultative anaerobes, 9 produced amylase and 8 produced cellulase; no bacteria produced protease and lipase detected in aerobic condition. Under anaerobic conditions, there was no difference in amylase activity among 9 facultative anaerobic culture bacteria (F = 1.953, P > 0.05); among the cellulase activities of 8 facultative anaerobes, E. casseliflavus lead had the strongest cellulase activity, with a D/d value of 1.36 ± 0.06 (F = 3.367, P < 0.05); and only P. rettgeri produced lipase, with a D/d value of 2.28 ± 0.16. In the aerobic state, among the amylase activities of 9 facultative anaerobic bacteria, E. faecalis had the strongest amylase-producing activity, and the D/d value of 1.42 ± 0.06 (F = 3.881, P < 0.05). Among the cellulase activities of 8 facultative anaerobes, E. casseliflavus had the strongest cellulase activity, with a D/d value of 1.29 ± 0.01（F = 6.633, P < 0.05）. Among aerobic bacteria, P. aeruginosa and B. safensis produced protease, with D/d values of 3.67 ± 0.25 and 3.58 ± 0.31 (F = 0.087, P > 0.05); and only K. pneumoniae produced amylase, with a D/d value of 4.83 ± 0.12; no bacteria produced cellulase and lipase. Conclusion The composition of cultivable bacteria in the intestine of housefly larvae is relatively unitary, but most anaerobic bacteria and aerobic bacteria have the function of secreting digestive enzymes.

Key words: Housefly larvae, Intestinal anaerobic bacteria, Intestinal aerobic bacteria, Digestive enzyme-producing bacteria

中图分类号:

R384.21

张可心, 刘文娟, 张心雨, 张倩, 张瑞玲, 张忠. 实验室品系家蝇幼虫肠道可培养细菌的分离、鉴定与产消化酶活性分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(3): 355-361.

ZHANG Ke-xin, LIU Wen-juan, ZHANG Xin-yu, ZHANG Qian, ZHANG Rui-ling, ZHANG Zhong. Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(3): 355-361.

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细菌种类 Bacterial species	蛋白酶Protease		淀粉酶Amylase		纤维素酶Cellulase		脂肪酶Lipase
细菌种类 Bacterial species	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture	厌氧培养Anaerobic culture	需氧培养Aerobic culture
普罗威登斯菌属Providencia
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	-	-	+	+	+	+	-	-
雷氏普罗维登斯菌P. rettgeri	-	-	+	+	+	+	+	-
斯氏普罗威登斯菌P. stuartii	-	-	+	+	+	+	-	-
肠球菌属Enterococcus
铅黄肠球菌E. casseliflavus	-	-	+	+	+	+	-	-
粪肠球菌E. faecalis	-	-	+	+	+	+	-	-
假柠檬酸杆菌属Pseudocitrobacter
粪假柠檬酸杆菌 P. faecalis	-	-	+	+	+	+	-	-
摩根菌属Morganella
摩根摩根菌M. morganii	-	-	+	+	+	+	-	-
肠杆菌属Enterobacter
霍氏肠杆菌E. hormaechei	-	-	+	+	-	+	-	-
克雷伯氏菌属Klebsiella
肺炎克雷伯菌K. pneumoniae	-	-	+	+	+	-	-	-

细菌种类Bacterial species	蛋白酶Protease	淀粉酶Amylase	纤维素酶Cellulase	脂肪酶Lipase
肠杆菌属Enterobacter
霍氏肠杆菌E. hormaechei	-	-	-	-
阴沟肠杆菌E. cloacae	-	-	-	-
普罗威登斯菌属Providencia
斯氏普罗威登斯菌P. stuartii	-	-	-	-
居幼虫普罗威登斯菌P. vermicola	-	-	-	-
克雷伯氏菌属Klebsiella
肺炎克雷伯菌K. pneumoniae	-	+	-	-
不动杆菌属Acinetobacter
别雷斯不动杆菌A. bereziniae	-	-	-	-
假单胞菌属Pseudomonas
铜绿假单胞菌P. aeruginosa	+	-	-	-
乳球菌属Lactococcus
乳酸乳球菌L. lactis	-	-	-	-
赖氨酸芽胞杆菌属Lysinibacillus
纺锤形赖氨酸芽孢杆菌L. fusiformis	-	-	-	-
芽孢杆菌属Bacillus Cohn
沙福芽孢杆菌B. safensis	+	-	-	-

细菌种类 Bacterial species	厌氧培养的D/d值 D/d value of anaerobic culture	需氧培养的D/d值 D/d value of aerobic culture
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	1.31 ± 0.02^a	1.26 ± 0.05^b
粪假柠檬酸杆菌 P. faecalis	1.39 ± 0.08^a	1.26 ± 0.05^b
摩根摩根菌M. morganii	1.32 ± 0.05^a	1.32 ± 0.01^ab
霍氏肠杆菌E. hormaechei	1.31 ± 0.04^a	1.32 ± 0.08^ab
铅黄肠球菌E. casseliflavus	1.37 ± 0.03^a	1.35 ± 0.05^ab
雷氏普罗维登斯菌株 P. rettgeri	1.30 ± 0.08^a	1.27 ± 0.01^ab
肺炎克雷伯菌 K. pneumoniae	1.27 ± 0.00^a	1.26 ± 0.03^b
斯氏普罗威登斯菌 P. stuartii	1.43 ± 0.02^a	1.41 ± 0.02^a
粪肠球菌E. faecalis	1.33 ± 0.05^a	1.42 ± 0.06^a

细菌种类 Bacterial species	厌氧培养的D/d值 D/d value of anaerobic culture	需氧培养的D/d值 D/d value of aerobic culture
铅黄肠球菌E. casseliflavus	1.36 ± 0.06^a	1.29 ± 0.01^a
雷氏普罗维登斯菌株 P. rettgeri	1.23 ± 0.05^ab	1.13 ± 0.01^c
霍氏肠杆菌E. hormaechei	-	1.13 ± 0.02^c
肺炎克雷伯菌 K. pneumoniae	1.15 ± 0.01^b	-
粪肠球菌E. faecalis	1.20 ± 0.04^ab	1.24 ± 0.05^ab
普罗维登斯菌DSM 19967 P. sneebia DSM 19967	1.23 ± 0.04^ab	1.17 ± 0.03^bc
粪假柠檬酸杆菌P. faecalis	1.31 ± 0.06^ab	1.18 ± 0.04^bc
摩根摩根菌M. morganii	1.35 ± 0.08^a	1.17 ± 0.03^bc
斯氏普罗威登斯菌 P. stuartii	1.24 ± 0.07^ab	1.13 ± 0.04^c

实验室品系家蝇幼虫肠道可培养细菌的分离、鉴定与产消化酶活性分析

Isolation, identification and the activity of producing digestive enzymes of culturable bacteria from the larval intestine of laboratory line housefly

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