Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria

doi:10.3969/j.issn.1004-1524.2020.08.15

›› 2020, Vol. 32 ›› Issue (8): 1446-1456.DOI: 10.3969/j.issn.1004-1524.2020.08.15

• Foods Science • Previous Articles Next Articles

Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria

JIANG Yuhang^1,2, LI Hongwei^1,2, CAI Saibo^1,2, LIN Lianbing^1,2, ZHANG Qilin^1,2,*

1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China;
2. Engineering Research Center for Replacement Technology of Feed Antibiotics of Yunnan College, Kunming 650500, China

Received:2020-03-09 Online:2020-08-25 Published:2020-08-28

Abstract

Abstract: To isolate and identify intestinal bacteria from Dendrolimus punctatus larvae and to tap resources of protease-producing bacteria. D. punctatus larvae was used as experimental material in this paper, several intestinal bacteria were obtained by conventional bacterial culture method in vitro. 16S rDNA was used to identify these bacteria, and protease-producing strains were screened by casein protein selection medium. Protease activity was determined according to GB/T 23527—2009 “protease preparation” test method. The results showed that 18 different species of non-anaerobic bacteria were obtained, including Bacillus genera belonging to Firmicutes, and Sulfurobacillus, Staphylococcus, Psychrobacter, Pantoea, Pseudomonas, Acinetobacter and Citrobacter belonging to Proteobacteria. Six strains of the protease-producing bacteria all belonging to Bacillus genus were screened by casein selective medium. The results of enzyme activity showed that all of these protease-producing bacteria had high protease-producing ability, especially strain MW-39 (Bacillus licheniformis), the highest enzyme activity reached to 255.36 U·mL^-1, followed by MW-50 (Bacillus amyloliquefaciens) and MW-4 (Bacillus subtilis), their highest enzyme activity reached to 193.85 U·mL^-1 and 165.81 U·mL^-1, respectively. Enzymatic characteristics of protease produced by three bacterial species with the highest enzymatic activity were further analyzed, and their proteases showed a certain thermal stability and acid-base tolerance. One-way ANOVA analysis showed that protease activity of investigated bacteria were high at pH 5.0-9.0 and 30-50 ℃, and the optimum pH of protease produced by MW-39, MW-4 and MW-50 were 8.0, 7.5 and 8.0, respectively, and the optimum temperatures were 45 ℃, 40 ℃ and 40 ℃. This study revealed various species of intestinal bacteria in larvae of D. punctatus, enriched resources of protease-producing bacteria, and could provide reference for further development and utilization of protease-producing bacteria in insect intestine.

Key words: Dendrolimus punctatus, intestinal bacteria, Bacillus, protease, enzymological characteristics

CLC Number:

S816.7

JIANG Yuhang, LI Hongwei, CAI Saibo, LIN Lianbing, ZHANG Qilin. Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria[J]. , 2020, 32(8): 1446-1456.

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Isolation and identification of intestinal culturable bacteria of Dendrolimus punctatus and screening of protease-producing bacteria

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