1株耐高温热嗜淀粉芽孢杆菌的分离鉴定和产蛋白酶条件优化

doi:10.16431/j.cnki.1671-7236.2025.06.036

摘要/Abstract

摘要： 【目的】从动物尸体化制副产物肉骨渣发酵反应堆中分离耐高温产蛋白酶菌株，研究其蛋白酶活性，以期为肉骨渣中蛋白降解开发利用提供理论基础和研究策略。【方法】设置高温培养条件，使用酪蛋白平板水解和蛋白酶福林酚法进行耐高温产蛋白酶菌株筛选，通过16S rRNA测序鉴定菌株；通过高温驯化、紫外诱变提升菌株的耐热性和蛋白酶活性，通过单因素试验和响应面试验探究菌株最佳产酶条件。【结果】从肉骨渣发酵反应堆中筛选出1株耐高温产蛋白酶的热嗜淀粉芽孢杆菌R4.1(Bacillus thermoamylovorans R4.1)，通过紫外诱变，蛋白酶活性提升了40.92%，高温驯化使菌株的最适生长和产酶温度由50 ℃提升至65 ℃；单因素试验结果显示，菌株蛋白酶活性受发酵时间、pH、NaCl浓度和Ca²⁺浓度的影响；响应面优化试验确定菌株的最适产酶条件为：65 ℃发酵96.36 h，初始pH 8.02，NaCl浓度0.59%，Ca²⁺浓度1.11%，接种量1%，在此条件下，蛋白酶活性为55.08 U/mL。相较于原始菌株，酶活性提升了1.96倍。此外，菌株在肉骨渣中发酵120 h后肉骨渣减重率为12.30%，粗蛋白质含量减少11.06%。【结论】热嗜淀粉芽孢杆菌R4.1具有较高的高温蛋白酶活力，能对肉骨渣中的蛋白质进行有效降解，具有良好的应用前景。

关键词: 热嗜淀粉芽孢杆菌; 耐高温筛选; 分离鉴定; 产蛋白酶条件

Abstract: 【Objective】 This study was conducted to isolate high-temperature protease-producing strains from the fermentation reactor of rendering by-product of animal carcass meat and bone residue,and study their protease activity,with the aim of providing a theoretical basis and research strategy for the degradation and utilization of proteins in meat and bone residues.【Method】 High-temperature culture conditions were established,and high-temperature tolerant protease-producing strain was assessed using casein plate hydrolysis and the Folin phenol method.Strains were identified through 16S rRNA sequencing.Thermal acclimation and UV mutation were employed to enhance the strain’s heat resistance and protease activity.Optimal enzyme production conditions were explored using single-factor and response surface methodology.【Result】 A high-temperature protease-producing strain,Bacillus thermoamylovorans R4.1,was isolated from rendering-processed meat and bone residues.UV mutation increased the protease activity by 40.92%.Thermal acclimation raised the optimal growth and enzyme production temperature of the strain from 50 to 65 ℃.Single-factor experiments indicated that the strain’s protease activity was significantly influenced by fermentation time,pH,NaCl concentration and Ca²⁺ concentration.Response surface optimization determined the optimal enzyme production conditions to be fermentation at 65 ℃ for 96.36 h,pH 8.02,NaCl concentration of 0.59%,Ca²⁺ concentration of 1.11%,and an inoculum size of 1%,under which the protease activity reached 55.08 U/mL.Compared to the original strain,the enzyme activity increased by 1.96 times.Additionally,the strain demonstrated a weight reduction rate of 12.30% in meat and bone meal after 120 h,with a crude protein content decrease of 11.06%.【Conclusion】 Bacillus thermoamylovorans R4.1 exhibited high protease activity at elevated temperatures and could effectively degrade proteins in meat and bone meal,indicating promising application potential.

Key words: Bacillus thermoamylovorans; high-temperature tolerant screening; isolation and identification; protease production conditions

中图分类号:

S476

杨钦鸿, 杨佳, 段文洁, 张永仙, 朱尤帅, 杨绍红, 和塍, 尹红斌, 李素华. 1株耐高温热嗜淀粉芽孢杆菌的分离鉴定和产蛋白酶条件优化[J]. 中国畜牧兽医, 2025, 52(6): 2839-2850.

YANG Qinhong, YANG Jia, DUAN Wenjie, ZHANG Yongxian, ZHU Youshuai, YANG Shaohong, HE Cheng, YIN Hongbin, LI Suhua. Isolation and Identification of a High-temperature Tolerant Bacillus thermoamylovorans and Optimization of Its Enzyme Production Conditions[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2839-2850.

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