1株耐高温且高产蛋白酶的苍白空气芽孢杆菌的分离鉴定及培养条件优化

doi:10.16431/j.cnki.1671-7236.2026.02.044

Abstract

Abstract:

Objective This study aimed to screen microbial resources with high-temperature tolerance and high protease production from meat and bone residue， optimize the enzyme production process， and provide a theoretical basis for industrial development and application. Method Using protein-rich meat and bone residue as the source for strain isolation， thermotolerant and protease-producing strains were screened based on their high-temperature survival ability and casein-degrading activity. Species identification was performed through morphological observation， physiological and biochemical characteristics， and 16S rRNA sequence analysis. Using protease activity as the indicator， culture conditions were optimized by single-factor tests and response surface methodology （RSM）. Ultraviolet （UV） mutagenesis and high-temperature acclimation were applied to enhance the enzyme activity and thermotolerance of the strain. Result A strain identified as Aeribacillus pallidus 60 was isolated from fermented meat and bone residue. The optimal enzyme production conditions were determined as follows： 0.89% NaCl， 1.37% Ca²⁺， 0.5% Mg²⁺， pH 8.87 and incubation time 86.29 h. Under these conditions， protease activity increased by 59.51% compared to the non-optimized strain. After three rounds of UV mutagenesis， protease activity was significantly increased by 24.65% （P<0.01） on the basis of the optimized conditions. Subsequent high-temperature acclimation further enhanced the strain’s heat tolerance to 70 °C， and protease activity was significantly increased by an additional 24.16% （P<0.01）. Conclusion Aeribacillus pallidus 60 exhibited excellent high-temperature tolerance and protease-producing capacity. Culture condition optimization， coupled with UV mutagenesis and thermal acclimation， significantly enhanced its enzyme activity. Results provided a theoretical foundation and microbial resource for industrial applications.

Key words: Aeribacillus pallidus; protease activity; cultural characteristics; UV mutagenesis; high-temperature acclimation

CLC Number:

S476

YANG Qinhong, YANG Jia, JI Jia, ZHANG Yongxian, ZHU Youshuai, YANG Shaohong, HE Cheng, YIN Hongbin, LI Suhua. Solation， Identification， and Culture Condition Optimization of a Thermotolerant and High-protease-producing Strain of Aeribacillus pallidus[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(2): 1009-1022.

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References 33

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因素 Factors	水平 Levels
因素 Factors	－1	0	1
培养时间 Incubation time/h	72	84	96
pH	8.5	9.0	9.5
NaCl浓度 NaCl concentration/%	0.5	1	2
Ca²⁺浓度 Ca²⁺concentration/%	0.5	1	2

项目 Items	结果 Results	项目 Items	结果 Results
pH	7.0~10.0	氧化酶 Oxidase	+
温度 Temperature/℃	50~75	过氧化氢酶 Catalase	+
NaCl浓度 NaCl concentration/%	0.5~4.0	明胶水解 Gelatin hydrolysis	+
革兰染色 Gram stain	+	MR-VP试验 MR-VP test	－

来源 Source	平方和 Sum of squares	自由度 df	均方 Mean square	F 值 F-value	P 值 P-value
模型 Model	958.85	14	68.49	195.27	<0.001
A	5.07	1	5.07	14.46	0.003
B	37.36	1	37.36	106.50	<0.001
C	121.85	1	121.85	347.41	<0.001
D	2.46	1	2.46	7.01	0.023
AB	4.83	1	4.83	13.76	0.003
AC	9.63	1	9.63	27.47	<0.001
AD	0.0883	1	0.0883	0.25	0.626
BC	0.3806	1	0.3806	1.09	0.320
BD	3.25	1	3.25	9.27	0.011
CD	3.43	1	3.43	9.77	0.010
A²	107.52	1	107.52	306.55	<0.001
B²	174.09	1	174.09	496.35	<0.001
C²	218.2	1	218.20	622.12	<0.001
D²	151.29	1	151.29	431.35	<0.001
残差 Residual	3.86	11	0.3507
失拟值 Lack of fit	3.07	7	0.4386	2.23	0.229
纯误差 Pure error	0.7876	4	0.1969
总和Cor total	962.71	25

Solation， Identification， and Culture Condition Optimization of a Thermotolerant and High-protease-producing Strain of Aeribacillus pallidus

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编号 No.	时间 Time/h	pH	NaCl浓度 NaCl concentration/%	Ca²⁺浓度 Ca²⁺ concentration/%	酶活 Enzyme activity/（U/mL）
1	84	9.5	1	2	61.29
2	72	9.0	2	1	59.48
3	96	9.0	2	1	58.11
4	72	9.0	0.5	1	64.95
5	96	8.5	1	1	70.93
6	84	9.0	1	1	76.32
7	84	9.5	2	1	56.23
8	72	9.0	1	2	64.47
9	96	9.5	1	1	64.50
10	84	9.0	1	1	75.30
11	84	8.5	1	0.5	66.22
12	72	9.0	1	0.5	65.01
13	84	8.5	1	2	67.08
14	72	8.5	1	1	65.13
15	84	9.0	1	1	76.10
16	84	8.5	0.5	1	66.40
17	84	8.5	2	1	58.89
18	84	9.0	1	1	75.65
19	72	9.5	1	1	63.10
20	84	9.0	2	2	56.23
21	84	9.0	0.5	2	70.34
22	84	9.0	1	1	76.29
23	96	9.0	0.5	1	69.42
24	84	9.0	2	0.5	59.69
25	96	9.0	1	2	65.42
26	84	9.0	0.5	0.5	65.37
27	96	9.0	1	0.5	66.76
28	84	9.5	1	0.5	64.16
29	84	9.5	0.5	1	64.05

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