Screening， Identification， and Fermentation Optimization of a Protease-producing Bacterial Strain

doi:10.13560/j.cnki.biotech.bull.1985.2024-0945

Abstract

Abstract:

Objective To isolate and screen protease-producing bacteria from sauerkraut juice, clarify the species classification status of the strains, optimize the fermentation enzyme production process, enrich the resources of protease strains, and improve the efficiency of fermentation enzyme production. Method Using the sauerkraut juice of Chinese cabbage in a sauerkraut workshop as the isolation source, the ten-fold layer dilution method was used to screen the casein plate, and the folinol method was further re-screened to obtain the protease-producing strains. Morphological observation and 16S rRNA gene phylogenetic analysis were combined to complete the species identification. Single factor test was to optimize the composition and conditions of the fermentation medium, and the response surface method was further to optimize the enzyme-producing process for improving the efficiency of enzyme production. Result A total of 15 protease-producing strains were obtained by initial screening, and strain P-133 with acidic, neutral and alkaline protease abilities was obtained by re-screening with forinol method. The acidic, neutral and alkaline protease activities were 1.00, 52.50 and 48.50 U/mL, respectively, and were identified as Pseudomonas sp. The results via single factor and response surface tests showed that the optimal conditions for enzyme production of strain P-133 were soluble starch 5.0 g/L, rapeseed meal 5.9 g/L, NaCl 3.3 g/L, K₂HPO₄ 5.0 g/L, pH 7.8, temperature 28℃, inoculation volume 5%. and liquid-loading capacity 75 mL/250 mL. Under these conditions, the acidic, neutral and alkaline protease activities were 4.14, 185.19 and 177.30 U/mL, respectively, which were 4.14, 3.53 and 3.66 times higher than those before optimization. Conclusion Pseudomonas sp. P-133 with acidic, neutral and alkaline proteases is isolated and screened from sauerkraut juice. The activities of acidic, neutral and alkaline proteases were 4.14, 3.53 and 3.66 times higher than those before optimization, respectively.

Key words: protease, screening, identification, Pseudomonas sp., fermentation optimization, single factor method, response surface method

LIU Shuang, JIANG Zhou, ZHAO Shuai, ZHAO Lei-zhen, HUANG Feng, ZHOU Jia, QU Jian-hang. Screening， Identification， and Fermentation Optimization of a Protease-producing Bacterial Strain[J]. Biotechnology Bulletin, 2025, 41(4): 335-344.

Figures/Tables 12

References 30

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水平 Level
水平 Level	A pH	B菜粕浓度 Rapeseed meal concentration/（g·L^-1）	C NaCl浓度 NaCl concentration/（g·L^-1）
-1	5	1	0.5
0	7	5	1
1	9	10	5

水平 Level
水平 Level	A pH	B菜粕浓度 Rapeseed meal concentration/（g·L^-1）	C NaCl浓度 NaCl concentration/（g·L^-1）
-1	5	1	0.5
0	7	5	1
1	9	10	5

编号 Number	水解圈直径Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）	编号 Number	水解圈直径 Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）
P-6	20	11	1.8	P-18	12	6	2.0
P-7	11	6	1.8	P-25	18	7	2.6
P-8	13	6	2.7	P-34	24	5	4.8
P-9	12	5	2.4	P-35	20	10	2.0
P-12	14	6	2.3	P-36	14	7	2.0
P-13	20	5	4.0	P-37	10	5	2.0
P-14	12	6	2.0	P-133	20	4	5.0
P-15	10	6	1.7

编号 Number	水解圈直径Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）	编号 Number	水解圈直径 Diameter of hydrolysis ring （D）/mm	菌落直径 Diameter of colony （d）/mm	圈径比Ring diameter ratio （D/d）
P-6	20	11	1.8	P-18	12	6	2.0
P-7	11	6	1.8	P-25	18	7	2.6
P-8	13	6	2.7	P-34	24	5	4.8
P-9	12	5	2.4	P-35	20	10	2.0
P-12	14	6	2.3	P-36	14	7	2.0
P-13	20	5	4.0	P-37	10	5	2.0
P-14	12	6	2.0	P-133	20	4	5.0
P-15	10	6	1.7

方差来源 Source of variance	平方和 Sum of squares	自由度 Freedom	均方 Mean	F值 F value	P值 P value	显著性 Significance
pH	64 659.717	4	16 164.929	1 349.359	1.30×10^-13	**
菜粕浓度	21 586.241	6	3 597.707	203.065	8.62×10^-13	**
NaCl浓度	20 247.864	6	3 374.644	126.434	2.24×10^-11	**
装液量	2 923.535	5	584.707	208.34	3.15×10^-11	**
温度	53 335.824	4	13 333.96	349.142	1.08×10^-10	**
接种量	1 205.128	4	301.282	64.771	4.12×10^-7	**
可溶性淀粉浓度	3 331.388	6	555.231	4.269	0.012	*