高产蛋白酶菌株的筛选、鉴定及其在方斑东风螺养殖中的应用

doi:10.11978/2022263

Abstract

Abstract:

Babylonia areolata is an important marine aquaculture variety in China and Southeast Asia. Due to the pollution of the breeding substrate, the disease of Babylonia areolata happens frequently. In this study, high-yielding protease strains were screened from the bottom sand of the healthy Babylonia areolata breeding pond. The high-yielding protease strain was identified based on morphology, physiological, biochemical and molecular characteristics. Its safety was preliminarily evaluated through strain hemolytic detection and drug sensitivity test, and its fermentation medium was optimized by single-factor and orthogonal tests. Chemicalization, and under indoor aquaculture conditions, the effects of the water bodies (ammonia nitrogen, nitrite nitrogen, COD), sediment (total nitrogen, organic carbon) and the growth (survivability rate and specific growth rate) of snails were investigated. The results showed that one strain YZS02 of high-yielding protease bacillus was isolated from the bottom sand of one healthy Babylonia areolata breeding pond, and was identified as Rossellomorea vietnamensis. The strain has no hemolysis activity. Sensitive to 24 antibiotics, the optimal fermentation medium is composed of 15 g·L^-1 yeast powder, soluble starch 20 g·L^-1, MgCl₂ 8 g·L^-1, sodium chloride 30 g·L^-1. This optimized liquid medium is used to shake bottle fermentation for 24 hours at 30℃ and 200 r·min^-1. Under this conditions, the bacterial density can reach 7.19×10⁹cfu·mL^-1, which is 2.89 times than that before optimization. YZS02 can effectively reduce the level of ammonia nitrogen and COD in the water cultured by Babylonia areolata. It also has a postive effect on removing the total nitrogen in the bottom sand, which can effectively improve the survival rate and specific growth rate of Babylonia areolata. At the end of the experiment, the ammonia nitrogen concentration in the control group was 2.33 mg·L^-1, COD was 278.37 mg·L^-1, and the total nitrogen content of sis was 538.31 μg·g^-1, while the ammonia nitrogen concentration in the water body of the experimental group 3 was 0.78 mg·L^-1 and COD was 128.47 mg·L^-1. The total nitrogen content of sediment was 128.71 μg·g^-1. YZS02 can also effectively improve the survival rate and specific growth rate of Babylonia areolate.

Key words: Babylonia areolata, aquaculture, Rossellomorea vietnamensis, protease, Medium optimization

WU Huiyu, GAO Xiuju, GE Dongzhen, ZHU Jun, ZOU Xiaoxiao, HUANG Huiqin, GU Zhifeng, BAO Shixiang. Screening and identification of high protease producing strain and its application in culture of Babylonia areolata[J].Journal of Tropical Oceanography, 2023, 42(5): 124-133.

Figures/Tables 19

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References 20

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菌株编号	蛋白酶活性/(U∙mL^-1)
YZS01	298.3
YZS02	324.0
PWK02	277.5
P398	199.0
P401	203.0

指标	结果	最佳生长状态
酶试验	W
酪蛋白水解	+
pH 5~9	+	7~8
NaCl浓度0~10%	+	3%~5%
温度10~45℃	+	30~37℃
明胶液化	-
荧光色素	W
甲基红试验	W
V-P试验	+
运动性	+
氧化酶	+
硝酸盐还原	-
吲哚产生	-
脲酶	-
精氨酸脱羧酶	-
H₂S产生	-

抗性药物	纸片含药量	是否敏感
青霉素	10µg	+
氨苄	10µg	+
头孢氨苄	30µg	+
头孢唑林	30µg	+
头孢拉定	30µg	+
头孢呋辛	30µg	+
头孢他啶	30µg	+
头孢曲松	30µg	+
头孢哌酮	75µg	+
多西环素	30µg	+
丁胺卡那	30µg	+
庆大霉素	10µg	+
卡那霉素	30µg	-
新霉素	30µg	+
四环素	30µg	-
米诺环素	30µg	+
红霉素	15µg	+
麦迪霉素	30µg	+
氧氟沙星	5µg	+
环丙沙星	5µg	+
万古霉素	30µg	+
多粘霉素	300IU	+
复方新诺明	1.25µg	+
痢特灵	300µg	+
霉素	30µg	+
克林霉素	2µg	+

试验号	因子			OD₆₀₀
试验号	可溶性淀粉浓度/(g·L^-1)	酵母粉浓度/(g·L^-1)	MgCl₂浓度/(g·L^-1)	OD₆₀₀
1	10	15	4	4.652
2	10	20	8	4.280
3	10	25	6	3.975
4	15	15	6	4.930
5	15	20	4	3.903
6	15	25	8	4.191
7	20	15	8	5.244
8	20	20	6	4.390
9	20	25	4	4.208
K1	12.907	14.826	12.763
K2	13.024	12.573	13.295
K3	13.842	12.374	13.715
R	0.935	2.452	0.952

项目	对照	试验组1	试验组2	试验组3
初始平均体重/g	0.62±0.02^a	0.61±0.01^a	0.62±0.01^a	0.60±0.01^a
终末平均体重/g	2.17±0.04^b	2.24±0.09^b	2.30±0.12^b	2.53±0.11^a
增重率/%	249.72±6.24^b	264.34±14.64^b	270.76±19.34^b	319.40±18.98^a
特定生长率/%	8.32±0.21^b	8.81±0.49^b	9.03±0.64^b	10.65±0.63^a
存活率/%	78.00±5.29^bc	72.67±8.33^c	87.33±4.16^ab	93.33±3.06^a

Screening and identification of high protease producing strain and its application in culture of Babylonia areolata

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