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

热带海洋学报 ›› 2023, Vol. 42 ›› Issue (5): 124-133.doi: 10.11978/2022263CSTR: 32234.14.2022263

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

武慧宇¹^,²(), 高秀菊¹^,², 葛东振², 朱军²^,³^,⁴, 邹潇潇²^,³^,⁴, 黄惠琴²^,³^,⁴, 顾志峰¹, 鲍时翔²^,³^,⁴()

1. 海南大学海洋学院, 海南海口 570228
2. 中国热带农业科学院热带生物技术研究所/海南热带农业资源研究院海南省热带农业资源保护与利用重点实验室, 海南海口 571101
3. 中国热带农业科学院湛江实验站, 广东湛江 524013
4. 海南省海洋生物功能性成分研究与利用重点实验室, 海南海口 571101

收稿日期:2022-12-26 修回日期:2023-02-27 出版日期:2023-09-10 发布日期:2023-03-08
作者简介:
武慧宇(1995—), 男, 山西省太原市人, 硕士研究生, 从事水生动植物疫病防控研究。email: 1820920812@qq.com
基金资助:
农业农村部财政专项(NFZX2021); 农业农村部财政专项(NHYYSWZZZYKZX2020); 中央级公益性科研院所基金项目(1630052019014)

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

WU Huiyu¹^,²(), GAO Xiuju¹^,², GE Dongzhen², ZHU Jun²^,³^,⁴, ZOU Xiaoxiao²^,³^,⁴, HUANG Huiqin²^,³^,⁴, GU Zhifeng¹, BAO Shixiang²^,³^,⁴()

1. College of Oceanography, Hainan University, Haikou 570228, China
2. Institute of Tropical Bioscience and Biotechnology, CATAS & Key Laboratory of Biology and Genetic Resources of Tropical Crops of Hainan Province, Hainan Institute for Tropical Agricultural Resources, Haikou 571101, China
3. Zhanjiang Experimental Station, CATAS, Zhanjiang 524013, China
4. Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bio-resources, CATAS, Haikou 571101, China

Received:2022-12-26 Revised:2023-02-27 Online:2023-09-10 Published:2023-03-08
Supported by:
Ministry of Agriculture and Rural Affairs, P. R. of China(NFZX2021); Ministry of Agriculture and Rural Affairs, P. R. of China(NHYYSWZZZYKZX2020); Central Public-Interest Scientific Institution Basal Research Fund(1630052019014)

摘要/Abstract

摘要：

方斑东风螺是我国及东南亚地区重要的海水养殖品种, 由于养殖底质污染, 导致方斑东风螺病害频发。文章从健康方斑东风螺养殖池底沙中筛选蛋白酶高产菌株, 基于形态、生理生化和分子特征对高产蛋白酶菌株进行鉴定, 通过菌株溶血性检测和药敏试验初步评价其安全性, 在单因素试验的基础上通过正交试验对其发酵培养基碳源、氮源及金属盐进行优化, 并在室内养殖条件下考察了其对方斑东风螺养殖水体(氨氮、亚硝酸盐氮、化学需氧量)、底沙(总氮、有机碳)以及方斑东风螺生长(存活率、特定生长率)的影响。结果表明: 从健康方斑东风螺养殖池底沙分离得到一株高产蛋白酶芽孢菌, 其蛋白酶活性为324.0U·mL^-1, 命名为YZS02, 经鉴定菌株YZS02为越南蔷薇菌(Rossellomorea vietnamensis), 该菌株没有溶血活性, 对24种抗生素敏感, 其最优发酵培养基为酵母粉15g·L^-1、可溶性淀粉20g·L^-1、MgCl₂ 8g·L^-1、NaCl 30g·L^-1, 用此优化培养基在30℃, 200r·min^-1条件下摇瓶发酵24h, 菌体密度可达到7.19×10⁹个·毫升^-1, 该密度是优化前的2.89倍, 同时优化后蛋白酶活性可达753.3U·mL^-1, 为原来的2.3倍。YZS02能够有效降低方斑东风螺养殖水体氨氮和化学需氧量水平, 对底沙中的总氮也有很好的去除效果, 试验结束时对照组水体氨氮浓度为2.33mg·L^-1、化学需氧量为278.37mg·L^-1、底沙总氮含量为538.31μg·g^-1, 而试验组水体氨氮浓度为0.78 mg·L^-1、化学需氧量为128.47mg·L^-1、底沙总氮含量为128.71μg·g^-1。YZS02还能有效提高方斑东风螺的存活率及特定生长率。

关键词: 方斑东风螺, 养殖, 越南蔷薇菌, 蛋白酶, 培养基优化

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

武慧宇, 高秀菊, 葛东振, 朱军, 邹潇潇, 黄惠琴, 顾志峰, 鲍时翔. 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[J]. 热带海洋学报, 2023, 42(5): 124-133.

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.

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参考文献 20

[1]	东秀珠, 蔡妙英, 2001. 常见细菌系统鉴定手册[M]. 北京: 科学出版社.
	DONG XIUZHU, CAI MIAOYING, 2001. Common Bacterial System Identification Manual[M]. Beijing: Science Press (in Chinese with English abstract).
[2]	何志伟, 董起钰, 刘锐, 等, 2021. 白斑蛾蚋幼虫肠道可培养细菌的分离鉴定与产消化酶活性分析[J]. 中国媒介生物学及控制杂志, 32(2): 224-229. doi: 10.11853/j.issn.1003.8280.2021.02.021
	HE ZHIWEI, DONG QIYU, LIU RUI, et al, 2021. Isolation and identification of culturable intestinal bacteria from Clogmia albipunctata larvae and analysis of their digestive enzyme-producing activities[J]. Chinese Journal of Vector Biology and Control, 32(2): 224-229 (in Chinese with English abstract).
[3]	梁飞龙, 毛勇, 余祥勇, 等, 2005. 方斑东风螺人工育苗试验[J]. 海洋湖沼通报, 34(1): 79-85.
	LIANG FEILONG, Mao YONG, et al, 2005. Experiment on artificial breeding of Babylonia areolata (lamrck)[J]. Haiyang Huzhao Tongbao, 34(1): 79-85 (in Chinese with English abstract).
[4]	宋俊男, 2020. 水产养殖用益生菌的分离鉴定及其应用潜力评估[D]. 广州: 华南理工大学.
	SONG JUNNAN, 2020. Genotyping of MLST and type 3 secretion system of a strain of Salmonella enteritidis[D]. Guangzhou: South China University of Technology (in Chinese with English abstract).
[5]	袁艳超, 2017. 蛋白酶高产菌株的筛选及其发酵芝麻粕制备芝麻多肽的研究[D]. 南昌: 江西师范大学.
	YUAN YANCHAO, 2017. Screening of protease high yield strain and its application in preparation of Sesame Polypeptide from Sesame Meal[D]. Nanchang: Jiangxi Normal University (in Chinese with English abstract).
[6]	AMOAH K, DONG XIAOHUI, TAN BEIPING, et al, 2021. In vitro assessment of the safety and potential probiotic characteristics of three bacillus strains isolated from the intestine of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)[J]. Frontiers in Veterinary Science, 8: 675962. doi: 10.3389/fvets.2021.675962
[7]	DOBSON G T, DUY N D Q, PAUL N A, et al, 2020. Assessing potential for integrating sea grape (Caulerpa lentillifera) culture with sandfish (Holothuria scabra) and Babylon snail (Babylonia areolata) coculture[J]. Aquaculture, 522: 735135. doi: 10.1016/j.aquaculture.2020.735135
[8]	GUPTA R S, PATEL S, SAINI N, et al, 2020. Robust demarcation of 17 distinct bacillus species clades, proposed as novel bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus bacillus limiting it only to the members of the Subtilis and Cereus clades of species[J]. International Journal of Systematic and Evolutionary Microbiology, 70(11): 5753-5798. doi: 10.1099/ijsem.0.004475
[9]	KIM M J, KU S, KIM S Y, et al, 2018. Safety evaluations of bifidobacterium bifidum BGN4 and bifidobacterium longum BORI[J]. International Journal of Molecular Sciences, 19(5): 1422. doi: 10.3390/ijms19051422
[10]	KUEBUTORNYE F K A, ABARIKE E D, LU YIAHAN, 2019. A review on the application of bacillus as probiotics in aquaculture[J]. Fish Shellfish Immunol, 87: 820-828. doi: 10.1016/j.fsi.2019.02.010
[11]	LEE R D, JOSPIN G, LANG J M, et al, 2015. Draft genome sequence of bacillus vietnamensis strain UCD-SED5 (Phylum Firmicutes)[J]. Genome Announcements, 3(6): e01376-15.
[12]	LIU YANBO, FU JUNYING, WANG LINLIN, et al, 2022. Isolation, identification, and whole-genome sequencing of high-yield protease bacteria from Daqu of Zhang Gong Laojiu[J]. PLoS One, 17(4): e0264677. doi: 10.1371/journal.pone.0264677
[13]	NOGUCHI H, UCHINO M, SHIDA O, et al, 2004. Bacillus vietnamensis sp. nov., a moderately halotolerant, aerobic, endospore-forming bacterium isolated from Vietnamese fish sauce[J]. International Journal of Systematic and Evolutionary Microbiology, 54(Pt6): 2117-2120. doi: 10.1099/ijs.0.02895-0
[14]	RUANGSRI J, THAWONSUWAN J, WANLEM S, et al, 2018. Effect of body size and sub-optimal water quality on some hemato-immunological parameters of spotted babylon snail babylonia areolata[J]. Fisheries Science, 84: 513-522. doi: 10.1007/s12562-018-1191-8
[15]	TAMURA K, STECHER G, KUMAR S, 2021. MEGA11: molecular evolutionary genetics analysis version 11[J]. Molecular Biology and Evolution, 38(7): 3022-3027. doi: 10.1093/molbev/msab120 pmid: 33892491
[16]	VERSCHUERE L, ROMBAUT G, SORGELOOS P, et al, 2000. Probiotic bacteria as biological control agents in aquaculture[J]. Microbiology and Molecular Biology Reviews, 64(4): 655-671. doi: 10.1128/MMBR.64.4.655-671.2000 pmid: 11104813
[17]	YI YANGLEI, ZHANG ZHENHUA, ZHAO FAN, et al, 2018. Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus[J]. Fish Shellfish Immunol, 78: 322-330. doi: 10.1016/j.fsi.2018.04.055
[18]	ZHAO WANG, HUANG XINGMEI, DENG ZHENGHUA, et al, 2022. Growth performance, digestive and immune enzyme activities, and the environmental effects of the ivory shell, Babylonia areolata (Link 1807) in integrated multi trophic aquaculture systems[J]. Aquaculture Research, 53(11): 4168-4177. doi: 10.1111/are.v53.11
[19]	ZHOU JIANCONG, LIU CHUANG, YANG YUMEI, et al, 2023. Effects of long-term exposure to ammonia on growth performance, immune response, and body biochemical composition of juvenile ivory shell, Babylonia areolata[J]. Aquaculture, 562: 738857. doi: 10.1016/j.aquaculture.2022.738857
[20]	ZOKAEIFAR H, BABAEI N, CHE R S, et al, 2014. Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei[J]. Fish & Shellfish Immunology, 36(1): 68-74.

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

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

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

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