Isolation, Identification and Biological Characteristics of Probiotic Strains Derived from Intestine of Rockfish Sebastes schlegelii
LIU Danni, YE Bo, DONG Ying, WANG Xiaoyue, WANG Xuda, ZHAO Zhenjun, ZHANG Qian, YANG Boxue, LI Shilei
Key Laboratory of Germplasm Improvement and Fine Seed Breeding for Marine Aquatic Animals, Liaoning Province, Key Laboratory of Protection and Utilization of Aquatic Germplasm Resource, Ministry of Agriculture and Rural Affairs, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China
Abstract：In order to isolate potential probiotics from the intestinal tract of rockfish Sebastes schlegelii for application in its aquaculture production, two strains of Bacillus were isolated from the intestinal tract of wild rockfish with body weight of 62.96 g caught in the sea,and identified by morphological observation, biochemical identification, 16S rDNA gene sequencing and phylogenetic tree.The growth curves, growth characteristics, enzyme production, self-agglutination ability and hydrophobicity of two strains of Bacillus were investigated, and the drug sensitivity test and hemolysis test of two strains were carried out. The two strains of Bacillus were identified as B. licheniformis A1M1 and B. subtilis A3M3, respectively, and Gram-positive and rod-shaped, with logarithmic growth stage in 3 h of culture, and stable growth stage in 21 h and 24 h of culture, respectively. The most suitable cultivation conditions were shown to be temperature of 30 ℃, pH 7, salinity about 5—24 for strains A1M1 and A3M3. B. licheniformis A1M1 and B. subtilis A3M3 had the ability to produce amylase, cellulase, lipase and protease, and showed strong autoagglutination at 24 h (52.78% and 77.29%, respectively). The hydrophobicity of the both strains to different solvents was found to be significantly different, with the best to xylene (81.04% and 84.92%, respectively). B. subtilis A3M3 was sensitive to penicillin, ampicillin, ceftriaxone, gentamicin, tetracycline, erythromycin, ciprofloxacin, cotrimoxazole and lincomycin. B. licheniformis A1M1 had different degree of resistance to 9 kinds of antibiotics. The hemolytic test showed that the two strains had no hemolytic ability, and were featured by wide temperature and salinity, and acid and alkali resistance, fast growth rate, good enzyme production performance, strong self-agglutination ability and good hydrophobicity, indicating that they can be used as a probiotic candidate for the development of aquaculture and probiotics preparations of S. schlegelii. The findings provide data support for their application in aquaculture,especially in aquaculture of S. schlegelii.
[1] 孙敏敏,宋伟豪,吴鹏,等.许氏平鲉(Sebastes schlegelii)DAZ基因家族的鉴定及表达分析[J].海洋湖沼通报,2022,44(5):9-17. [2] 周海欧, 李兆龙, 李翠婷,等.牦牛源肠道芽孢杆菌的分离、鉴定及生物学特性研究[J]. 中国草食动物科学,2024,44(2):32-38 [3] 农业农村部渔业渔政管理局,全国水产技术推广总站,中国水产学会.2023中国渔业统计年鉴[M].北京:中国农业出版社,2023:21. [4] CAO M, YAN X, LI Q, et al. Genome-wide identification and analysis of NOD-like receptors and their potential roles in response to Edwardsiella tarda infection in black rockfish (Sebastes schlegelii)[J]. Aquaculture,2021,541:736803. [5] BEN HAMED S, TAPIA-PANIAGUA S T, MORIÑIGO M Á, et al. Advances in vaccines developed for bacterial fish diseases, performance and limits[J]. Aquaculture Research,2021,52(6):2377-2390. [6] 浦岩松.抗生素在水产养殖应用存在的问题及对策[J].黑龙江水产,2023,42(4):291-293. [7] KIM S, JEON H, HAN H S, et al. Evaluation of Bacillus albus SMG-1 and B. safensis SMG-2 isolated from Saemangeum Lake as probiotics for aquaculture of white shrimp (Litopenaeus vannamei)[J]. Aquaculture Reports,2021,20:100743. [8] KONG Y D, LI M, LI R M, et al. Evaluation of cholesterol lowering property and antibacterial activity of two potential lactic acid bacteria isolated from the intestine of snakehead fish (Channa argus)[J]. Aquaculture Reports,2020,17:100342. [9] BANERJEE G, RAY A K. The advancement of probiotics research and its application in fish farming industries[J]. Research in Veterinary Science,2017,115:66-77. [10] NAYAK S K. Probiotics and immunity:a fish perspective[J]. Fish & Shellfish Immunology,2010,29(1):2-14. [11] HASAN M T, JE JANG W, LEE J M, et al. Effects of immunostimulants, prebiotics, probiotics, synbiotics, and potentially immunoreactive feed additives on olive flounder (Paralichthys olivaceus):a review[J]. Reviews in Fisheries Science & Aquaculture,2019,27(4):417-437. [12] 孙博超,杨运楷,李玉宏,等.饲料中添加复合芽孢杆菌对凡纳滨对虾抗病毒感染能力及抗病基因表达的影响[J].渔业科学进展,2019,40(3):113-121. [13] CHEN B, PENG M F, TONG W T, et al. The quorum quenching bacterium Bacillus licheniformis T-1 protects zebrafish against Aeromonas hydrophila infection[J]. Probiotics and Antimicrobial Proteins,2020,12(1):160-171. [14] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:62-65. [15] 张昊,孙愉,李畅洋,等.牛源地衣芽孢杆菌的分离培养及生物学特性分析[J].中国畜牧杂志,2023,59(7):281-286. [16] 中华人民共和国卫生部.纸片法抗菌药物敏感试验标准:WS/T 125—1999[S].北京:中国标准出版社,2000:5-16. [17] MATHUR S, SINGH R. Antibiotic resistance in food lactic acid bacteria—a review[J]. International Journal of Food Microbiology,2005,105(3):281-295. [18] 杨红玲,孙云章,叶继丹,等.2株鱼源乳酸菌的生物学特性研究[J].西北农林科技大学学报(自然科学版),2008,36(8):25-30. [19] RINGØ E, OLSEN R E, MAYHEW T M, et al. Electron microscopy of the intestinal microflora of fish[J]. Aquaculture,2003,227(1/2/3/4):395-415. [20] 王旭达,关晓燕,王鉴,等.地衣芽孢杆菌对仿刺参生长、消化和免疫功能的影响[J].水产科学,2021,40(2):151-158. [21] 关晓燕,陈仲,赵春霞,等.一株芽孢杆菌的分离、鉴定及其对仿刺参免疫的影响[J].水产科学,2015,34(9):565-570. [22] 谢柳,鲁平才,赵钎.一株净水枯草芽孢杆菌的分离、鉴定及应用[J].环保科技,2019,25(6):1-4. [23] 彭友莲.复合微生物生态制剂在水产养殖中的应用效果[J].农业工程技术,2020,40(11):82-83. [24] MUJEEB RAHIMAN K M, JESMI Y, THOMAS A P, et al. Probiotic effect of Bacillus NL110 and Vibrio NE17 on the survival, growth performance and immune response of Macrobrachium rosenbergii (de Man)[J]. Aquaculture Research,2010,41(9):e120-e134. [25] 王成强,李宝山,王际英,等.饲料中添加枯草芽孢杆菌和酵母培养物对珍珠龙胆石斑鱼幼鱼生长、血清生化指标及抗氧化能力的影响[J].渔业科学进展,2019,40(4):47-56. [26] SUN Y Z, YANG H L, MA R L, et al. Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides[J]. Fish & Shellfish Immunology,2010,29(5):803-809. [27] 王娟,封永辉,蔡立胜,等.来自大黄鱼(Pseudosciaena crocea)肠道的弧菌拮抗菌的筛选与鉴定[J].海洋与湖沼,2010,41(5):707-713. [28] 孙娜,王腾腾,韩慧宗,等.两株海水鱼肠道芽孢杆菌的分离鉴定及特性分析[J].海洋渔业,2019,41(5):606-615. [29] 王晓杰,张秀梅,李文涛.盐度胁迫对许氏平鲉血液免疫酶活力的影响[J].海洋水产研究,2005,26(6):17-21. [30] 王晓杰,张秀梅,姜明.盐度胁迫对许氏平鲉鳃、头肾、脾脏超微结构的影响[J].中国海洋大学学报(自然科学版),2006,36(增刊):85-90. [31] 雷娟娟,梁玲,蒋顺进,等.猪肠源型枯草芽孢杆菌的分离鉴定及生物学特性研究[J].黑龙江畜牧兽医,2023(17):83-90. [32] 董文豪,宫晓炜,郑福英,等.猪源地衣芽孢杆菌的分离筛选及其特性[J].甘肃农业大学学报,2021,56(5):8-17. [33] 孙尹双,陈甜甜,白冬红,等.瘤胃源枯草芽孢杆菌BX1-12的筛选鉴定及其产酶抑菌活性[J].中国饲料,2022(19):62-66. [34] 陶荣霞.17株芽孢杆菌产酶特性及益生特性的研究[D].武汉:华中农业大学,2012. [35] MONTEAGUDO-MERA A, RASTALL R A, GIBSON G R, et al. Adhesion mechanisms mediated by probiotics and prebiotics and their potential impact on human health[J]. Applied Microbiology and Biotechnology,2019,103(16):6463-6472. [36] SHIN J H, LEE J S, SEO J G. Assessment of cell adhesion, cell surface hydrophobicity, autoaggregation, and lipopolysaccharide-binding properties of live and heat-killed Lactobacillus acidophilus CBT LA1[J]. The Korean Journal of Microbiology,2015,51(3):241-248. [37] GRIGORYAN S, BAZUKYAN I, TRCHOUNIAN A. Aggregation and adhesion activity of lactobacilli isolated from fermented products in vitro and in vivo:a potential probiotic strain[J]. Probiotics and Antimicrobial Proteins,2018,10(2):269-276. [38] KIM D H. Gut microbiota-mediated pharmacokinetics of ginseng saponins[J]. Journal of Ginseng Research,2018,42(3):255-263. [39] 赵浩航,康旭,黄子威,等.大口黑鲈肠道益生菌的分离与鉴定[J].广东海洋大学学报,2023,43(4):62-68. [40] ZENG Z B, ZHANG J B, LI Y, et al. Probiotic potential of Bacillus licheniformis and Bacillus pumilus isolated from Tibetan yaks, China[J]. Probiotics and Antimicrobial Proteins,2022,14(3):579-594. [41] LIU H T, WANG S F, CAI Y, et al. Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus[J]. Fish & Shellfish Immunology,2017,60:326-333. [42] 刘晓燕,王玲玲,栾会妮,等.一株枯草芽孢杆菌的分离鉴定、生物学特性及其对水质净化的作用[J].微生物学通报,2021,48(2):449-461. [43] 李旭,李雪,胡秀彩,等.鲫鱼肠道中枯草芽孢杆菌的分离鉴定及药敏试验[J].江苏农业科学,2014,42(7):244-245.
2025, Vol. 44 
