|
|
Screening, Identification and Biological Characteristics Research of Digestive Enzyme Secreting Probiotics from Gibel Carp (Carassius auratus gibelio) CASⅤ |
YU Peng1, MA Jing-Jing1,*, CHENG Yu1, ZHANG Cheng-Shuo1, WANG Kai1, ZHOU Zheng-Hai2 |
1 Jiangsu Coastal Area Institute of Agricultural Sciences, Yancheng 224001, China; 2 Jiangsu Zheng Yuan Chuang Hui Agricultural Science and Technology Development Co., Ltd., Yancheng 224700, China |
|
|
Abstract The low feed digestibility and absorption rate is an important reason for high feed cost and low profit of fish farming. Gibel carp (Carassius auratus gibelio) is one of the main current crucian carp breeding in China, the aim of this study was to screen safe and efficient probiotics which can secret digestive enzymes from healthy culture system of gibel carp CASⅤ, so as to improve feed digestion and absorption capacity of CASⅤ and reduce feed cost. Ten strains with protease, lipase and amylase activities were isolated from CASⅤ and pond water by spot inoculation method. Two highly efficient, non-hemolytic, sensitive to most antibiotics and non-pathogenic strains, ZKW7 and ZKF17, were obtained by further analysis of enzyme secretion ability and safety test (hemolysis, antibiotics susceptibility and intraperitoneal infection). Based on 16S rDNA sequence analysis, both strains had the highest similarity to Bacillus subtilis and B. halotolerans. The results of biological characteristics analysis showed that both strains had strong degrading activities for NH4+, NO2- and NO3-. The optimum growth conditions of ZKW7 were pH 8, salinity 15 and Fe2+ in culture medium, respectively. By contrast, the optimum growth conditions of ZKF17 were pH 7, salinity 10~30 and the presence of Mg2+. In addition, these 2 strains were resistant to acid, bile salt, intestinal fluid and succus gastricus to some extent. In the present study, 2 strains of safe and efficient enzyme-producing Bacillus sp. were obtained, which could be used as potential probiotics and would be conducive to the healthy farming of C. auratus gibelio var. CASⅤ.
|
Received: 28 August 2023
|
|
Corresponding Authors:
*mjj-1981@163.com
|
|
|
|
[1] 陈书畅, 徐晔, 曲乐天, 等. 2022. 鲤肠道内产消化酶益生菌的分离与筛选[J]. 水产学杂志, 35(03): 58-62. (Chen S C, Xu Y, Qu L T, et al.2022. Isolation and screening of probiotics producing digestive enzymes in intestine of common carp Cyprinus carpio[J]. Chinese Journal of Fisheries, 35(03): 58-62.) [2] 陈薇, 丁祥力, 吴迎奔, 等. 2013. 水产养殖用复合微生态制剂的安全性评价[J]. 贵州农业科学, 41(02): 138-140. (Chen W, Ding X L, Wu Y B, et al.2013. Security evaluation of a compound probiotics used for aquaculture[J]. Guizhou Agricultural Sciences, 41(02): 138-140.) [3] 成钰, 李秋芬, 费聿涛, 等. 2016. 海水异养硝化-好氧反硝化芽胞杆菌SLWX2的筛选及脱氮特性[J]. 环境科学, 37(07): 2681-2688. (Chen Y, Li Q F, Fei Y T, et al.2016. Screening and nitrogen removing characteristics of heterotrophic nitrification-aerobic denitrification bacteria SLWX2 from sea water[J]. Environmental Science, 37(7): 2681-2688.) [4] 董昭然. 2023. CyHV-2酵母表面展示口服疫苗对异育银鲫的免疫效果评价[D]. 硕士学位论文, 华中农业大学, 导师: 苏建国, 徐震. pp. 1-2. (Dong Z R.2023. Evaluating the immune responses to CyHV-2 yeast surface display oral vaccine in Carassius auratus gibelio[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Su J G, Xu Z. pp. 1-2.) [5] 窦春萌, 左志晗, 刘逸尘, 等. 2016. 凡纳滨对虾肠道内产消化酶益生菌的分离与筛选[J]. 水产学报, 40(04): 537-546. (Dou C M, Zuo Z H, Liu Y C, et al.2016. Isolation and screening of digestive enzyme producing probiotics from intestine of Litopenaeus vannamei[J]. Journal of Fisheries of China, 40(04): 537-546.) [6] 樊丹, 邓福容, 李绍戊, 等. 2022. 一株虹鳟源枯草芽胞杆菌产胞外蛋白发酵条件优化[J]. 江西农业大学学报, 44(2): 452-460. (Fan D, Deng F R, Li S W, et al.2022. Fermentation condition optimization for extracellular protein of Bacillus subtilis from rainbow trout (Oncorhynchus mykiss)[J]. Acta Agriculturae Universitatis Jiangxiensis, 44(2): 452-460.) [7] 蒋翠翠, 尚昊, 张素芳, 等. 2023. 产脂肪酶菌株的筛选、酶学特性及其接种对酸肉风味物质的影响[J]. 食品科学, 44(10): 106-113. (Jiang C C, Shang H, Zhang S F, et al.2023. Screening of lipase-producing strains, enzymatic properties and effects of inoculation on flavor compounds of fermented sour meat[J]. Food Science, 44(10): 106-113.) [8] 康兴娇, 贾招闪, 申红妙, 等. 2016. 内生枯草芽胞杆菌CN181发酵条件优化[J]. 山东农业大学学报(自然科学版), 47(5): 647-653. (Kang X J, Jia Z S, Shen H M, et al.2016. Optimization of fermentation conditions for endophytic Bacillus subtilis CN181[J]. Journal of Shandong Agricultural University (Natural Science Edition) , 47(5): 647-653.) [9] 李静, 陈维新, 邓毛程. 2015. 白鲳鱼肠道中产蛋白酶菌的筛选、鉴定及产酶条件[J]. 河南工业大学学报 (自然科学版), 36(06): 37-41, 50. (Li J, Chen W X, Deng M C.2015. Screening, identification and protease-producing conditions of a strain isolated from gastrointestinal tract of white pomfret[J]. Journal of Henan University of Technology (Natural Science Edition), 36(06): 37-41, 50.) [10] 廖梦香. 2023. 百草霜对异育银鲫“中科5号”幼鱼生长及消化性能的影响[J]. 中国饲料, (07): 72-78. (Liao M X. 2023. Feeding plant soot affecting growth performance and enzyme activities in juvenile Carassius auratus gibelio “CASⅤ”[J]. China Feed, (07): 72-78.) [11] 刘吉丹, 樊英, 刘洪军, 等. 2022. 斑节对虾(非洲群体)肠道中哈维氏弧菌拮抗菌的筛选、鉴定及生理特性分析[J]. 水生生物学报, 46(12): 1876-1887. (Liu J D, Fan Y, Liu H J, et al.2022. Screening, identification and physiological characteristics of intestinal bacteria against Vibrio harveyi from Penaeus monodon[J]. Acta Hydrobiologia Sinica, 46(12): 1876-1887.) [12] 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. 2022. 中国渔业统计年鉴[M]. 中国农业出版社, 北京.pp. 25. (Ministry of Agriculture and Rural Affairs of the People's Republic of China, National Fisheries Technology Extension Center, China Society of Fisheries. 2022. China Fishery Statistical Yearbook[M]. Chinese Agricultural. Beijing. pp. 25.) [13] 彭小倩, 任朝颖, 邓雪玥, 等. 2023. 杂交鲟源温和气单胞菌的分离鉴定及生物学特性的研究[J]. 淡水渔业, 53(03): 62-70. (Peng X Q, Ren C Y, Deng X Y, et al.2023. Isolation, identification, and biological characterization of Aeromonas sobria from hybrid sturgeon[J]. Freshwater Fisheries, 53(03): 62-70.) [14] 孙亮, 刘文波, 杨廷雅, 等. 2013. 枯草芽胞杆菌HAB-1产生抗菌物质的最优发酵条件[J]. 热带生物学报, 4(3): 225-235. (Sun L, Liu W B, Yang Y T, et al.2013. Optimization of fermentation conditions for Bacillus subtilis HAB-1 to produce antimicrobial substances[J]. Journal of Tropical Biology, 4(03): 225-235.) [15] 王家川, 张德锋, 王亚军, 等. 2021. 一株鳜鱼源产酶芽胞杆菌的筛选、鉴定及其生物学特性[J]. 农业生物技术学报, 29(03): 558-570. (Wang J C, Zhang D F, Wang Y J, et al.Screening, identification and biological characteristics of an enzyme-producing Bacillus strain isolated from Siniperca chuatsi[J]. Journal of Agricultural Biotechnology, 29(03): 558-570.) [16] 王建建, 施兆鸿, 高权新, 等. 2015. 野生银鲳消化道内潜在产酶益生菌产酶条件的初步研究[J]. 海洋渔业, 37(06): 533-540. (Wang J J, Shi Z H, G Q X, et al.2015. On enzyme-production conditions of potential probiotics in digestive tracts of wild Pampus argenteus[J]. Marine Fisheries, 37(06): 533-540.) [17] 王学文, 于存, 潘洪祥. 2023. 高知芽胞杆菌Cytobacillus kochii H产蛋白酶条件优化及抑菌效应[J]. 福建农业学报, 38(1), 47-57. (Wang X W, Yu C, Pan H X.2023. Process optimization and antimicrobial effect of Cytobacillus kochii H protease[J]. Fujian Journal of Agricultural Sciences, 38(1), 47-57.) [18] 吴建军, 邱权, 付大波, 等. 2017. 水产复合酶在不同蛋白水平下对鲫鱼生长性能及表观消化率的影响[J]. 饲料工业, 38(06): 16-19. (Wu J J, Qiu Q, Fu D B, et al.2017. Effects of compound enzymes on growth performance and apparent digestibility of Gibel carp at different protein levels[J]. Feed Industry, 38(06): 16-19.) [19] 吴振超, 陈玉珂, 高永生, 等. 2020. 一株黄金鲫鱼源蜡状芽胞杆菌的筛选、鉴定及耐性研究[J]. 大连海洋大学学报, 35(06): 883-892. (Wu Z C, Chen Y H, Gao Y S, et al.2020. Isolation,identification and probiotic properties of a Bacillus cereus strain from Golden crucian carp[J]. Journal of Dalian Ocean University, 35(06): 883-892.) [20] 徐英杰, 戎华南, 陈雨, 等. 2023. 两种池塘养殖模式下异育银鲫(Carassius auratus gibelio)的形质特征差异分析[J]. 海洋与湖沼, 54(01): 204-212. (Xu Y J, Rong H N, Chen Y, et al.2023. Analysis of the differences in morphology and quality of Carassius auratus gibelio under two pond aquaculture modes[J]. Oceanologia et Limnologia Sinica, 54(01): 204-212.) [21] 许文婕, 雨夏, 朱晓鸣, 等. 2022. 饲料中添加椭圆栅藻对异育银鲫中科5号越冬后抗病力的影响[J]. 水生生物学报, 46(04): 457-465. (Xu W J, Xia Y, Zhu X M, et al.2022. Effects of dietary Scenedesmus ovalternus on the disease resistance of overwintering gibel carp (Carassius gibelio var. CASⅤ)[J]. Acta Hydrobiologia Sinica, 46(04): 457-465.) [22] 于鹏, 叶海斌, 单洪伟, 等. 2020. 凡纳滨对虾养殖体系中群体感应淬灭菌的筛选、安全性评估及发酵条件优化[J]. 渔业科学进展, 41(05): 82-91. (Yu P, Ye H B, Shan H W, et al.2020. Screening, safety evaluation and fermentation conditions optimization of quorum quenching bacteria from Litopenaeus vannamai culture system[J]. Progress in Fishery Sciences, 41(05): 82-91) [23] Abatenh E, Gizaw B, Tsegay Z, et al.2018. Health benefits of probiotics[J]. Journal of Bacteriology and Infectious Diseases, 2(1): 17-27. [24] Abdel-Latif H M R, Chaklader M R, Shukry M, et al.2023. A multispecies probiotic modulates growth, digestive enzymes, immunity, hepatic antioxidant activity, and disease resistance of Pangasianodon hypophthalmus fingerlings[J]. Aquaculture, 563: 738948. [25] Agrawal V, Sastry K, Kaushab S.1975. Digestive enzymes of three teleost fishes[J]. Acta Physiologica Academiae Scientiarum Hungaricae, 46(02): 93-98. [26] Cao S P, Mo P, Xiao Y B, et al.2021. Dietary supplementation with fermented plant meal enhances growth, antioxidant capacity and expression of TOR signaling pathway genes in gibel carp (Carassius auratus gibelio var. CAS V)[J]. Aquaculture Reports, 19: 100559. [27] Cerezuela R, Guardiola F A, Meseguer J, et al.2012. Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damsela[J]. Fish & Shellfish Immunology, 32: 1032-1040. [28] Dawood M A O, Koshio S, Ishikawa M, et al.2016. Probiotics as an environment-friendly approach to enhance red sea bream, Pagrus major growth, immune response and oxidative status[J]. Fish & Shellfish Immunology, 57: 170-178. [29] Fan Y, Liu L T, Zhao L H, et al.2018. Influence of Bacillus subtilis ANSB060 on growth, digestive enzyme and aflatoxin residue in Yellow River carp fed diets contaminated with aflatoxin B1[J]. Food Chemical Toxicology, 113: 108-114. [30] Guo X H, Li D F, Lu W Q, et al.2006. Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs[J]. Antonie Van Leeuwenhoek, 90: 139-146. [31] Han B, Long W Q, He J Y, et al.2015. Effects of dietary Bacillus licheniformis on growth performance, immunological parameters, intestinal morphology and resistance of juvenile Nile tilapia (Oreochromis niloticus) to challenge infections[J]. Fish & Shellfish Immunology, 46: 225-231. [32] Hu Q, Wu Q, Dai B H, et al.2022. Fermentation optimization and amylase activity of endophytic Bacillus velezensis D1 isolated from corn seeds[J]. Journal of Applied Microbiology, 132(5): 3640-3649. [33] Husain F, Duraisamy S, Balakrishnan S, et al.2022. Phenotypic assessment of safety and probiotic potential of native isolates from marine fish Moolgarda seheli towards sustainable aquaculture[J]. Biologia, 77: 775-790. [34] Liu H T, Wang S F, Cai Y, et al.2017. 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, 60: 326-333. [35] Mohammadian T, Monjezi N, Peyghan R, et al.2022. Effects of dietary probiotic supplements on growth, digestive enzymes activity, intestinal histomorphology and innate immunity of common carp (Cyprinus carpio): A field study[J]. Aquaculture, 549: 737787. [36] Nayak S K.2010. Probiotics and immunity: A fish perspective[J]. Fish & Shellfish Immunology, 29: 2-14. [37] Pedersen C, Jonsson H, Lindberg J E, et al.2004. Microbiological characterization of wet wheat distillers' grain, with focus on isolation of lactobacilli with potential as probiotics[J]. Applied and Environmental Microbiology, 70: 1522-1527. [38] Xue M, Cui Y B.2001. Effect of several feeding stimulants on diet preference by juvenile gibel carp (Carassius auratus gibelio), fed diets with or without partial replacement of fish meal by meat and bone meal[J]. Aquaculture, 198: 281-292. [39] Yu P, Ye H B, Ma S, et al.2021. Study of quorum quenching bacteria Cobetia sp. reducing the mortality of Penaeus vannamei infected with Vibrio harveyi: In vitro and in vivo tests[J]. Aquaculture, 534: 736244. [40] Zhou Y J, Yang X X, Li Q, et al.2023. Optimization of fermentation conditions for surfactin production by B. subtilis YPS-32[J]. BMC Microbiology, 23(1):117. |
|
|
|