Screening and Identification of Algal Polysaccharide-degrading Bacteria in the Intestinal Contents of Sea Cucumber

doi:10.13560/j.cnki.biotech.bull.1985.2020-1113

Abstract

Abstract:

In order to promote the application of algae in sea cucumber culture,algae polysaccharide-degrading bacteria were screened. In this study,algal powder was used to enrich the algal polysaccharide-degrading bacteria in the intestinal tract of sea cucumbers. Alginate and carrageenan were used as the sole carbon source to screen alginate- and carrageenan-degrading bacteria,respectively. The alginate- and carrageenan-biodegrading activities of the strain were determined by DNS method,and the strains were identified by 16S rDNA sequencing combined with physiological and biochemical index detection. Concurrently,the growth characteristics of the strains,the abilities of bacteria to degrade other main components of sea cucumber feed and the biosafety were detected. The results showed that two strains of algae polysaccharide-degrading bacteria with high activity were screened. Both strains belonged to Bacillus,which grew rapidly and had strong adaptability to acid,alkali and salt concentration,and had no significant effect on the health of sea cucumbers. The alginate-degrading bacterium HZ-1,which was the most similar with Bacillus altitudinis,had an alginate-degrading activity of 43.2 U/mL,and it also presented amylase,cellulase and protease activities. Carrageenan-degrading bacterium KL-6,which was the most similar with Bacillus megaterium,had a carrageenan-degrading activity of 1.45 U/mL,and it also had amylase and protease activity. Two strains of Bacillus with algae polysaccharide-degrading ability are screened. The two strains demonstrate fast growth speed and high enzyme activity,and can be used as breeding probiotics or used in the fermentation process of algae feed.

Key words: alginate, carrageenan, degradation, screening, Bacillus

BAI Xin-feng, JIA Ai-rong, LIU Xue, ZHANG Mian-song, CUI Ting-ting, LIU De-ting, LIU Chang-heng. Screening and Identification of Algal Polysaccharide-degrading Bacteria in the Intestinal Contents of Sea Cucumber[J]. Biotechnology Bulletin, 2021, 37(6): 147-153.

Figures/Tables 5

Fig.1 Some biological phenotypes of the strains A:Degradation activity of alginate. B:Degradation activity of carrageenan. C:Colony morphology of Hz-1. D:Colony morphology of KL-6. E:Cell morphology of Hz-1. F:Cell morphology of KL-6

Table1 Some physiological and biochemical characteristics of strains HZ-1 and KL-6

检测项目Test item	HZ-1	KL-6	检测项目Test item	HZ-1	KL-6
运动	+	+	葡萄糖 Glucose	+	+
革兰氏染色 Gram Staining	阳 Positive	阳 Positive	果糖 Fructose	+	+
形状 Shape	杆状 Rod	杆状 Rod	木糖 Xylose	+	+
形成芽孢 Sporulation	+	+	甘露糖 Mannose	+	+
VP实验 VP test	-	-	阿拉伯糖 arabinose	-	+
2% NaCl生长 Grown in 2% NaCL	+	+	蛋白酶 Protease activity	+	+
7% NaCl生长 Grown in 7% NaCL	+	+	脂酶 Lipase enzyme activity	-	-
10% NaCl生长 Grown in 10% NaCL	+/-	-	淀粉酶 Amylase activity	+	+
柠檬酸盐利用 Utilization of citrate	+	+	接触酶 Contact enzyme activity	+	+
硝酸盐还原 Nitrate reduction	+	+

Fig. 2 Phylogenetic tree of strains HZ-1 and KL-6 The numerical value on the branch node represents the confidence percentage of the branch. The length shown on the scale represents 0.01% nucleotide replacement rate

Fig. 3 Growth and enzyme production characteristic curves of strains HZ-1(A)and KL-6(B)

Fig. 4 Detection of some biological activities of the strain A:Amylase activity of the strain. B:Protease activity of the strain. C:Cellulase activity of the strain. D:Hemolytic enzyme activity of the strain

References 24

[1]	农业农村部渔业渔政管理局. 2020中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2020.
	Fisheries and fisheries administration of the ministry of agriculture and rural affairs of China China fisheries statistics yearbook 2020[M]. Beijing: China Agriculture Press, 2020.
[2]	郑娜. 海参养殖常见病害及防治技术[J]. 齐鲁渔业, 2017(34):25-26.
	Zheng N. Common diseases in sea cucumber breeding and their control techniques[J]. Qilu Fishery, 2017(34):25-26.
[3]	Guo X, Wang Y, Qin Y, et al. Structures, properties and application of alginic acid:A review[J]. International Journal of Biological Macromolecules, 2020, 162:618-628. doi: 10.1016/j.ijbiomac.2020.06.180 URL
[4]	喻虎. 海藻多糖制备与应用研究[J]. 农业与技术, 2018, 38(20):255.
	Yu H. Research on preparation and application of seaweed polysaccharide[J]. J Agr Sci Tech, 2018, 38(20):255.
[5]	Hentati F, Tounsi L, Djomdi D, et al. Bioactive polysaccharides from seaweeds[J]. Molecules, 2020, 25:3152. doi: 10.3390/molecules25143152 URL
[6]	Besednova N, Zaporozhets T, et al. Extracts and marine algae polysaccharides in therapy and prevention of inflammatory diseases of the intestine[J]. Marine Drugs, 2020, 18(6):289. doi: 10.3390/md18060289 URL
[7]	Cheng C, Yang W, et al. Transcriptomically revealed oligo-fucoidan enhances the immune system and protects hepatocytes via the ASGPR/STAT3/HNF4A axis[J]. Biomol, 2020, 10(6):898. doi: 10.3390/biom10060898 URL
[8]	Liu W, Zhou S, Balasubramanian B, et al. Dietary seaweed(Enteromorpha)polysaccharides improve growth performance involved in regulation of immune responses, intestinal morphology and microbial community in banana shrimp Fenneropenaeus merguiensis[J]. Fish Shellfish Immunol, 2020, 104:202-212. doi: 10.1016/j.fsi.2020.05.079 URL
[9]	Cheng W, Yu J. Effects of the dietary administration of sodium alginate on the immune responses and disease resistance of Taiwan abalone, Haliotis diversicolor supertexta[J]. Fish & Shellfish Immunology, 2013, 34:902-908.
[10]	Zhu B, Yin H. Alginate lyase:Review of major sources and classification, properties, structure-function analysis and applications[J]. Bioengineered Bugs, 2015, 6(3):125-131.
[11]	周敏, 王海英, 冷凯良, 等. 基于宏基因组学的海带降解菌群微生物多样性及其褐藻多糖降解酶系分析[J]. 海洋湖沼通报, 2018, 6:109-117.
	Zhou M, Wang HY, Leng KL, et al. Analysis of microbial diversity of laminaria degrading bacteria and its fucoidan degrading enzyme system based on metagenomics[J]. Marine Limnology Bulletin, 2018, 6:109-117.
[12]	管斌, 倪雪朋, 李悦明, 等. 海藻多糖降解酶的研究进展[J]. 中国酿造, 2010, 29(9):8-12.
	Guan B, Ni XP, et al. Research progress of seaweed polys-accharide degrading enzymes[J]. Chinese Brew, 2010, 29(9):8-12.
[13]	田良. 仿刺参消化道中产酶菌株的筛选鉴定及其在仿刺参饲料中的应用[D]. 厦门:集美大学, 2015.
	Tian L. Screening and identification of enzyme-producing strains in the digestive tract of sea cucumber and its application in sea cucumber feed[D]. Xiamen:Jimei University, 2015.
[14]	李凤辉. 刺参消化道微生物组成及其产酶功能研究[D]. 上海:上海海洋大学, 2014.
	Li FH. The composition and enzyme-producing function of the microflora in the digestive tract of the sea cucumber Apostichopus japonicus[D]. Shanghai:Shanghai Ocean University, 2014.
[15]	Cheng C, Yang W, Hsiao M, et al. Transcriptomically revealed oligo-fucoidan enhances the immune system and protects hepatocytes via the ASGPR/STAT₃/HNF₄A axis[J]. Biomolecules, 2020, 10(6):898. doi: 10.3390/biom10060898 URL
[16]	Wu S, Yang W, Cheng C, et al. Low molecular weight fucoidan prevents radiation-induced fibrosis and secondary tumors in a zebrafish model[J]. Cancers(Basel), 2020, 12(6):1608.
[17]	Van Doan H, Tapingkae W, Moonmanee T, et al. Effects of low molecular weight sodium alginate on growth performance, immunity, and disease resistance of tilapia, Oreochromis niloticus.[J]. Fish & Shellfish Immunology, 2016, 55:186-194.
[18]	Gurpilhares D, Cinelli L, Simas N, et al. Marine prebiotics:Polysaccharides and oligosaccharides obtained by using microbial enzymes[J]. Food Chemistry, 2019, 280:175-186. doi: S0308-8146(18)32118-6 pmid: 30642484
[19]	林清菁. 一株海藻多糖降解菌的分离与鉴定[J]. 微生物学通报, 2014, 41(11):2208-2215.
	Lin QJ. Isolation and identification of a seaweed polysaccharide degrading bacteria[J]. Microbiology Bulletin, 2014, 41(11):2208-2215.
[20]	朱大玲, 唐啸龙, 张宝玉, 等. 一株海藻多糖降解菌的分离鉴定及产酶条件优化[J]. 海洋科学, 2017, 41(8):101-109.
	Zhu DL, Tang XL, et al. Isolation and identification of a seaweed polysaccharide degrading bacteria and optimization of its enzyme production conditions[J]. Marine Sci, 2017, 41(8):101-109.
[21]	潘爱红, 李江, 等. 南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化[J]. 微生物学通报, 2018, 45(9):2022-2034.
	Pan AH, Li H, Wang L, et al. Optimization of fermentation conditions for carrageenase production by alteromonas antarctica R11-5[J]. Microbiology Bulletin, 2018, 45(9):2022-2034.
[22]	Wang XT, Wang LL, Che J, et al. Improving the quality of Laminaria japonica-based diet for Apostichopus japonicus through degradation of its algin content with Bacillus amyloliquefaciens WB1[J]. App Microb Biotech, 2015, 99:5843-5853. doi: 10.1007/s00253-015-6583-4 URL
[23]	Sha YJ, Liu M, Wang BJ, et al. Gut bacterial diversity of farmed sea cucumbers Apostichopus japonicus with different growth rates[J]. Microbiology, 2016, 85(1):109-115. doi: 10.1134/S0026261716010112 URL
[24]	李昌明. 海参肠道微生物多样性分析及菌株s12~T褐藻胶裂解酶研究[D]. 济南, 山东大学, 2019.
	Li CM. Diversity analysis of sea cucumber intestinal microorganisms and study on S12~T alginate lyase[D]. Ji'nan:Shandong University, 2019.