海参肠道内容物中海藻多糖降解菌的筛选及鉴定

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

摘要/Abstract

摘要：

为促进藻类在海参养殖中的应用,筛选藻类海藻多糖降解菌。采用藻粉诱导的方法,促进海参肠道中藻类多糖降解菌的富集,分别以褐藻胶和卡拉胶为唯一碳源,进行褐藻胶和卡拉胶降解菌的筛选。采用DNS法测定菌体降解褐藻胶和卡拉胶的活性,通过16S rDNA测序结合生理生化指标检测对菌种进行种属鉴定,同时检测菌株的生长特性、降解饵料其他主要成分的活性和生物安全性。结果筛选到两株高活性海藻多糖降解菌,两菌株属于芽孢杆菌,菌体生长迅速,具有较强的酸碱和盐浓度适应能力,并且对海参健康均没有明显影响。褐藻胶降解菌HZ-1与Bacillus altitudinis相似性最高,褐藻胶降解酶活力为43.2 U/mL,具有淀粉酶、纤维素酶和蛋白酶活性。卡拉胶降解菌KL-6与Bacillus megaterium相似性最高,卡拉胶降解酶活力为1.45 U/ mL,具有淀粉酶和蛋白酶活性。筛选到两株具有海藻多糖降解能力的芽孢杆菌,两菌株生长速度快、酶活力高,可用作养殖益生菌或用于藻类饲料加工。

关键词: 褐藻胶, 卡拉胶, 降解, 筛选, 芽孢杆菌

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

白新峰, 贾爱荣, 刘雪, 张绵松, 崔婷婷, 刘德亭, 刘昌衡. 海参肠道内容物中海藻多糖降解菌的筛选及鉴定[J]. 生物技术通报, 2021, 37(6): 147-153.

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.

图/表 5

图1 菌株部分生物学表型 A:褐藻胶降解活性;B:卡拉胶降解活性;C:HZ-1 菌落形态;D:KL-6菌落形态;E:HZ-1 菌体形态;F:KL-6 菌体形态

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

表1 菌株HZ-1和KL-6的部分生理生化特征

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	+	+

图2 菌株HZ-1和KL-6的系统发育树分支节点上的数值表示分支的置信度百分数;标尺所示长度为 0.01% 核苷酸置换率

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

图3 菌株HZ-1(A)和KL-6(B)的生长和产酶特征曲线

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

图4 菌株部分生物学活性检测 A:菌株淀粉酶活性;B:菌株蛋白酶活性;C:菌株纤维素酶活性;D:菌株溶血酶活性

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

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