珊瑚益生菌筛选及其益生特性分析

Screening of coral probiotics and their probiotic characteristics

  • 摘要: 【目的】挖掘珊瑚潜在益生菌并明确其益生特性,为珊瑚益生菌资源库提供新材料,也为珊瑚疾病的微生物防治研究提供理论依据。【方法】分别以蛋白酶和β-葡萄糖苷酶活性筛选胞外酶产生菌株,以溶珊瑚弧菌为指示菌筛选具拮抗活性的菌株,以紫色杆菌为指示菌筛选有群体淬灭活性的菌株,通过16S rRNA测序对所获菌株进行种属鉴定,然后利用缸内添加试验验证益生菌的抗病功能,并通过全基因组分析预测益生菌的益生特性。【结果】通过原位模拟培养法,从7种珊瑚样品中共分离纯化获得271株细菌,其中,136株具有蛋白酶活性,87株具有β-葡萄糖苷酶活性,20株具有群体淬灭活性,28株对溶珊瑚弧菌有拮抗活性。既能产生胞外酶又具有拮抗活性或群体淬灭活性的菌属有芽孢杆菌属、假芽孢杆菌属、微小杆菌属、鲁杰氏菌属、鞘脂菌属、假交替单胞菌属、弧菌属、微球菌属、诺卡菌属和鞘氨醇单胞菌属。益生菌抗病作用缸内验证试验发现,芽孢杆菌GXU-Z9株能有效维持鹿角杯形珊瑚的光合效率和虫黄藻密度,保护珊瑚免受溶珊瑚弧菌引起的白化现象。基于全基因组数据分析发现,芽孢杆菌GXU-Z9株具有非常活跃的氨基酸代谢和运输能力,具备为珊瑚宿主提供必需和非必需氨基酸的潜力;通过产生对珊瑚有益的化合物(抗生素等)而抵御弧菌感染,并通过降解纤维类或蛋白类颗粒物质来补充珊瑚能量需求;同时,其全基因组中包含多种氨基酸生物合成相关基因及B族维生素合成相关基因,具有为珊瑚宿主提供氨基酸和维生素以获得能量的潜力。【结论】芽孢杆菌GXU-Z9株能有效维持鹿角杯形珊瑚的光合效率和虫黄藻密度,展现出良好的抗病功能;芽孢杆菌GXU-Z9株全基因组中含有编码几丁质酶和β-葡萄糖苷酶的相关基因,能大量合成珊瑚的必需氨基酸和维生素等营养物质,具有辅助珊瑚消化碳水化合物的酶类物质、提供氨基酸和维生素等益生特性。

     

    Abstract: 【Objective】This study investigated potential probiotics in coral and elucidated their probiotic characteristics,thereby contributing novel materials to the coral probiotic resource library and offering theoretical foundation for microbial control research in coral disease management.【Method】Extracellular enzyme producing strains were screened using protease and β-glucosidase activities,antagonistic active strains was screened with Vibrio coralliilyticus as indicator strain,quorum quenching active strains was screened with Chromobacterium violaceum as indicator strain,and strain identification on species and genus was performed via 16S rRNA sequencing. Then,the anti-disease function of probiotics was verified by in-tank addition test,and the probiotic properties of probiotics were predicted by whole genome analysis. 【Result】A total of 271 bacterial strains were isolated and purified from 7 coral samples using an in situ simulated cultivation method. Activity screening indicated that 136 strains exhibited protease activity,87 strains exhibited β-glucosidase activity,20 strains exhibited quorum quenching activity,and 28 strains exhibited antagonistic activity against V. coralliilyticus. The bacterial genera capable of producing extracellular enzymes and displaying antagonistic or quorum quenching activities included Bacillus sp.,Fictibacillus sp.,Exiguobacterium sp.,Ruegeria sp.,Sphingobium sp.,Pseudoalteromonas sp.,Vibrio sp.,Micrococcus sp.,Nocardia sp.,and Sphingomonas sp. The in-tank validation test of probiotic disease resistance demonstrated that the Bacillus sp. GXU-Z9 strain could effectively sustain the photosynthetic efficiency and zooxanthellae density of Pocillopora damicornis,thereby protecting the coral from bleaching induced by V. coralliilyticus. Whole genome data analysis revealed that Bacillus sp. GXU-Z9 strain possessed highly active amino acid metabolism and transport capabilities,indicating its potential to supply both essential and non-essential amino acids to coral hosts. Additionally,it could produce compounds beneficial to corals,such as antibiotics,which helped in resisting Vibrio infections,and could supplement the coral’s energy requirements by degrading fibrous or protein particulate matter. Concurrently,its genome encompassed multiple genes associated with amino acid biosynthesis and B Vitamin synthesis,which had the potential to supply essential amino acids and vitamins to the coral hosts,thereby facilitating their energy acquisition.【Conclusion】Bacillus sp. GXU-Z9 strain can effectively sustain the photosynthetic efficiency and zooxanthellae density of P. damicornis,exhibiting remarkable disease resistance. The whole genome of Bacillus sp. GXU-Z9 strain includes genes encoding chitinase and β-glucosidase,enabling the synthesis of essential amino acids and vitamins in substantial amounts. These capabilities assist the coral in digesting carbohydrate enzymes and the provision of amino acids and vitamins.

     

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