Screening And Identification Of Symbiotic Microorganisms Of Starfish With Antibacterial And Antioxidant Activity And Separation And Identification Of Active Substances

Symbiotic microorganisms are a kind of microorganisms that have a symbiotic,parasitic,or epiphytic relationship with host organisms.Some can produce bioactive substances that are the same as or similar to host cells,and have now become an important source for finding functional microorganisms.As a marine animal with pharmacological effects,starfish has long-term survival in the intertidal zone and the deep sea.It colonizes a large number of symbiotic flora,which is a huge treasure trove of biologically active marine symbiotic microorganisms.In this experiment,the starfish samples from the sea area of Naozhou Island in Zhanjiang City were collected,and the population,abundance and microbial community diversity of the starfish symbiotic bacteria were analyzed using culture-free High-throughput sequencing technology;The isolation of cultivable symbiotic microorganisms and screening of antibacterial active strains by traditional separation techniques,and identification of 16 S r RNA of active strains;Then,the antioxidation ability of the strains with antibacterial activity was measured in vitro,and the High-quality strains with antibacterial activity and antioxidant activity were selected;Isolate and purify the active metabolites of High-quality strains,and use LC-MS/MS to identify the active substances.The main research results are as follows:1.Through Illumina Miseq High-throughput sequencing technology,a total of 2384 OTUs were generated.After dilution curve and Alpha diversity analysis,a total of 5 gates,11 classes,15 orders,26 families,30 genera,among which were superior There are 11 genera,namely Psychrobacter,Lactococcus,Shewanella,Hydrogenophaga,Pseudomonas,Parvibaculum,Mesorhizobium,Phenylobacterium,Vibrio,Prolixibacter,Enterococcus.The above sequencing results show that the sea star contains extremely rich symbiotic flora.2.A total of 109 symbiotic bacteria were isolated from starfish of Naozhou Island using 8 mediums,and Staphylococcus aureus,Bacillus subtilis,Listeria monocytogenes,Escherichia coli,Vibrio parahaemolyticus were used as antagonistic strains.The cup agar diffusion method screened out 24 strains with antibacterial activity,of which 9 symbiotic strains had inhibitory effects on five pathogenic bacteria,accounting for 29.2% of the total active strains.According to 16 S r RNA identification,they belong to three phylum Firmicutes,Proteobacteria,Bacteroidetes,9 families and 9 genera(Exiguobacterium,Psychrobacter,Pseudomonas,Bacillus,Shewanella,Enterococcus,Lactobacillus,Lactococcus,Myroides).3.Taking 24 symbiotic antimicrobial active strains as screening materials,they were tested for in vitro antioxidant activity by measuring their DPPH radical scavenging ability,hydroxyl radical scavenging ability,ABTS radical scavenging ability,and total reducing ability 3 strains(BL-15,BL-17,BL-27)were screened out,the scavenging ability of the three free radicals were all above 40%,and the total reducing ability was above 1.0.Lactobacillus pentosus BL-15 was used to separate and identify active substances.4.Using ethyl acetate to extract the active crude extract of the fermentation broth of Lactobacillus pentosus BL-15,the active crude extract was treated with different p H,temperature,ultraviolet light and enzyme preparation.The results showed that: The antibacterial and antioxidative activity of the crude extract can maintain good activity stability under the conditions of weak acid,low temperature,high temperature and ultraviolet light,especially 77.9% and 67.3% are retained at a high temperature of 120 ?Papain and pepsin are more sensitive in the protease sensitivity experiment,so as to determine the active substances as peptides or proteins.The fermentation supernatant was extracted with ethyl acetate,SP-Sepharose Fast Flow cation column,Sephadex LH-20 gel chromatography column,and semi-preparative reversed-phase high-performance liquid chromatography were separated and purified step by step to obtain an active single peak.After LC-MS/MS determined that the molecular weight of bacteriocin Peptide BL-15 was1279.66 Da,and the amino acid sequence was analyzed by secondary mass spectrometry to LEVQAVSKNKL.Comparison with the Universal Protein Database(Uni Prot)revealed that bacteriocin Peptide BL-15 and a protein G2/mitotic-specific cyclin-The similarity of B(login number: P15206)is 90.0%.