| The frequent occurrence of aquatic animal diseases and the abuse of antibiotics have seriously hindered the development of the aquaculture industry.Therefore,it is urgent to look for efficient and chemical residue-free microbial agents to replace antibiotics.Probiotics represented by lactic acid bacteria can be used to improve and maintain the ecological balance of the host's intestinal microflora,thus promoting the health of the host organisms.Therefore,they are an ideal alternative to antibiotics.The purpose of this study is to screen out the lactic acid bacteria that can inhibit pathogenic bacteria from the intestine of sea cucumber.To some extent,the study will provide certain resources and basis for the development of China's sea cucumber aquaculture industry.Firstly,Samples from the intestine of the sea cucumber were obtained in the culture circle of the Yellow Sea in Dalian.After enrichment and cultivation of the samples,1397 single colonies were picked on the MRS and SL agar media according to the colony morphology.And the first screening was done by the calcium lysis circle method,and then re-screening was carried out by the acid-resistant and salt-tolerant,which simulated as the sea cucumber intestine,as well as antibacterial experiments.Finally,16 strains with obvious inhibitory effects on pathogenic bacteria were obtained.Furthermore,the antibacterial activities of these strains were measured and compared by using the Oxford cup method.As a result,five strains with strong and stable antibacterial activity were selected,namely HS-R9,HS-R13,HS-R14 and HS-R51.Further analysis was done using molecular biology methods to identify these four strains.Firstly,the genomic DNA of the four strains were extracted and the 16 S rDNA gene sequences were successfully amplified using common bacterial primers,respectively.The sequences were then compared with related sequences in the GenBank database,and the phylogenetic trees were constructed using the Neighbor-Joining method by the MEGA 6.0.The analysis showed that the HS-R9 was highly homologous with Lactobacillus plantarum,the HS-R13 and HS-R14 were highly homologous with Pediococcus lolii and Pediococcusacidilactici,and the HS-R51 was highly homologous with Enterococcus faecium.In this project,the active substances produced by strains HS-R9 and HS-R13 were further studied.By measuring the growth curves of the two strains,it was found that they had the strong inhibitory effect at 40 to 48 hours of fermentation,and the fermentation broth had a broad-spectrum antibacterial activity.The antibacterial active substances in the fermentation supernatants of HS-R9 and HS-R13 strains were extracted by different methods,such as organic solvent extraction,ammonium sulfate precipitation,ultrafiltration and cell adsorption,respectively.The results showed that the organic solvent extraction had the best effect.The active substances extracted from the two strains were determined to be antibacterial proteins according to the antibacterial activity of the acid-expelling test,the hydrogen peroxide-eliminating test,and the protease-sensitive test.Analysis of Tricine-SDS-PAGE electrophoresis revealed that the fermented extracts of strains HS-R9 and HS-R13 had a molecular weight of about 6.5 kDa.For their biological characteristics,the active substances produced by HS-R9 and HS-R13 can maintain high activity after treatment at different temperatures and pH conditions.However,after treatment with pepsin,trypsin,papain,and proteinase K,the activity of the fermented extracts of the two strains decreased to varying degrees,of which trypsin and proteinase K had the most significant effect on the activity of the fermented extracts.This research provides an important theoretical basis for the study and application of marine probiotics and marine organisms for aquatic feeding. |
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