공배양 세균의 세포간 교신 기전 연구를 통한 2차 대사산물의 기능성 분석

Abstract

This study was to determine the increase of secondary metabolites production such as astaxanthim by using co-culture method. The optimal culture conditions for Paracoccus sp. were established and Lactobacillus fermentum was selected as the strain to co-culture with Paracoccus sp. In order to optimize the production of astaxanthin in co-culture, various mediums were cultured, and PMF medium was selected. The mass culture process was performed using the optimum production conditions and the optimal astaxanthin production medium. As a result of mass culture, the time to reach maximum yield in the mass culture was almost the same. Cell mass produced by the 5 L fermentor were used for cell experiments and biological activity experiments using fish. Increased production of the secondary metabolite, astaxanthin in co-cultures of Paracoccus sp. and L. fermentum indicates that there was an interaction affecting the production of astaxanthin between the two strains. To determine the communication between the two strains, transcriptome and metabolome analysis of co-culture cells of Paracoccus sp. and L. fermentum was performed. To compare transcriptome and metabolome analysis from co-culture, RNA seq, LC-MS, and CE-MS were performed. Changes in Paracoccus sp. were analyzed by the KEGG database, and up-regulation were related to inositol phosphate metabolism, ABC transporter, and carotenoid biosynthesis pathway. On the other hand, down-regulation were involved in sulfur metabolism. In L. fermentum, up-regulation were involved in thiamine metabolic pathway, purine metabolic pathway, pyrimidine metabolic pathway, amino acid metabolism and ABC transporter, and down-regulation were involved in DNA replication, transcription, translation. When Paracoccus sp. is co-cultured with L. fermentum, it is thought to be supplied with factors necessary for growth and production of astaxanthin while inhibiting the growth of L. fermentum. We confirmed the cytotoxic and anti-inflammatory activity of co-culture extracts of Paracoccus sp. and L. fermentum using the RAW264.7 cell inflammatory model, and animal experiments with mice were performed to evaluate the toxicity of co-culture cell mass. Co-culture cell extract did not show significant toxicity in the cell viability test in normal cells such as RAW264.7, HEK293 and HaCat cells, but down-regulated the production of inflammatory factors in LPS-stimulated RAW264.7 cells and phosphorylation of Akt, p38, and JNK in cell signaling pathways was inhibited. In addition, phosphorylation of IκBα and NF-κB was inhibited. This result indicates that co-culture extracts have anti-inflammatory activity. Animal experiment using mice to confirm the toxicity of co-culture cell mass of Paracoccus sp. and L. fermentum showed no abnormalities in body weight change, mortality and autopsy. Feeding co-culture cell mass as a feed additive to Epinephelus akaara and Pagrus major showed no significant results in growth. As a result of analyzing the color difference in the skin using the color meter, the red color of the skin was higher in the experimental group fed the co-culture cell mass. RNA was extracted from E. akaara and P. major hemocytes and measured for antioxidant activity using real-time qPCR. The RNA levels of CAT, SOD and GPx genes involved in oxidative stress were reduced. These results suggest that oxidative stress was reduced by co-culture cell mass of Paracoccus sp. and L. fermentum. In conclusion, co-culture of Paracoccus sp. and L. fermentum increases the production of astaxanthin, a secondary metabolite, and it confirmed that promotes the growth and astaxanthin production by vitamins, amino acids and bases produced by L. fermentum. Co-culture of Paracoccus sp. and L. fermentum cells with enhanced production of astaxanthin was found to have anti-inflammatory, antioxidant and body color enhancement effects in fish. These studies provide basic research data for increasing astaxanthin production from carotenoid biosynthetic strains and potential as a novel functional feed additive using co-culture cells of Paracoccus sp. and L. fermentum at the same time.