Study On The Structure Identification And Biological Activity Of Prodigiosin,a Secondary Metabolite Of Serratia Marcescens

Prodigiosin is a natural red pigment with anti-cancer,anti-malarial,and antibacterial effects.Due to the complex steps and high cost of chemical synthesis of prodigiosin,biosynthesis of prodigiosin is of great significance.At present,the research on prodigiosin generally focuses on the mechanism,and there are not many studies on the effect on the body's metabolic growth and development.There are no reports on the biological function of the body from the perspective of intestinal microbial composition.Its producing strains are mainly Serratia marcescens,Pseudomonas and some actinomycetes.In this paper,a red pigment-producing strain was selected from the bacterial residue compost,and was determined to be Serratia marcescens through 16 S r DNA molecular biology identification.The red pigment was extracted from the fermentation broth by Ultrasoundassisted acidic methanol.The red pigment is identified by HPLC-MS.The result shows that the red pigment m/z is 324.2160,which is consistent with the prodigiosin standard product.The red pigment was determined to be prodigiosin.It was also found in the study that in addition to prodigiosin,the ZPG19 bacteria can stably produce two other substances with m/z of 272.1995,272.1937.The fermentation medium was optimized by response surface method to obtain the best formula for producing prodigiosin.In order to study the effect of prodigiosin on the body of mice,the crude prodigiosin extract was orally fed to mice for 18 days,and the contents of the cecum were taken for highthroughput sequencing.The main conclusions are as follows:(1)A red pigment-producing strain was selected from the compost of the anaerobic fermentation of Flammulina velutipes which produce biogas.After Gram staining,scanning electron microscopy and 16 S r DNA molecular biology identification,the strain was determined to be Serratia marcescens ZPG19,deposited in CCTCC,deposit number M 2019645.The prodigiosin was extracted from the fermentation broth by ultrasonicassisted acid methanol after the ZPG19 was fermented,and was performed by UV fullband scanning,high-performance liquid chromatography and liquid-mass spectrometry.The results showed that prodigiosin was synthesized during the logarithmic growth period in this study,reaching a maximum value of about 23.67 mg/L at 48 h.,the retention time of high performance liquid chromatography is 4.356 min under acidic conditions,and HPLC-MS shows that m/z is 324.2160,which is consistent with the prodigiosin standard.In addition,we found that ZPG19 can stably produce two other substances in HPLC,with retention times of RT2=5.236 and RT3=6.436,respectively.HPLC-MS indicated that its m/z were 272.1997 and 272.1995.(2)The culture medium for fermenting to produce prodigiosin was optimized by single factor experiment,Plackett-Burman design,the most shaking climbing experiment and the center combination design.The best medium was obtained: wheat flour 59.35 g/L and magnesium sulfate 0.56 g/L,Tween 80 1.61 g/L.Under these conditions,the predicted conversion of the concentration of prodigiosin was 462.47 mg/L,and the verification test showed that the reagent concentration of prodigiosin was 510.35 mg/L,which was 20 times higher than that under the initial medium condition.(3)The Kunming mice were treated with 1 mg/L prodigiosin crude extract for 18 days.The effect of the crude prodigiosin(95% purity)on the internal organs of mice was observed by hematoxylin-eosin staining.The effect of the crude extract of prodigiosin(95% purity)on the intestinal flora of mice was analyzed by high-throughput sequencing.The results show that 1 mg/L prodigiosin has no obvious toxicity to the internal organs of mice and is beneficial to the intestinal flora of mice.(4)Scanning electron microscope was used to observe the inhibitory effect of prodigiosin on Staphylococcus aureus.The Kunming mice were fed with Staphylococcus aureus liquid to get a poisoning mouse.The poisoning mice were treated with different concentrations of prodigiosin for 18 days.The intestinal flora was analyzed for the therapeutic effect of prodigiosin on the poisoning mice.The results showed that prodigiosin can destroy the cell membrane of Staphylococcus aureus,and eventually lead to the fragmentation of the bacteria.Prodigiosin at doses of 90 ?g/kg/d and 150 ?g/kg/d has varying degrees of therapeutic effects on poisoning mice,but both can increase the abundance of beneficial bacteria,reduce the abundance of harmful bacteria,and improve enteritis.