| Objective:Through purification,identification and biomass determination,the basic characteristics of Rhodopseudomonas palustris were systematically verified.Bacteriostatic test,artificial simulation of the survival rate of gastrointestinal fluid and cell adhesion test were used to evaluate the probiotics characteristics of R.palustris.Through the detection of gastrointestinal regulatory peptides in the serum of mice,the analysis of intestinal morphology,the high-flux sequencing of intestinal microorganisms in the feces,and the detection of short-chain fatty acids by gas chromatography,the mechanism of R.palustris as probiotics was explored.This study laid the foundation for realizing the application of R.palustris as probiotics in clinical practice.Methods:Experiment 1:The basic characteristics of the strain were systematically verified by PCR amplification gene sequencing,Gram staining,full-wavelength scanning,growth curve determination,and the corresponding relationship between the results of spectrophotometry and plate counting method.Experiment 2:The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of the strain were determined by means of filter paper method and enzyme-plate method.The survival rate of intestinal fluid and the cell adhesion of human colorectal adenocarcinoma cell(Caco-2)were simulated in vitro to determine whether the strain had probiotic characteristics.Experiment 3:The body weight,diet and water intake of mice were measured every 2days to monitor the health status of mice.The effects of erythromonas swamp on the gastrointestinal related indexes of healthy mice were investigated by detecting the gastrointestinal regulating peptide gastrin(GAS)and somatostatin(SS)in serum,detecting the intestinal morphology of colon and ileum,and measuring the villus height of ileum.High-throughput sequencing of intestinal microorganisms in mouse feces were conducted to explore the effects on intestinal microorganisms in healthy mice.Results:Experiment 1:Cluster analysis showed that the strain had the closest genetic relationship with R.palustris(sequence number:AF184625.1).In the measured growth curve,R.palustris 24 h entered the logarithmic growth phase,and 72 h entered the stable phase.The linear relationship between the concentration(y)and the absorbance value(x)was as follows:y(10~8CFU/mL)=29.123x-1.2844(R~2=0.9999).Experiment 2:The strain could tolerate for 3 h in artificial simulated gastric juice(p H3.0)and the survival rate was greater than 50%.The strain could tolerate 8 h in artificial simulated intestinal fluid(p H8.0),and the survival rate reached great than70%.When the sample concentration was 5×10~9CFU/mL,the inhibitory zone against Escherichia coli reached 10.40 mm,the MIC value was 1.25×10~9CFU/mL,and the MBC value was 2.5×10~9CFU/mL.The inhibitory zone against Bacillus subtilis was 14.70 mm,the MIC value was 1.25×10~9CFU/mL,and the MBC value was 2.5×10~9CFU/mL.The adhesion rate to Caco-2 was 160 cells.Experiment 3:By monitoring the health indicators of mice,the results showed that the body weight trend of blank group,low-dose group and medium-dose group was highly similar within 4 weeks.The body weight of high-dose group was lower than that of the other three groups from 14 d,and significantly lower than that of blank group from 26 d.At the same time,the weight gain of the medium dose group and the low dose group was larger than that of the blank group,while the increase value of the high dose group was smaller.The food intake of the high-dose group from day 14 was lower than that of the other three groups.The amount of water in the four groups was basically the same.Low dose group had a significant effect on the secretion of GAS(P<0.01),medium dose group and high dose group also had a promoting effect on the secretion of GAS.High dose group had significant inhibitory effect on SS secretion(P<0.05),low dose group and medium dose group also had inhibitory effect on SS secretion.Compared with the blank group,the height of villus in ileum of experimental group was slightly higher(P>0.05).After 4 weeks of strain intervention,the relative abundance of Bacteroidetes in the experimental group decreased significantly at the phylum level.The abundance of Firmicutes and Cyanobacteria increased observably.At the family level,the abundance of Muribaculaceae decreased observably.The relative abundance of Lachnospiraceae,Prevotellaceae,Rikenellaceae,Lactobacillaceae,Bacteroidaceae and Ruminococcaceae increased significantly.At the genus level,the relative abundance of Lachnospiraceae NK4A136 group,Lactobacillus,Bacteroidetes,Alistipes and Prevotellaceae UCG-001increased significantly.Conclusion:The logarithmic growth period of R.palustris was 24-72 h.The linear relationship between spectrophotometric method and plate counting method was obvious in the determination of bacterial liquid concentration.It has strong antibacterial ability,acid resistance and bile salt resistance,and has a strong adhesion ability,with probiotic properties.It can maintain the gastrointestinal hormone homeostasis and intestinal morphology structure;To some extent,the relative abundance of beneficial bacteria in the intestines was increased by improving the composition of intestinal microflora of healthy mice,R.palustris played a probiotic role. |
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