| As one of the most non-wood trees,persimmon fruit(Diospyros kaki L.f)has been consumed for a long time in China as well as the world.This tree has important economic values because it yields more fruits which are rich in active ingredients.In this work,Diospyros kaki L.cv.Mopan,one of variants that widely is grown in the north part of China,was used as test material for the extraction and structure analysis of persimmon polysaccharides.The growth and acid-producing ability of selected probiotci strains on persimmon polysaccharides-containing MRS medium were investigated.Transcriptomics was used to analyze the differentially expressed genes(DEGs)of the selected strains in the presence or absence of persimmon polysaccharides.Moreover,the growth and metabolic performance,pathological observation,and intestinal microbial flora diversity of mice were evaluated after different dosages of persimmon polysaccharides were orally administrated to mice.This study was designed to provide a theoretical basis for the fine processing of persimmon fruits in terms of the relationship between polysaccharides and intestinal microbes.The following are main findings.1.The optimal extraction and structure analysis of persimmon polysaccharides.Factors affecting the extraction of persimmon polysaccharides were investgated.The optimal extraction parameters of persimmon polysaccharides by hot water extraction method were 1: 3 ratio of material to liquid at 100 ? for 4 h extraction time.In this case,the yield of polysaccharide was 9.61%.The optimal extraction conditions of persimmon polysaccharides by ultrasonic-assisted method were ultrasonic time of 40 min,extraction temperature of 90? and extraction time of 3 h,in which the polysaccharide yield was 12.22%.The optimal extraction conditions of persimmon polysaccharides bycompound enzymatic method were hydrolysis at 45?C with 120 min and 3% compound enzyme.In this case,yield of the polysaccharide was 12.55%.Among the three tools used for extracting persimmon polysaccharides the compound enzymatic method was chosen for further study because of high sugar yield.It was seen that persimmon polysaccharides mainly consists of rhamnose,fructose,arabinose,xylose,glucose,galactose,and mannose according to gas chromatography-mass spectrometry(GC-MS).2.Effect of persimmon polysaccharide on the growth of selected probiotic bacteria.Two commercial probiotics strains Lactobacillus acidophilus NCFM and 6075 were selected to deal with the possible roles of persimmon polysaccharides in favoring the bacterial growth.It was seen that two strains incubated in modified MRS medium containing 50% persimmon polysaccharide grew rapidly and entered the logarithm stage of their growth quickly.The viable bacterial counts of two strains NCFM and 6075 were 9.527 ± 0.031 and 9.453 ± 0.071 CFU/m L(calculated in logarithm),respectively.Compared with the control,Lactobacillus acidophilus NCFM and 6075 had stronger acid-producing ability in the modified MRS medium.p H of the medium was 4.0 for strain NCFM after incubated for 16 hours,and 4.3 for strain 6075 after incubated for 18 h.3.Transcriptomic analysis into the effect of persimmon polysaccharide on the growth of probiotics.Compared with the control,there were 21 differential genes(DEGs)expressed significantly in strain NCFM treated group in the presence of persimmon polysaccharide,of which 16 were up-regulated genes and 5 were down-regulated genes.For Lactobacillus acidophilus strain 6075,265 differential genes were expressed significantly in the experimental group,in which 127 genes were up-regulated,and 138 genes were down-regulated compared with the control group.GO enrichment analysis showed that DEGs annotated to biological process(BP)for strain NCFM are mainly responsible for the transport of some ions,the biosynthesis and transport of amino acids,and the metabolic process.DEGs annotated to cell component(CC)are mainly involved in organelles and enzyme complexes in the cell.Genes annotated to molecular function(MF)are mainly responsible for enzyme activities related to transmembrane transport and magnesium ion transport.Among the genes annotated to biological process(BP)for strain 6075,the up-regulated genes are mainly involved in amino acid biosynthesis and regulating cell proliferation while the down-regulated genes are mainly responsible for material metabolism.The genes annotated to cell component(CC)are mainly enrichedin ribosome;and genes annotated to molecular function(MF)are mainly responsible for structural molecular activities and enzyme activities.Analysis of KEGG showed that there were two significant up-regulation pathways in strain NCFM genes.Three significant up-regulation pathways and two significant down-regulation pathways took place in strain 6075 genes.These data proved that the presence of persimmon polysaccharides greatly favors the growth of probiotics.4.Effect of persimmon polysaccharide on mouse body.Mice were treated experimentally for 28 days.The growth of mice fed with two dosages of persimmon polysaccharides(150 mg/kg BW and 450mg/kg BW)was observed.It was shown that oral consumption of persimmon polysaccharides did not affect the body weight,food intake and water drinking of mice.Monitoring short-chain organic acids of treated mouse feces showed that when 150 mg/kg BW of persimmon polysaccharide was ingested,acetic acid content in the mice feces decreased but propionic acid and butyric acid increased.Detection of enzyme activity showed that consumption of 150 mg/kg BW persimmon polysaccharide could promote the activity of the enzymes T-AOC,CAT and T-SOD in the tested mice,especially obvious for male mice.It was observed that the consumption of persimmon polysaccharides had not any pathological effects on the colon tissues of the tested mice.5.Effect of persimmon polysaccharide on intestinal flora of mice.16 S r DNA sequencing showed that oral adminstration of 150 mg/kg BW persimmon polysaccharides increased the total intestinal flora by 8,and increased the numbers of unique OTUs by 30.The intestinal microbiota richness of mice was improved,and small differences in ? diversity between the treated and control groups was observed.As the phylum of intestinal microbiota of treated mice was concerned,the relative abundance of Proteobacteria was reduced from 5.5% to1.8%,and Bacteroides and fermicutes remained within the normal range of 30% to 70%.Regarding the genus,the relative abundance of Aerococcus was reduced from 0.4% to 0.2%.Considering the species,the relative abundance of Aerococcus viridans was reduced from 0.4% to 0.2%,but Lactobacillus(intestinalis Lactobacillus)was greatly increased from 0.3% to 1.6%.Obviously,consumption of persimmon polysaccharide increases significantly the proliferation of intestinal probiotics,and reduces the abundance of harmful bacteria,maintaining the healthy environments of intestinal microflora. |
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