【作者】 李琪；

【导师】 张柏林；

【作者基本信息】 北京林业大学 ， 农业推广硕士（专业学位）， 2020， 硕士

【摘要】 作为药食同源性经济林果,柿子(Diospyros kaki L.f)种植面积大,产量较高,柿果中含有较多的生物活性成分,具有重要的经济利用价值和研究意义。本文以磨盘柿(Diospyros kaki L.cv.Mopan)为原料,开展了柿子多糖提取工艺及多糖结构分析工作;研究了益生菌在含有柿子多糖的MRS培养基上的生长和产酸能力,并用转录组学方法分析了菌株的相关基因,对柿子多糖与益生菌增殖的关系进行了探究;在此基础上,基于小鼠的生长和代谢表现、病理切片、肠道微生物菌群多样性等指标,评价了饲喂不同剂量的柿子多糖对小鼠生长及其肠道菌群的影响,旨在为柿子的精深加工提供理论依据。主要研究结果如下:(1)不同柿子多糖的提取工艺优化及结构分析。通过单因素与正交实验,得到热水浸提法的最佳提取条件:料液比1:3、浸提温度100℃、浸提时间4 h,多糖得率为9.61%。超声辅助法的最佳提取条件:超声时间40 min、浸提温度90℃、浸提时间3 h,多糖得率为12.22%。复合酶法的最佳提取条件:酶解温度45℃、酶解时间120 min、复合酶添加量3%,多糖得率为12.55%。经柿子多糖得率的比较选择复合酶法提取柿子多糖。基于气相色谱-质谱联用(GC-MS)技术研究柿子单糖的组成成分,其多糖中包含了鼠李糖、甘露糖、果糖、葡萄糖、阿拉伯糖、半乳糖、木糖。(2)柿子多糖对益生菌的生长和产酸能力的影响。选择商业用的益生菌嗜酸乳杆菌(Lactobacillus acidophilus)NCFM和6075作为供试材料,在含有柿子多糖(浓度为50%)的改良MRS培养基上,两株菌生长迅速,提前进入对数生长期,菌株NCFM和6075活菌数对数值分别9.527±0.031 CFU/m L和9.453±0.071 CFU/m L。与对照组相比,嗜酸乳杆菌NCFM和6075在改良MRS培养基中产酸能力更强,培养16 h时,菌株NCFM培养基的p H为4.0,培养18 h,菌株6075培养基的p H为4.3。(3)柿子多糖对益生菌生长影响的转录组分析。与对照相比,在柿子多糖存在下,差异分析表明,菌株NCFM实验组相较于对照组总的差异基因中有21个显著,其中上调基因有16个,下调基因有5个;菌株CICC 6075实验组相较于对照组总的差异基因有265个显著,其中上调基因有127个,下调基因有138个。GO富集分析表明,菌株NCFM生物过程(BP)注释到的基因主要富集在一些离子的转运、氨基酸的生物合成及转运、代谢过程中;细胞组分(CC)注释到的基因主要富集在酶复合物及细胞内部的细胞器上;分子功能(MF)注释到的基因主要富集在镁离子的转运和跨膜运输相关酶活。菌株6075生物过程(BP)注释的基因中,上调基因主要富集在氨基酸的生物合成、调节细胞增殖方面;下调基因主要富集在物质代谢;细胞组分(CC)的注释的基因主要富集在核糖体;分子功能(MF)注释到的基因主要富集在结构分子活性及酶活方面。KEGG通路分析表明,菌株NCFM基因中上调通路有2条显著;菌株6075基因中上调通路有3条显著,下调通路有2条显著。这表明,柿子多糖对益生菌生长的影响与菌株的存在密切关联。(4)柿子多糖对小鼠生长的影响。对小鼠进行28天的实验处理,饲喂不同剂量(150 mg/kg BW和450 mg/kg BW)的柿子多糖的小鼠生长表明,摄入柿子多糖不会影响小鼠的体重、摄食和饮水量。小鼠粪便短链有机酸监测表明,摄入柿子多糖150 mg/kg BW时,小鼠粪便中乙酸含量降低,丙酸和丁酸上升,雄性小鼠短链脂肪酸含量上升明显。酶活力监测表明,摄入柿子多糖150 mg/kg BW会促进小鼠T-AOC、CAT活力和T-SOD活力的提高,雄性小鼠更加明显。小鼠结肠切片观察表明,摄入不同剂量的柿子多糖对小鼠的结肠组织没有病理性影响。(5)柿子多糖对小鼠的肠道菌群变化的影响。16S r DNA测序表明,摄入柿子多糖150 mg/kg BW,小鼠肠道菌群共有OTUs增加了8个,特有OTUs增加了30个。小鼠肠道微生物的菌群丰富度改善,?多样性差异小。在门水平上,变形菌门(Proteobacteria)相对丰度从5.5%降低到1.8%,拟杆菌门(Bacteroides)和厚壁菌门(Pachyphyte)在正常范围(30%-70%)内波动。在属水平上,气球菌属(Aerococcus)相对丰度从0.4%降低到0.2%。在种水平上,绿色气球菌(Aerococcus＿viridans)相对丰度从0.4%降低到0.2%,肠道乳酸杆菌(Lactobacillus＿intestinalis)相对丰度从0.3%提高到1.6%,显然,柿子多糖可以提高有益菌的增殖,降低有害菌的丰度,维持小鼠肠道微生物的菌群平衡。本文系统探讨了柿子中多糖提取技术,阐明了柿子多糖成分在改善益生菌与肠道微生物中的作用,这一工作为开发柿子相关产品以及提升柿子的附加值提供了坚实的依据。更多还原

【Abstract】 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.更多还原