【作者】 王娜；

【导师】 赵智慧； 刘孟军； 贾彦丽；

【作者基本信息】 河北农业大学 ， 农业硕士（专业学位）， 2022， 硕士

【摘要】 肠道菌群的作用主要是维持肠道微生态的稳定性,据报道天然多糖具有调节人体肠道菌群及维护肠道健康。课题组前期研究发现,枣果实中酸性多糖分子具有免疫促进作用。为研究枣酸性多糖对人体粪便菌群的影响,本试验以河北省沧县金丝小枣为原料,通过响应面法优化碱提金丝小枣多糖的提取工艺,并探究不同分子量酸性多糖的理化性质、结构表征及体外酵解过程中对肠道菌群结构的影响,为进一步明确碱提金丝小枣多糖的益生元作用,开发枣多糖功能性食品及研究碱提枣多糖的构效关系提供参考依据。1.碱提金丝小枣多糖的制备:通过单因素和响应面试验得到碱提多糖的最佳提取工艺为:提取温度80℃、料液比1:35(g/mL)、提取时间120 min、NaOH浓度为0.2 mol/L,多糖得率达到11.44%。2.碱提金丝小枣多糖结构分析:利用超滤膜分离技术获得两种不同分子量的酸性多糖AJP1和AJP2组分。并采用紫外扫描光谱、高效液相色谱、气相色谱-质谱联用仪(GC-MS)、SEM扫描电镜及红外光谱分析了碱提多糖的纯度、分子量、单糖组成、结构表征及糖苷键的类型。结果表明:AJP1和AJP2在260 nm～280 nm之间出现特征吸收峰,表明碱提金丝小枣多糖中含有蛋白质物质。AJP1分子量为75465 Da,主要是由鼠李糖、阿拉伯糖、甘露糖、木糖、半乳糖和葡萄糖组成,其摩尔比为0.1:0.31:0.36:0.16:0.01:0.20。AJP2分子量为268678 Da,主要由鼠李糖、阿拉伯糖、甘露糖、核糖、木糖、半乳糖和葡萄糖组成,摩尔比分别为0.78:1.11:1.36:0.13:0.25:0.51:0.18,碱提金丝小枣多糖均为杂多糖。在SEM扫描电镜下观察到AJP1和AJP2多糖具有不同的形态学特征,在放大1000倍和2000倍下,AJP1多糖表面相对粗糙、疏松多孔,多糖更容易与水结合增大其表面积,提高溶解性;AJP2多糖内部紧实,呈片状。红外光谱显示,两组碱提多糖均具有C=O、C-O和C-H等酸性多糖的特征吸收峰,表明碱提金丝小枣多糖是含有糖醛酸的吡喃型酸性多糖。3.体外抗氧化活性试验表明:碱提多糖具有一定的清除自由基活性,AJP1对DPPH、羟基和超氧阴离子自由基的IC50分别为1.754 mg/mL、1.755 mg/mL、2.884 mg/mL;AJP2对DPPH、羟基和超氧阴离子自由基的IC50分别到达1.057 mg/mL、1.050 mg/mL、4.195 mg/mL。4.体外发酵法研究碱提金丝小枣多糖对人体粪便菌群的影响:通过分析体外消化及酵解过程中菌液浓度、pH值及总糖消耗率的变化,结果表明:在体外酵解48 h后,添加多糖组的细菌浓度均高于空白组;在产酸方面,AJP1和AJP2可显著降低pH,酸性环境中更有利于益生菌的生长;AJP1和AJP2在体外酵解48 h后总糖消耗率分别达到65.40%和56.24%。通过16S rRNA高通量测序技术分析体外消化酵解过程中肠道菌群的结构变化:枣多糖提取物在体外酵解过程逐步被肠道细菌降解和利用。在体外酵解48 h后,添加多糖组和空白组的肠道菌群结构存在差异;菌群多样性结果表明:碱提金丝小枣多糖具有显著(P<0.05)促进有益微生物巨单胞菌属(Megamonas)、阿里松氏菌属(Allisonella)等相对丰度的增加,同时显著(P<0.05)降低有害微生物嗜胨菌属(Peptoniphilus)、嗜胆菌属(Bilophila)及脱硫弧菌属(Desulfovibrio)的数量,枣多糖提取物能通过调节人体肠道菌群成为人类健康和疾病预防的益生元多糖。更多还原

【Abstract】 The role of intestinal flora is mainly to maintain the stability of intestinal microecology.It is reported that natural polysaccharides can regulate human intestinal flora and maintain intestinal health.Our previous research revealed that the acid polysaccharides in the jujube fruit had immune promotion effect.In order to study the effect of acid polysaccharide from jujube on human fecal flora,’Jinsixiaozao’ fruits from Cang County,Hebei Province were used as the raw material and we optimized the extraction process of alkali extracted polysaccharide by response surface methodology,and studied the physical and chemical properties,structural characterization of acid polysaccharides with different molecular weights,and their impact on intestinal flora structure during in vitro fermentation.This study will provide a reference for further clarifying the prebiotic effect of alkali extracted polysaccharides from jujube,developing functional food of jujube polysaccharide and studying the structure-activity relationship of alkali extracted polysaccharides.1.The optimum extraction conditions of alkali extracted jujube polysaccharide were obtained by single factor tests and response surface methodology as extraction temperature 80℃,material liquid ratio 1:35(g/mL),extraction time 120 min,NaOH concentration 0.2 mol/L.Under these conditions,the yield of polysaccharide was 11.44%.2.Two acidic polysaccharides AJP1 and AJP2 with different molecular weights were obtained by ultrafiltration membrane separation technology.The purity,molecular weight,monosaccharide composition,structural characterization and glycosidic bond types were analyzed by ultraviolet scanning spectrum,high performance liquid chromatography,gas chromatography-mass spectrometry(GC-MS),SEM scanning electron microscope and infrared spectroscopy,respectively.The absorption peaks between 260 nm～280 nm indicated that the polysaccharides contained protein substances.AJP1 had a molecular weight of 75465 Da and was mainly composed of rhamnose,arabinose,mannose,galactose and glucose with the molar ratio of 0.1:0.31:0.36:0.16:0.01:0.20.The molecular weight of AJP2 was 268678 Da,mainly composed of rhamnose,arabinose,mannose,xylose,galactose and glucose with molar ratios of 0.78:1.1 1:1.36:0.13:0.25:0.51:0.18,respectively.The morphological characteristics of AJP1 and AJP2 were different under SEM.Under the magnification of 1000× and 2000×,the surface of AJP1 was relatively rough,loose and porous,and could combine with water more easily to increase its surface area and improve solubility,whereas the interior of AJP2 was firm and flaky.The infrared spectra showed that these two alkali-extracted polysaccharides had the characteristic absorption peaks of acidic polysaccharides such as C=O,C-O and C-H.The results indicated that the alkaliextracted polysaccharide was acid polysaccharides of pyran-type.3.In vitro antioxidant activity test showed that the alkali extracted polysaccharide had certain free radical scavenging activity.The IC50 of AJP1 on DPPH,hydroxyl and superoxide anion radicals were 1.754 mg/mL,1.755mg/mL and 2.884 mg/mL,respectively;The IC50 of AJP2 to DPPH,hydroxyl and superoxide anion radicals reached 1.057 mg/mL,1.050 mg/mL and 4.195 mg/mL respectively.4.In vitro fermentation method was used to study the effect of alkali extracted polysaccharides from jujube on human fecal flora by analyzing the changes of bacterial concentration,pH value and total sugar consumption rate in the process of digestion and fermentation.The results showed that after 48 h of in vitro fermentation,the bacterial concentration in polysaccharides was higher than that in the blank group.In terms of acid production,AJP1 and AJP2 could significantly reduce the pH,and the acidic environment was more favorable for the growth of probiotic bacteria.After 48 h fermentation,the total sugar consumption rates of AJP1 and AJP2 reached 65.40%and 56.24%,respectively.The 16S rRNA high-throughput sequencing technology was used to analyze the structural changes of intestinal flora in the process of in vitro digestion and fermentation.The alkali extracted polysaccharides were gradually degraded and utilized by intestinal bacteria in the process of in vitro fermentation.After 48 h in vitro fermentation,the intestinal flora compositions of the treatment group and control group were different.The results of microbial diversity showed that the polysaccharides could significantly increase the relative abundance of beneficial microorganisms such as Megamonas and Allisonella,and meanwhile significantly reduce the number of harmful microorganisms such as Peptoniphilus,Bilophila and Desulfovibrio.Alkali extracted polysaccharides from jujube can regulate human intestinal flora,and has the potential to become a prebiotic product for promoting human health and preventing diseases.更多还原