【作者】 王海松；

【导师】 乐国伟；

【作者基本信息】 江南大学 ， 食品营养与安全， 2015， 博士

【摘要】 类虫草多糖巨寡糖具有良好的生理功能。而微波在有机合成反应中表现出快速、高效、低能耗、环境友好等特点。因此本文应用微波辅助、杂多酸催化技术快速制备类虫草多糖巨寡糖;并对巨寡糖的结构进行解析,明确巨寡糖的结构特征,推导巨寡糖的合成机理;然后通过体外发酵实验研究类虫草多糖巨寡糖对肠道菌群的影响;并进一步研究类虫草多糖巨寡糖对长期高脂膳食诱导小鼠肥胖的影响,论文的主要内容包括:1.类虫草多糖巨寡糖的合成及结构研究聚葡萄糖(PDX)的平均聚合度(DP)为13,是以葡萄糖和山梨醇(摩尔比8.9:1)为底物、水为引发剂(12%,v/w)、杂多酸(1.0%,v/w)为催化剂、120oC微波辐照2.5 min合成,葡萄糖转化率为92.31%。PDX含有T-b-D-Glcp,→2)-b-D-Glcp,→6)-a-D-Glcp,→4)-a-D-Glcp和T-a-D-Glcp片段,在O-2,O-4,O-6位产生分支。聚半乳糖(PGal)的DP为17,是以半乳糖为底物、0.25 mol/L氯化钠溶液为引发剂、1.1%(v/w)杂多酸为催化剂、130oC微波辐照4.5 min合成,半乳糖转化率为97.22%。PGal含有→6)-b-D-Galp,→4)-b-D-Galp,→4)-a-D-Galp,→2,4)-a-D-Galp和T-a-D-Galp片段,在O-2,O-4,O-6位产生分支。聚甘露糖(PMan)的DP为15,是以甘露糖为底物、2.5 mol/L杂多酸催为催化剂、115oC微波辐照4.5 min合成,甘露糖转化率为89.42%。PMan含有→3)-b-D-Manp,→6)-a-D-Glcp和→3,6)-a-D-Glcp片段,在O-3,O-6位产生分支。以甘露糖为底物制备的不同DP的聚甘露糖,其DP分别为:15,27和36。类虫草多糖巨寡糖(CM)的DP为14,是以葡萄糖、半乳糖和甘露糖为底物,杂多酸(1.2%,v/w)为催化剂,120oC微波辐照3.5 min合成,单糖转化率为87.79%。CM单糖摩尔比为葡萄糖:半乳糖:甘露糖=1.93:1:1.73。CM含有→4)-b-D-Galp,→2,6)-b-D-Galp,→,2)-a-D-Galp,T-b-D-Glcp,T-a-D-Glcp,→6)-a-D-Glcp,→3)-b-D-Manp和T-a-D-Manp片段,在O-2,O-6位产生分支。2.类虫草多糖巨寡糖对肠道菌群的影响体外消化实验表明,巨寡糖不被胃液和小肠液水解。体外发酵实验表明,巨寡糖可降低发酵液p H,增加乳酸及短链脂肪酸含量。限制性末端酶切(T-RFLP)研究发现,巨寡糖降低了肠道菌群多态性,并促进优势细菌出现。RT-PCR研究表明CM促进双歧杆菌和乳杆菌增殖、抑制肠杆菌生长的效果优于PDX、PGal和PMan。CM产生短链脂肪酸的量高于PDX、PGal和PMan。巨寡糖显著增加长期高脂日粮小鼠肠道Bif164、Lab158、Bac303、Mib724的含量,对双歧杆菌(Bif164)的作用效果为CM>PMan>PDX>PGal,对乳酸菌(Lab158)的作用效果为PDX>CM>PMan>PGal。巨寡糖显著促进小鼠肠道乳杆菌和双歧杆菌的增殖、抑制肠杆菌的生长。显著增加与肥胖相关的拟杆菌与厚壁菌的比值。3.类虫草多糖巨寡糖对长期高脂日粮小鼠肥胖的影响巨寡糖显著抑制长期高脂日粮小鼠体重的增加。显著降低高脂日粮小鼠Lee指数、脂肪指数和肝脏指数。显著降低血液内毒素水平、血浆TG和LDL-C水平,PDX、PMan和CM能够显著降低血浆TC水平。巨寡糖可以调节机体氧化还原状态,降低机体自由基(ROS)和脂质过氧化物(MDA)水平,增强机体总抗氧化能力(T-AOC),提高过氧化氢酶(CAT)表达。对肠道氧化还原状态及抗氧化基因表达研究发现,巨寡糖显著降低高脂膳食诱导的回肠和结肠的ROS水平,减少MDA含量,增强T-AOC,及抗氧化酶CAT及GSH/GSSG的活性。其中CM在降低ROS水平,减少MDA含量,增强T-AOC及抗氧化酶活性均显著高于PDX、PGal和PMan,高脂膳食中添加巨寡糖后,结肠Nrf2基因表达显著上调。对肠道炎性细胞因子及炎性相关基因表达的研究发现,巨寡糖显著降低了高脂膳食小鼠肠道TNF-a水平,显著提高了IL-10的水平。其中PMan和CM显著降低了肠道IL-6的水平。高脂膳食添加巨寡糖后结肠IL-6和TNF-a的基因表达显著下调,IL-10的基因表达显著上调,IL-12的基因表达下调,其中仅为添加CM的高脂膳食组小鼠结肠IL-12的基因表达下调显著。组织形态学研究发现,巨寡糖显著增强高脂膳食小鼠结肠的肌层和粘液层厚度。对肠道紧密连接蛋白相关基因表达的研究发现,巨寡糖显著上调高脂膳食小鼠结肠上皮细胞紧密连接相关蛋白Claudin-1、JAM和Zo-1的基因表达。而对于Occludin的基因表达仅在高脂膳食中添加CM后才显著上调。对肝脏脂肪酸代谢的研究发现,巨寡糖显著降低了长期高脂膳食小鼠肝细胞脂肪浸润。显著上调小鼠肝脏Nrf2的基因表达,提高肝脏抗氧化能力。显著上调高脂膳食小鼠肝脏Cyp7al的基因表达,促进肝脏脂肪酸分解。下调高脂膳食小鼠肝脏Lpl的基因表达,并显著下调Scd1的基因表达。综上所述,葡萄糖、半乳糖、甘露糖,在微波辅助、杂多酸催化条件下,控制合成反应温度,可制备多分支结构的PDX、PGal和PMan,以及具有类虫草多糖结构的巨寡糖。研究表明,四种巨寡糖不被胃液和小肠液水解;在结肠调节肠道菌群并发酵产生乳酸和SCFA,降低肠道p H;四种巨寡糖均可促进双歧杆菌和乳杆菌增殖、抑制肠杆菌生长;SCFA中的丁酸被肠道上皮细胞利用增强了肠道屏障功能;巨寡糖抑制肠杆菌等革兰氏阴性菌生长,降低血液内毒素水平,从而抑制全身慢性炎性反应,并进一步改善机体氧化还原状态;丙酸通过抑制肝脏脂肪酸合成,从而降低肝细胞脂肪浸润,进一步抑制由于长期高脂膳食诱导的小鼠肥胖发生。更多还原

【Abstract】 Cordyceps Megalosaccharide(CM) has good physiological function. Microwave has been widely used in organic synthesis due to its rapid, high efficient, lower energy consumption, and environment friendly features. Therefore, we used heteropolyacid as catalyst under the condition of microwave irradiation to rapidly and high efficiently synthesize CM. Then we studied the structure of megalosaccharides synthesized by microwave irradiation, and inferred the formation of glycosidic bond in the chemical reaction. Furthermore, we studied the effect of CM on gut microbiota and its inhibition of obesity on long-term high-fat diet fed mice. The main results of this study are as follow: 1. Preparation of Cordyceps Megalosaccahrides and its structure analysisPolydextrose(PDX) was synthesized using glucose and sorbitol(molar ratio 8.9:1) as substrate, and water as initiator, heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 120 oC, microwave irradiation time 2.5 min, the amount of initiator and catalyst were 12% and 1.0%(v/w), respectively. The actual maximum yield was 92.31%. The average DP was 13. The results of methylation analysis and NMR spectra assignments indicated that the structure of PDX had a branched backbone composed of T-b-D-Glcp, →2)-b-D-Glcp, →6)-a-D-Glcp, →4)-a-Glcp, and T-a-D-Glcp, the backbone were branched at the O-2, O-4, O-6 position.Poly-Galactose(PGal) was synthesized using galactose as substrate, sodium chloride solution as initiator, and heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 130 oC, microwave irradiation time 4.5 min, initiator concentration 0.25 mol/L, and catalyst 1.1%(v/w). The actual maximum yield was 97.22%. The average DP was 17. The structure of PGal had a branched backbone composed of →6)-b-D-Galp,→4)-b-D-Galp,→4)-a-D-Galp,→2,4)-a-D-Galp, and T-a-D-Galp,the backbone of PGal were branched at the O-2, O-4, O-6 position.Poly-Mannose(PMan) was synthesized under the condition of reaction temperature 115 oC, microwave irradiation time 4.5 min, and catalyst concentration 2.5 mol/L. The actual maximum yield was 89.42%. The average DP was 15. The structure of PMan had a branched backbone composed of →3)-b-D-Manp, →6)-a-D-Glcp, and →3,6)-a-Glcp,and PMan backbone were branched at the O-3, O-6 position. Using mannose as substract we synthesized PMan with the DP of 15, 27 and 36, respectively.Cordyceps Megalosaccharide(CM) was synthesized using glucose, galactose, and mannose as substrate, heteropolyacid as catalyst, under the condition of microwave irradiation. The optimum conditions were reaction temperature 120 oC, microwave irradiation time 3.5 min, the amount of catalyst 1.2%(v/w). The actual maximum yield was 87.79%. The average DP was 14. The structure of CM had a branched backbone composed of →4)-b-D-Galp,→2,6)-b-D-Galp,→,2)-a-D-Galp,T-b-D-Glcp,T-a-D-Glcp,→6)-a-D-Glcp,→3)-b-D-Manp, and T-a-D-Manp,the main chain were branched at the O-2, O-6 position. 2. Effect of Cordyceps Megalosaccharide on the gut microbiotaMegalosaccharides reduced the p H of fermentation liquid, and increase lactate acid and the amount of SCFA. The Terminal-restriction fragment length polymorphism(T-RFLP) analysis of gut microbiota in SHIME showed that megalosaccharides reduced the polymorphism of microbiota and promote advantage bacteria proliferation. RT-PCR studied showed that CM is better in proliferation of Bifidobacteria spp and Lactobacteria spp, and inhabitation of Enterobacteria spp compared with PDX, PGal, and PMan. Megalosaccharides fermentated by gut microbiota produced different amount of SCFA, CM produced much more SCFA than PGal, PMan and PDX.Fluorescence in situ hybridization(FISH) combined with flow cytometry(FCM) analysis the gut microbiota showed that megalosaccharides can significant increase the relative abundance of Bif164、Lab158、Bac303、Mib724, the effect of megalosaccharides on Bif164 was CM>PMan>PDX>PGal, and Lab158 was PDX>CM>PMan>PGal. Megalosaccharides significantly increased the ratio of Firmcuties to Bacterodies in HFD fed mice. 3. Effect of Cordyceps Megalosaccharide on HFD induced obese miceIn this study, megalosaccharides significantly reduced body weight(BW), Lee obesity index, fat to BW, and liver to BW of mice fed with HFD. It significantly reduced blood LPS, TG, and LDL-C level coampared with HFD fed mice. PDX, PMan, and CM significantly reduced blood TC level of HFD fed mice. Megalosaccharides can modulate the redox state of mice fed HDF, decrease ROS and MDA level, and improve total antioxidant capacity(TAOC) and antioxidant enzyme secretion.The effect of megalosaccharides on gut functional study showed that megalosaccharides decreased ileum and colon ROS and MDA level, improved the T-AOC, and increased CAT and GSH/GSSG activity. CM has higher ability than PDX, PGal and PMan in decrease ROS and MDA level, and improves T-AOC. Additionaly, megalosaccharides significantly up regulated the Nrf2 gene expression in HFD fed mice.Studied on the inflammatory cytokines and its relative gene expression showed that megalosaccharides significantly decrease TNF-a level and increased IL-10 level in HFD fed mice. PMan and CM significantly decreased the colon IL-6 and TNF-a level. Megalosaccharides significantly down regulate colon IL-6 and TNF-a relative gene expression, and significantly up regulate IL-10 gene expression. Althought megalosaccharides down regulated IL-12 gene expression, only CM significantly down regulted IL-12 gene expression compared with HFD fed mice. Furthermore, megalosaccharides significantly improved the thickness of muscular and mucous layers in HFD fed mice.Gut tight junction(TJ) protein relative gene expression analysis showed that megalosaccharide significantly up regulated the gene expression of Claudin-1, JAM, and Zo-1. But only CM up regulated Occludin gene expression in HFD fed mice.Live fatty acid metabolism study showd that megalosaccharides significantly decreased hepar cell fatty infiltration, up regulated liver lipolysis gene Cyp7 al expression, and down regulated LPl and SCD-1 gene expression. In addition, megalosaccharides up reglulated antioxidant gene Nrf2 expression in liver compared with HFD fed mice.In summary, PDX 、 PGal 、 PMan and CM were synthesized under the condition of microwave-assisted and heteropolyacid catalyst. The megalosaccharides were anti-digested in stomach and small intestinal, and reached to the colon, then fermented by gut microbiota to produce lactate and SCFA, which reduced the colon p H. The four megalosaccharides proliferated Bifidobacteria spp and Lactobacteria spp, and inhibitd Enterobacteria spp. Furthermore, the metabolites of megalosaccharides could regulate the body redox states, decrease gut inflammatory, improve gut barrier function, and reduce blood LPS level and heper cell fatty infiltration. Accordingly, megalosacchardes could alleviate obesity which was induced by long-term HFD.更多还原