【作者】 李敏；

【导师】 胡冰；

【作者基本信息】 南京农业大学 ， 食品科学与工程， 2021， 硕士

【摘要】 肥胖是威胁全人类健康的公共卫生问题,其与慢性低度炎症密切相关,会引起胰岛素抵抗、2型糖尿病、心血管疾病及癌症等代谢性疾病。因此,预防或治疗肥胖尤为重要。膳食成分干预是预防或治疗肥胖的有效举措,同时可以减少因药物治疗引起的不良反应。表没食子儿茶素-3-没食子酸酯(EGCG)作为绿茶多酚中含量最高且活性最强的儿茶素类化合物,具有良好的抗肥胖作用。但是由于EGCG的稳定性较差且生物利用率较低,限制了其在食品加工、保健和生物医药等众多领域的发展。为了提高EGCG的稳定性而充分发挥EGCG的生理活性,目前基于EGCG的纳米载体递送体系已被广泛研究。淀粉样纤维具有优异的结构特性,可作为生物活性分子的载体用来构建复合纳米材料。且已有研究证实,将EGCG与鸡蛋清溶菌酶(HEWL)淀粉样纤维混合自组装成水凝胶可以提高EGCG的载量,进而提高其稳定性及生物利用率。故本研究将EGCG负载到HEWL淀粉样纤维上,制备成EGCG-HEWL淀粉样纤维复合水凝胶,对其微观结构和流变学进行了分析,并通过构建肥胖小鼠模型研究该水凝胶对高脂饮食小鼠的抗肥胖作用及肠道菌群调节作用。主要研究结果如下:1、EGCG-HEWL淀粉样纤维复合水凝胶的构建与表征将HEWL淀粉样纤维作为模板,加入不同浓度的多酚混合形成多酚-淀粉样纤维复合水凝胶,研究水凝胶的结构特性和流变学性质。结果发现,EGCG、表儿茶素没食子酸酯(EGC)和桑色素(morin)都能够与淀粉样纤维混合形成水凝胶。EGCG和EGC分别与淀粉样纤维混合所形成的水凝胶在偏光条件下检测显示出双折射现象,说明多酚的加入并不会破环淀粉样纤维的结构,依然保留了向列相取向。EGCG-淀粉样纤维复合水凝胶的原子力显微镜(AFM)分析结果显示,EGCG能够不断地富集到淀粉样纤维表面而形成复合纳米丝,且浓度越大所形成的复合纳米丝直径越大。此外,不同浓度的EGCG、EGC和morin分别与不同浓度的淀粉样纤维混合所制备的水凝胶表现出相似的流变学特性。总而言之,在一定浓度范围内,淀粉样纤维浓度一定时,水凝胶强度随着多酚浓度增加而增加;多酚浓度一定时,水凝胶强度随着淀粉样纤维浓度的增加表现为先增加后降低。由此说明水凝胶的形成是多酚与淀粉样纤维相互作用的结果。2、EGCG-HEWL淀粉样纤维复合水凝胶对肥胖小鼠的降脂减肥作用构建高脂饮食诱导的肥胖小鼠模型,研究EGCG-HEWL淀粉样纤维复合水凝胶干预对高脂饮食小鼠的降脂减肥作用。结果表明,水凝胶干预组表现出良好的降脂减肥作用,且其抗肥胖作用主要源于EGCG的抗肥胖活性,淀粉样纤维的作用效果较弱。主要表现为:水凝胶干预显著抑制了高脂饮食引起的小鼠体重增加、脂肪堆积及肝脏质量增加;能够缓解肝脏脂肪变性,缩小附睾脂肪细胞尺寸。同时,水凝胶干预可以显著降低血清中T-CHO和LDL-C含量,降低血清炎症因子TNF-α、IL-1β、IL-6和CRP水平;缓解肝脏过氧化损伤,显著降低肝脏中COMT活性。此外,水凝胶干预可以显著降低肝脏中脂肪合成相关基因LXRα和FAS的表达水平,调节PPARγ、CD36和Scl27a4的表达;显著降低附睾脂肪中炎症相关基因IL-1β、IL-6、TNF-α和TLR4的表达,调节脂代谢相关基因LXRα、LPL、Fiaf、PPARα、UCP-1和CPT-1的表达;显著降低结肠中脂肪吸收相关基因CD36和NFIL3的表达,增加紧密连接蛋白Claudin-1的表达,抑制IL-1β的表达水平。综上可知,水凝胶可调节肝脏、附睾脂肪和结肠组织中脂代谢水平,减轻附睾脂肪组织炎症,提高肠道屏障完整性,进而缓解由高脂饮食引起的体重增加、脂肪肝及慢性组织炎症等相关症状,抵抗高脂饮食诱导的肥胖的发展。3、EGCG-HEWL淀粉样纤维复合水凝胶对肥胖小鼠的肠道菌群调节作用以高脂饮食喂养诱导的肥胖小鼠为模型,研究EGCG-HEWL淀粉样纤维复合水凝胶干预对小鼠肠道微生物的影响。研究发现,水凝胶干预可增加小鼠肠道微生物多样性,并且调节肠道微生物的整体结构;门水平上,显著增加Bacteroidetes的相对丰度而降低Proteobacteria的相对丰度,降低Firmicutes与Bacteroidetes的比值;科水平上,主要降低了在HFD组中显著富集的Desulfovibrionaceae与Deferribacteraceae的相对丰度;在OTUs水平上,逆转了与肥胖表型相关的OTUs,主要抑制了属于Ruminiclostridium＿9、Oscillibacter(Clostridium＿leptum)、Ruminiclostridium＿5(Ruminiclostrium＿sp.＿KB18)、Ruminococcaceae＿UCG-009、Ruminiclostrium、Lachnospiraceae＿NK4A136＿group和Bilophila的OTUs的增殖,促进了属于Alistipes、Parabcteroides(Parabacteroids＿distasonis)、Blautia(Blautia＿coccoide)和Bacteroides(Bacteroides＿uniformis)的OTUs的生长。综上可知,水凝胶可通过调节肠道微生物结构来影响宿主代谢,进而减轻高脂饮食诱导的肥胖。更多还原

【Abstract】 Obesity is a public health problem threatening the health of all human beings.It is closely related to chronic low-grade inflammation and can cause metabolic diseases such as insulin resistance,type 2 diabetes,cardiovascular disease and cancer.Therefore,prevention or treatment of obesity is particularly important.Dietary intervention is an effective measure to prevent or treat obesity,and can reduce the adverse reactions caused by drug therapy.As a catechin compound with the highest content and the strongest activity in green tea polyphenols,(-)epigallocatechin-3-gallate(EGCG)has an excellent anti-obesity effect.However,due to the poor stability and bioavailability of EGCG,its development in many fields such as food processing,health care and biomedicine has been limited.In order to improve the stability of EGCG and give full play to the physiological activity of EGCG,EGCG-based nanocarrier delivery system has been widely studied.Amyloid fibrils have excellent structural properties and can be used as carriers of bioactive molecules to construct nanohybrids.It has been confirmed that the self-assembly of EGCG binding HEWL amyloid fibrils into hydrogels can improve the loading capacities of EGCG,and thus improve its stability and bioavailability.Therefore,in this study,we prepared EGCG-HEWL amyloid fibrils hydrogels and analyzed the microstructure and rheological properties.Furthermore,the anti-obesity effect of the hydrogels on high-fat diet-induced obesity and modulation of gut microbiota were investigated.The main results are as follows:1.Construction and characterization of EGCG-HEWL amyloid fibrils hydrogelsThe HEWL amyloid fibrils were used as a template,and polyphenols of different concentrations were mixed to form polyphenols-HEWL amyloid fibrils hydrogels.The structural characteristics and rheological properties of hydrogels were studied.The results showed that EGCG,Epicatechin gallate(EGC)and morin were mixed with amyloid fibrils to form hydrogels.The hydrogels formed by the mixture of EGCG or EGC with amyloid fibrils respectively showed birefringence under the condition of polarized light,indicating that the addition of polyphenols did not destroy the structure of amyloid fibrils,and the nematic orientation was still retained.The Atomic Force Microscope(AFM)analysis of EGCG-amyloid fibrils hydrogels showed that EGCG could continuously enrich on the surface of amyloid fibrils to form hybrid nanofilaments,and the diameter of the hybrid nanofilaments increased with the increase of EGCG concentration.In addition,the hydrogels prepared by mixing different concentrations of EGCG,EGC and morin with different concentrations of fibrils showed similar rheological properties.In general,within a certain concentration range,when the concentration of amyloid fibrils was constant,the strength of hydrogels increased with increasing polyphenols content.When the concentration of polyphenols was constant,the strength of hydrogels increased firstly and then decreased with the increasing concentration of amyloid fibrils.This indicated that the formation of hydrogels was the result of the interaction between polyphenols and amyloid fibrils.2.The effect of EGCG-HEWL amyloid fibrils hydrogels on lipid reduction and weight loss in obese mice induced by high fat dietTo study the effects of EGCG-HEWL amyloid fibrils hydrogels intervention on lipid reduction and weight loss in mice fed with high fat diet.The results showed that the hydrogels intervention group showed excellent anti-obesity effect,and its anti-obesity effect was mainly due to the anti-obesity activity of EGCG,while the effect of amyloid fibrils was weak.The main results were as follows: the hydrogels intervention significantly inhibited the weight gain,fat accumulation and the increase of liver mass of mice induced by high fat diet,and it could relieve hepatic steatosis and reduce the size of epididymal adipocytes.At the same time,the hydrogels intervention could significantly reduce serum T-CHO and LDL-C content and serum inflammatory cytokines TNF-α,IL-6,IL-1β and CRP levels.Furthermore,it could alleviate liver peroxidation injury and reduce liver COMT activity.In addition,the hydrogels intervention could significantly reduce the expression level of liver lipogenesis-related genes(LXRα and FAS),regulate the expression of PPARγ,CD36 and Scl27a4;and significantly reduce the expression level of the epididymal fat inflammation-related genes(IL-6,IL-1β,TNF-α and TLR4),regulating the expression of lipid metabolism genes(LXRα,LPL,Fiaf,PPARα,UCP1 and CPT-1);and significantly reduce the expression of fat absorption-related genes(CD36 and NFIL3),increase the expression of Claudin-1 and inhibit the expression of IL-1β in the colon.In conclusion,the hydrogels could regulate lipid metabolism in liver,epididymal fat and colon,reduce inflammation of epididymal adipose tissue,improve the integrity of intestinal barrier,and then relieve weight gain,fatty liver and chronic tissue inflammation caused by high fat diet,and eventually resist the development of obesity.3.EGCG-HEWL amyloid fibrils hydrogels alleviate obesity by modulating gut microbiotaThe effect of EGCG-HEWL amyloid fibrils hydrogels on gut microbiota were studied in the model of obese mice fed with high fat diet.It was found that hydrogels intervention could increase the diversity of gut microbiota and regulate the overall structure of gut microbiota in mice.The relative abundance of Bacteroidetes were significantly increased,while the relative abundance of Proteobacteria and the ratio of Firmicutes to Bacteroidetes were decreased.At the family level,the relative abundance of Desulfovibrionaceae and Deferribacteraceae enriched in the HFD group were reduced significantly.Moreover,OTUs associated with obesity phenotypes were reversed by hydrogels intervention,maily inhibiting the growth of Ruminiclostridium＿9,Oscillibacter(Clostridium＿leptum),Ruminiclostridium＿5(Ruminiclostrium＿sp.＿KB18),Ruminococcace UCG-009,Bilophila Ruminiclostrium,and Lachnospiraceae＿NK4A136＿group,while promoting the growth of Alistipes,Parabcteroides(Parabacteroids＿distasonis),Blautia(Blautia＿coccoide)and Bacteroides(Bacteroides＿uniformis).In conclusion,hydrogels could affect host metabolism by regulating gut microbiota structure,and thus play a key role in preventing or alleviating obesity.更多还原