【作者】 骞宇；

【导师】 阚建全；

【作者基本信息】 西南大学 ， 食品科学， 2013， 博士

【摘要】 抗消化淀粉(resistant starch, RS)作为低热量淀粉添加到食物中,对机体产生的生理功能与膳食纤维类似：控制体重,预防便秘、结直肠癌等肠道疾病,改善肠道菌群,降低血脂血糖等。但RS具有比膳食纤维更加优质的加工特性,近来被广泛添加到各类食品中。不同种类的RS在机体肠道中的发酵模式是不同的,可能对机体肠道生理环境和胃肠功能性作用的影响也不相同。国内外关于不同种类和含量的RS对机体肠道生理环境和胃肠功能性作用比较的研究成果较少。本研究以不同种类(RS2、RS3、RS4)和含量(5%、10%、15%)的RS为研究对象,并建立大鼠、小鼠的病理模型进行动物实验,研究分析RS对机体肠道生理环境和胃肠功能性作用的影响。为以后进一步研究RS对机体肠道健康作用机制提供实验数据。本文主要研究成果如下：(1)不同种类(RS2、RS3、RS4)和含量(5%、10%、15%)的RS对健康SD大鼠肠道代谢产物和血脂影响的研究结果表明：RS组均能增加大鼠的摄食量,减少体重增加量和饲料效率；均能使大鼠盲肠内容物和粪便湿润变软；降低盲肠内容物和粪便的pH值、游离氨含量；增加盲肠内容物和粪便的SCFAs含量；显著降低血清中TG、TC和LDL-C的浓度。总体效果为：RS组均能改善大鼠肠道生理环境、降低血脂。其中不同种类的RS组间有显著差异,相对于RS2和RS4组,RS3组对改善大鼠肠道生理环境、降低血脂作用最明显；而不同含量的RS组中,15%RS组控制体重、增加盲肠内容物湿重和水分含量的效果较好。10%RS组在增加肠道主要产物SCFAs、减少游离氨含量、降低pH值、增加粪便的湿重及水分含量和血脂方面的作用最佳。(2)不同种类(RS2、RS3、RS4)和含量(5%、10%、15%)的RS对大鼠肠道形态结构的研究结果表明：RS组均能增加大鼠的盲肠总重量、盲肠壁重量和盲肠内容物重量。但不同种类的RS组间有显著差异,相对于RS2和RS4组,RS3组增重效果最明显；而不同含量的RS组中,15%RS组增重效果最明显。改变大鼠小肠、盲肠和结肠的形态结构的效果比较：15%RS>10%RS>5%RS。与对照组相比,5%RS组对大鼠小肠、盲肠、结肠肠道形态结构的影响不明显；10%RS组中,RS3组改变肠道肠粘膜结构的效果比RS2、RS4组更明显；而15%RS组中,RS4组改变肠道肠粘膜结构的效果比RS3组更明显。但是高、中、低含量的RS组都没有引起大鼠小肠、盲肠、结肠形态结构的病理变化。(3)不同种类(RS2、RS3、RS4)和含量(5%、10%、15%)的RS在大肠发酵,通过Nested PCR-DGGE技术对大鼠粪便中微生物区系进行了分析,研究结果表明：RS组具有对照组没有的4种优势菌,分别为产纤维二糖梭菌(Clostridium cellobioparum)、解纤维素醋弧菌(Acetivibrio cellulolyticus)、青春双歧杆菌(Bifidobacterium adolescentis)和乳酸杆菌(Lactobacillus oligofermentans)对照组具有RS组没有的3种优势菌,分别为粪球菌属(Coprococcus catus)、瘤胃球菌属(Ruminococcus gauvreauii)和Blautia schinkii。Allobaculum stercoricanis只出现在RS2组和RS4组,而在对照组和RS3组中未出现。Real-time PCR反应的数据分析表明,相对于对照组,实验期间RS实验组均能促进双歧杆菌和乳酸杆菌的生长增殖,降低肠杆菌的生长增殖。其中不同种类的RS组间有显著差异,相对于RS2和RS4组,各含量组均为RS3组对双歧杆菌、乳酸杆菌的生长增殖的促进效果和对肠杆菌的数量的控制效果更好；而不同含量的RS组中,10%RS组对双歧杆菌和乳酸杆菌的生长增殖的促进效果、对肠杆菌数量的控制效果均优于5%RS组和15%RS组。在整个实验过程中,相对于对照组,不同含量(5%、10%、15%)的RS组对大鼠粪便中肠球菌的数量变化影响不显著。这与RS组大鼠的粪便样品的细菌DGGE水平电泳的显示结果是一致的。(4)比较不同种类(RS2、RS3、RS4)的15%RS与便秘治疗药物秘可舒(bisacodyl)对活性炭诱导小鼠便秘的影响,研究结果表明,在诱导小鼠便秘模型前,预先给小鼠喂食不同种类的15%RS,均能不同程度的缓解、改善便秘小鼠的便秘症状：三种RS组小鼠的第一次排黑便的时间均低于便秘对照组；小肠推进率明显高于便秘对照组；小鼠小肠绒毛的损伤均低于便秘对照组；能增加血清中MTL、Gas、ET、AchE、SP和VIP因子水平,降低SS因子水平。相对于RS2组和RS4组,RS3组最接近正常组和便秘药物bisacodyl治疗组,表现出最好的便秘预防效果。(5)不同种类(RS2、RS3、RS4)的15%RS对盐酸/乙醇诱导大鼠胃损伤影响的研究表明,在诱导大鼠胃损伤模型前,预先给大鼠喂食不同种类的15%RS,均能不同程度的预防、减轻胃损伤大鼠的胃损伤情况：三种RS组大鼠的血清IL-6和TNF-α水平均低于胃损伤对照组；胃损伤程度显著低于胃损伤对照组,而损伤抑制率显著高于胃损伤对照组；均能减少胃液分泌量并增加胃液pH值；均能降低胃组织的炎症基因iNOS, COX-2, TNF-α和IL-1β的mRNA和蛋白质表达。相对于RS2组和RS4组,RS3组对化学诱导大鼠胃损伤的预防作用最佳。本文通过Nested PCR-DGGE和实时荧光定量PCR技术对RS在大肠发酵过程中微生物区系的变化进行了分析,初步推测RS组的4种优势细菌：产纤维二糖梭菌(Clostridium cellobioparum)、解纤维素醋弧菌(Acetivibrio cellulolyticus)、青春双歧杆菌(Bifidobacterium adolescentis)、乳酸杆菌(Lactobacillus oligofermentans),和对照组中的3种优势细菌：粪球菌属(Coprococcus catus)、瘤胃球菌属(Ruminococcus gauvreauii)及Blautia schinkii共同作用,导致主要代谢产物SCFA的量发生变化,并选择性的促进有益菌群双歧杆菌和乳酸杆菌的生长增殖,控制条件致病菌肠球菌和肠杆菌的数量,而且因为不同种类和量的短链脂肪酸生理作用不同,最终导致其代谢结果的不同；间接影响肠道功能,起到改善肠道环境的作用。而只存在于RS2组和RS4组中的Allobaculum stercoricanis细菌可能是导致RS3组改善肠道环境优于RS2和RS4组的原因。但是,关于RS通过影响肠道中微生物区系的变化而间接影响肠道功能机理的研究还需要深入的试验进行探讨。大鼠肠道主要代谢产物SCFA、游离氨,肠道pH,血脂以及肠道形态结构的变化与肠道中微生物种类和数量变化基本一致。本实验建立小鼠便秘模型和大鼠胃损伤模型,运用逆转录-聚合酶链式反应(RT-PCR)、酶联免疫法(ELISA法)和蛋白质印迹(Western blot)等分子生物学方法,进一步来研究15%RS对肠胃功能性作用的影响,发现15%RS通过改变血清中MTL、Gas、ET、AchE、SP、VIP、SS胃肠激素的因子水平、降低组织炎症基因iNOS、COX-2、TNF-α和IL-1β的：mRNA和蛋白质表达来调节机体肠胃功能性作用,具有良好的改善肠道生理作用,跟前面的实验结果是一致的。说明RS确实对胃肠道具有保护作用,为进一步研究RS对肠道生理环境和胃肠道功能性作用提供实验依据；也为利用“药食两用”的天然、安全的RS食品资源,开发RS的保健功能性食品,研究通过食疗改善、缓解肠道疾病的不适症状提供实验数据。更多还原

【Abstract】 Resistant starch (RS) is added to foods as a low-calorie starch, which can play with the physiological functions of dietary fiber:such as weight control, prevention constipation colorectal cancer and other intestinal diseases, improving the intestinal flora, reducing blood lipid and glucose.Compared with dietary fiber, RS has become the central issue because of its good processing properties. It can be added into different kinds of foods.Different types of resistant starch have different effects on body intestinal physiological environment and gastrointestinal function because of different fermentation patterns. There are few dates about different types of resistant starch and contentsresistant starch on body intestinal physiological environment and gastrointestinal function in domestic and international studies.To examine the effect of different types(RS2、RS3、RS4) and contents (5%、10%、15%)of resistant starch on intestinal environment and gastrointestinal function, Sprague-Dawley rats and mice experimental model were fed diet containing resistant starch. This study provides theoretical basis on the mechanism of intestinal health for further study about resistant starch. The main conclusions of the research are as follows:(1) To examine the effect of different types and contents of resistant starch on intestinal metabolites and serum lipids, Sprague-Dawley rats were fed diet containing resistant starch for four weeks. The results showed that:compared to the control group, different types and contents of resistant starch can increase food intake, lower food efficiency and body weight gain. The pH of experimental groups was lower than the control group. The free ammonia concentration of caecal and fecal contents of experimental groups was lower than the control group. Cecal contents and feces of experimental groups was more moist and softer than the control group. The concentration of acetate, propionate, butyrate and total short-chain fatty acids of caecal and fecal contents of experimental groups were higher than the control group. It can lower the concentration of bad metabolites and higher the concentration of good metabolites. The AI and concentrations of TG, TC and LDL-C of experimental groups were lower than the control group. The overall effects were:compared with the other two RS groups, RS3group was more significant on reducing weight, improving the intestinal physiological environment and lowering serum lipids in rats.15%RS group was better at weight control, increasing the wet weight and moisture content of cecal contents;10%RS group was better at increasing intestinal main product SCFAs,reducing the free ammonia content, reducing the pH value, increasing fecal wet weight and moisture content and serum lipids.(2) To examine the effect of different types and contents of resistant starch on intestinal morphology, Sprague-Dawley rats were fed diet containing resistant starch for four weeks. The results showed that:the total cecal weight, cecal wall weight, weight of cecal contents of experimental groups were higher than the control group. But there was a significant difference between the different types of RS group, compared to the RS2and RS4group, RS3group had the most obvious effect with increasing their weight. Change of small intestine, cecum and colon morphology were:15%RS>10%RS>5%RS. Compared with the control group,5%RS group had no significant effect on the morphology of small intestine, cecum, colon. However,10%RS3group was better than RS2、RS4group at changing significantly the structure of the intestinal mucosa,and15%RS4group was better at changing significantly the structure of the intestinal mucosa.In short,high, medium and low contents of resistant starch group did not cause pathological changes in the small intestine, cecum and colon.(3) The impact of different types and contents of resistant starch on intestinal microbial flora by nested PCR-DGGE technique showed that compared to the control group, the resistant starch group had4dominant bacterias, they were Clostridium cellobioparum,Acetivibriocellulolyticus,Bifidobacterium adolescentis and Lactobacillus oligofermentans. The control group had3dominant bacterias, they were Coprococcus catus.Ruminococcus gauvreauii and Blautia schinkii. Allobaculum stercoricanis only existed in RS2and RS4group, not in the control group and RS3group.By real-time PCR technology, the results showed that:different types and contents of resistant starch could significantly increase the growth of Bifidobacteria and Lactobacilli, and reduce the growth of the Enterobacteriaceae. RS3showed the best acticity in three kinds of resistant starch.10%RS group was better than5%RS and15%RS group in the effect of promoting the growth of bifidobacteria and lactobacilli, and controlling the number of Enterobacteriaceae. During the whole experiment, compared to the control group, three types of resistant starch group could not significant impact on the change of Enterococcus in rat feces. The conclusions were consistent with the results of DGGE level electrophoresis.(4) Effect of different types of15%resistant starch in activated carbon-induced constipation mice showed that resistant starch had the preventive effect of active-carbon induced constipation in mice which was regarded as a functional food. According to the results of RS3, it found that first black stool defecation shorten time was only a little longer than bisacodyl, gastrointestinal transit was longer than control and similar to bisacodyl, various serum levels such as MTL, Gas, ET, AchE, SP and VIP in resistant starch dose mice were higher than control mice, the SS levels showed opposite tendency. These results suggested that resistant starch had a significant preventive effect on activated carbon induced constipation mice, and RS3showed the best acticity.(5) This study demonstrates that different types of15%resistant starch is effective in the prevention of HCl/ethanol-induced gastric injury in SD rats. The protective effects of resistant starch may be because of the decreased the proinflammatory cytokines such as IL-6and TNF-a. The stomach appearance also showed that resistant starch could prevent HCl/ethanol-induced injury in the stomach. Furthermore, mRNA and protein expression levels of inflammation-related genes in the stomach, including iNOS, COX-2, TNF-α and IL-1β were significantly reduced in the resistant starch-treated rats. These results suggested that resistant starch was potentially useful in the treatment or prevention of chemical-induced gastric injury in vivo. And the RS3showed the best activity.Through PCR-DGGE and and real-time quantitative PCR technology, this paper analyzed the changes of microbial flora in different types and contents of resistant starch fermentation process in the large intestine. The results showed that:4kinds of dominant flora (Clostridium cellobioparum,Acetivibrio cellulolyticus,Bifidobacterium adolescentis and Lactobacillus oligofermentans) in resistant starch groups, and3kinds of dominant flora in the control group (Coprococcus catus, Ruminococcus gauvreauii.and the Blautia schinkii interaction) were interacted and leaded the change of major metabolite (SCFA); indirectly, caused the change of intestinal pH value, contents of free ammonia, blood lipids as well as the changes of intestinal morphology. Those microbial flora played a important role in improving the intestinal environment and lowering blood lipids. Allobaculum stercoricanis bacteria that existed only in the RS2and RS4group may lead to the RS3group,which had the best activity in improving intestinal environment and reducing serum lipids. However, different types and contents of resistant starch affect the mechanism of intestinal function by changing the intestinal microbial flora also are required in-depth testing.The major metabolite in gut, such as SCFA、free ammonia content, serum lipids changing in pH and gut morphology consistented with the change of the type and quantity of intestinal microbial flora.This experiment made the model of activated carbon-induced constipation mice and HCl/ethanol-induced gastric injury rats, to study the impact of high concents of different types of resistant starch on gastrointestinal functional on body, through reverse transcription-polymerase chain reaction, enzyme-linked immuno sorbent assay and western blot. The results showed that:the high concentration of the different types of RS did have good improvement in gastrointestinal function of the body, Althought the effect of drug (bisacodyl) was better than RS, but resistant starch did improve symptoms of constipation in activated carbon-induced constipation mice.The effect of prevention and inhibition of gastrointestine was good in HCI/ethanol-induced gastric injury rats. In the three different types of resistant starch, the RS3showed the best activity, which consistented preferably with previous experimental results. Resistant starch did have a protective effect on the gastrointestine.The paper provided experimental evidences for study effects of resistant starch in physiological environment of the gut and gastrointestinal function. These results contributed to development health functional food containing resistant starch,and to alleviation the symptoms of intestinal diseases.更多还原