Structural Changes During In Vitro Fermentation Of Resistant Starch And Its Effects On The Intestinal Microbiota Of Mice

With the improvement of living standards,the large proportion of meat products consumption and irregular diet have led to an increasingly prominent intestinal flora disorder.Disorder of the microbiota will break the relatively stable state between the gut and the host,inducing such diseases as hypertension,diabetes,and obesity.Improving the dietary structure and increasing the intake of dietary fiber is one of the effective means to improve the disturbance of intestinal flora.Resistant starch（RS）,as a kind of dietary fiber,not only has biological functions such as regulating intestinal health and preventing diseases,but also has the industrialization characteristics of moderate cost and adjustable structure.Therefore,the development and utilization of RS has received more and more attention.Currently,physical or enzymatic methods have become a research hotspot because of the green and safe preparation process.Autoclaving,enzymatic debranching,microwave,ultrasound and compound treatment are increasingly used for the preparation of RS.However,data on the structural properties and effects of different preparation methods of RS on intestinal flora are still limited and need further study.Therefore,in this thesis,three different methods of autoclaving（ARS）,enzymatic debranching（DRS）and autoclaving-debranching（ADRS）were used to prepare RS to investigate the structure,in vitro fermentation characteristics and the effects of RS on the intestinal flora of mice.The study contents and results are as follows.（1）The differences in the structural properties of ARS,DRS and ADRS were investigated by scanning electron microscopy,X-ray diffraction,ion chromatography,Fourier transform infrared and thermogravimetric analysis.The results showed that:ADRS was irregular block;ADRS branched chain was dominated by B1 chain,with an average DP value of 19.97%,slightly higher than that of ARS.The crystallization types of HAMS and DRS were A-type and B-type,respectively,while ADRS and ARS are B+V crystals.ADRS had the highest R₁₀₄₇^-1-1_{cm/1022 cm} and R^-1-1_{995 cm/1022cm} value of 1.35 and0.94,indicating that its double helix structure was tightly bound.In addition,ADRS had a low thermal degradation rate,and excellent thermal stability.In summary,compared to other RSs,ADRS existed with the roughest particle surface and exhibited a favorable double helix structure and thermal stability properties,which may have an impact on its functional properties.（2）Bifidobacterium adolescentis and Escherichia coli in the adult intestinal tract were selected test strains,and RS was used as a medium component to explore the in vitro fermentation characteristics of RS.The findings suggested that extracellular amylase was produced in Bifidobacterium adolescentis and Escherichia coli media to utilize RS,at the same time organic acid produced to reduce the p H of the media.In ADRS medium,the OD_{600 nm} value and amylase activity of Bifidobacterium adolescentis were significantly higher than those in ARS and DRS medium.On the other hand,the growth of Escherichia coli was inhibited in the medium of ARS,DRS and ADRS,and OD_{600 nm} values were lower than those of bacteria on control medium.In addition,the acid resistance and bile salt resistance of Bifidobacterium adolescentis on ADRS medium were also better than HAMS,ARS and DRS medium.In conclusion,ADRS was more effective than HAMS,ARS and DRS in promoting the proliferation of beneficial bacteria and enhancing the gastrointestinal tolerance of beneficial bacteria,and it was also effective in inhibiting the growth of harmful bacteria.（3）RS was isolated after in vitro fermentation and its properties were determined to investigate the changes in structural properties of RS before and after fermentation.The results showed that Bifidobacterium adolescentis and Escherichia coli fermentation resulted in the ARS,DRS and ADRS particles degraded and some debris fragments appeared,with loose structures and rougher surfaces.The crystallization type of RS showed no change after in vitro fermentation,but the utilization of Bifidobacterium adolescentis resulted in the replacement of the double peaks of ARS and ADRS at 22°and 24°by diffuse peaks at 23°and a decrease in the intensity of the V-shaped crystallization peaks at diffraction angles 13°and 19.7°.The utilization of RS by Bifidobacterium adolescentis and Escherichia coli did not affect the chemical groups and chemical bonds of RS,but reduced the R_{1047 cm}^-1-1_{/1022 cm} and R^-1_{995 cm}^-1_/1022cm value of ADRS by 20.74%and 30.85%,respectively.（4）ADRS was used as a component of mouse feed for animal experiments,and HAMS was used as a positive control to explore the effects of RS and its dose on physiological and biochemical indicators and intestinal flora of mice.There were no significant changes in the organ indices of mice in all groups,indicating that ADRS had no adverse side effects on mice organism.After 4 weeks of feeding,the high-dose group significantly reduced the serum cholesterol content and atherosclerosis index in mice.The fecal butyric acid content of mice was significantly higher in the high-dose group,at 5.65μmo L/m L.The abundance and diversity of the gut microbial community showed a decreasing trend with the intake of ADRS.Differential analysis of gut microbiota in mice showed that different RS induced enrichment of different species.Among them,the bacteria of Bacteroidetes were significantly enriched in HRS group with a strong dose-dependence.The most affected species in the low-dose group was Ruminococcus＿2;in the middle-dose group,Actinobacteriaceae and Lachnospiriaceae were enriched.In summary,the microscopic morphology,chain length distribution,and crystalline properties of RS all affected its utilization by intestinal bacteria.In addition,the intake of RS can also affect the structure of the intestinal flora and the metabolism of short-chain fatty acids,thereby affecting the health of the body.Therefore,exploring the structural properties of RS and its impact on the gut microbiota is crucial for maintaining the relative stability between the gut microbiota and the host.