Molecular Mechanism Of Butyrylated Lotus Seed Starch Targeting The Colon To Produce Butyric Acid

Lotus seeds are high starchy food,with starch content accounting for more than 50%of solids content.It is specific starches with high amylose content up to 42%.Butyric acid is the main energy substance of colonic epithelial cells and has the highest utilization rate among short-chain fatty acids,which is crucial to human health.Butyrylated lotus seed starch formed from the esterification reaction of butyric anhydride and lotus seed starch is an important carbon sources for colonic microbial,which modulates the composition of intestinal flora and affects the production of short-chain fatty acids.Meanwhile,the esterified butyric acid can be targeted to the colon to promote intestinal health.In this thesis,the structural characteristics,anti-digestion characteristics,and in vitro fermentation ability of butyrylated lotus seed starch with different degrees of substitution were systematically studied.On this basis,16S r RNA high-throughput sequencing technology,targeted metabolomics technology,and proteomics technology were used to study the regulation mechanism of lotus seed starch on intestinal flora and butyric acid production in normal mice.The research findings provide new ideas for individualized precise nutrition design.It also has certain theoretical significance and practical value for the high-value processing of lotus seed starch and the development and utilization of new prebiotics with a high yield of butyric acid.(1)Study on structural characteristics and anti-digestion properties of butyrylated lotus seed starchButyrylated lotus seed starch was prepared from butyric anhydride and lotus seed starch by aqueous phase method.The structural characteristics and anti-digestion properties of butyrylated lotus seed starch with different degrees of substitution were determined.The result showed that with the increase of butyric anhydride content,the degree of substitution increased.But the degree of substitution did not change when the concentration of butyric anhydride increased to a certain extent.During the process of butyrylation,the surface of the starch ester began to become rough.FT-IR spectra proved the formation of ester bond.The XRD result showed that the high degree of butyrylation process destructed the crystal structure of natural starch turned the starch crystal form from C type to V type,whose relative crystallinity decreased with the increase of DS.NMR analysis results also confirmed that a new ester bond was connected to starch.During the process of butyrylation.M_w significantly decreased,and the starch granules expanded to form larger aggregates.The study results of in vitro anti-digestion characteristics showed that with the increase of degree substitution of butyrylated lotus seed starch,the content of resistant starch increased,which indicated the validity of butyrylated starch targeting to colon.The above results showed that the distribution ratio of butyryl groups at different carbon sites had a significant effect on the properties of butyrylated lotus seed starch.(2)Study on the changes of butyrylated lotus seed starch structural characteristics and microbial composition during the process of in vitro fermentation of fecal bacteriaTo study the structural variation of substrate and changes in bacterial composition in different fermentation times,lotus seeds resistant starch and unbutyrylated lotus seed starch were used as control,and butyrylated lotus seed starch with different degrees of substitution was used as fermentation substrate to ferment mouse fecal flora in vitro.The results showed that butyrylated lotus seed starch significantly promoted the abundance of Clostridium＿sensu＿stricto＿1 which produced butyric acids and inhibited the abundance of the harmful bacteria Enterococcus with the extension of fermentation time.It indicated that the composition of intestinal flora could be modulated by butyrylated lotus seed starch.A large amount of short-chain fatty acids was produced during the fermentation of fecal flora by butyrylated lotus seed starch.The content of butyric acid produced by high substitution degree of butyrylated lotus seed starch was the highest,which may be due to the release of esterified butyric acid on the starch ester.Spearman correlation index of dominant fecal bacteria and short-chain fatty acids showed that the content of butyric acid was positive correlated with Citrobacter(p<0.05),Lysinibacillus,Bifidobacterium,Faecalibacterium.After the fermentation of butyrylated starch by flora,the phenomenon of particle adhesion and aggregation was intensified,the surface crystals increased,and the surface of the particles was seriously damaged.The ordered structure showed that microbial fermentation could promote the increase of order degree of starch molecular chain and the decrease of double helix content.The results of crystal and molecular structure showed that with the prolongation of fermentation time,the crystallinity,the proportion of microcrystalline region,and the degree of the ordered structure of butyrylated lotus seed starch increased.While the proportion of double helix structure and the proportion of amorphous region decreased during fermentation,which indicated that the microbial community first utilized the amorphous region and sub-crystalline region of butyrylated lotus seed starch.According to the correlation analysis between structural characteristics,dominant genera,and short-chain fatty acids,the structural characteristics of lotus seed resistant starch showed more correlation with the dominant genera of Proteobacteria,while the structural characteristics of butyrylated lotus seed starch showed more correlation with the dominant genera of Firmicutes.(3)Effects of butyrylated lotus seed starch on physiological characteristics and butyric acid metabolism in miceIn this chapter,the effects of butyrylated lotus seed starch on the physiological characteristics of mice were studied.The body weight and food intake of mice were measured.And the histopathological characteristics of the small intestine and colon were observed.On this basis,the contents of short-chain fatty acids,especially butyric acid in the small intestine,colon,liver,and serum of different tissues were determined,and the metabolic profile of short-chain fatty acids was constructed.The results showed that the butyrylated starch had no adverse effects on the body weight and health status of mice.There was no significant difference in body weight among the normal group,lotus seed resistant starch group,and butyrylated lotus seed starch group.Histopathological observation showed that feeding butyrylated lotus seed starch could improve the morphology of the small intestine in mice,and the effect of LLSB group was the most significant.At the same time,the thickness of the colonic muscle layer,the number of goblet cells,and the intestinal barrier ability of mice fed with butyrylated lotus seed starch increased.The results of short-chain fatty acid determination showed that the content of butyric acid in different groups of resistant starch in the small intestine was low,and there was no significant difference.Butyrylated lotus seed starch could increase the content of butyric acid in the colon of mice,and the content of butyric acid increased with the increase of substitution degree,which confirmed the effectiveness of butyrylated lotus seed starch as a carrier to transport butyric acid to the colon.Butyric acid had the highest concentration in the colon,followed by blood,liver,and small intestine.Butyric acid was the preferred energy source for colon cells,propionic acid was metabolized in the liver,and acetic acid was the most abundant short-chain fatty acid in the blood circulation of mice.(4)Study on the effect of butyrylated lotus seed starch on intestinal flora of mice.On the basis of studying the effects of butyrylated lotus seed starch on the physiological characteristics and butyric acid metabolism in mice,the effects of butyrylated lotus seed starch on the diversity,community composition,dominant genera and differential flora of the intestinal flora of small intestine and large intestine in normal mice were investigated.By combining the metabolic profile of butyric acid in the small intestine and colon of mice,the relationship between the dominant flora in the small intestine and butyric acid,and the relationship between the dominant flora in the large intestine and butyric acid were established respectively.The purpose was to construct the correlation network map of structural characteristics-intestinal flora-short chain fatty acids.The results showed that the type of resistant starch and the change of substitution degree of resistant starch would affect the change of microbial community structure.The number of unique species of each group was lower in the colonic flora compared to the small intestine flora.The analysis of microbial taxonomic units shows that compared with the non-butyrylated starch group,the relative abundance of Firmicutes increased and the relative abundance of Bacteroidota decreased in the butyrylated lotus seed starch group in the small intestinal flora.At the genus level,the medium degree substitution of butyrylated lotus seed starch group promoted the abundance of acetogenic bacteria(Lactobacillales)and Bacteroidales.And the high degree substitution of butyrylated lotus seed starch group promoted the growth of butyric acid bacteria(Clostridiales).In the colon flora,Firmicutes and Bacteroideta were the dominant bacteria in the colon of mice.Compared with non-butyrylated starch,low,medium and high degree of substitution butyrylated starch promoted the abundance of Clostridia and Campylobacteria and inhibited the abundance of Bacilli and Desulfovibrionales.From the significance test of the differential flora between groups,the relative abundance of Lactobacillus,Streptococcus and Bacillus was significantly different in different carbon source groups in the small intestinal flora.Different carbon sources mainly affected the relative abundance of unclassified＿f＿Lachnospiraceae,Cladosporium(Alistipes),Lachnospiraceae＿NK4A136＿group in the colon flora.The correlation network diagram of structural index-intestinal flora-short chain fatty acids showed that BA,AA and SCFA were positively correlated with Mw,DD and Crystallinity value,and negatively correlated with DO,C6-DS,C2,3-DS,amorphous value and DS value.In the colon,BA,AA,PA,IBA and SCFA were negatively correlated with Mw value,DD value and Crystallinity value,and positively correlated with DO value,C6-DS value,C2,3-DS value,amorphous value and DS value.(5)Study on mechanism of butyric acid molecules produced by butyrylated lotus seed starch targeted to colonBased on the study of intestinal flora,coliform bacteria and butyric acid metabolism,to study the expression of butyrate-releasing enzyme in colon tissue contents of normal mice during feeding butyrate lotus seed starch.By proteomics bioconductivity analyzing the contents of colon tissue,the significantly up-regulated and down-regulated differential proteins were screened out,and various functions of the samples were analyzed(Cluster analysis,GO analysis,and KEGG pathway analysis).The results showed that there were 5 up-regulated proteins related to butyric acid production metabolism,including Q8N7N8(3-Hydroxy-3-methylglutaryl Co A synthetase),Q8BMS1(Trifunctional enzyme subunit),R9K6C2(Acetyl-coa acetyltransferase),Q80XN0(Hydroxybutyrate dehydrogenase),A0A7U6KDA3(Pyruvate-flavoxin oxidoreductase).There were 2 down-regulated proteins related to butyric acid production metabolism,including A0A2K4ZLR1(Formate acetyltransferase),A0A3D5TAP2(Ethylene reductase).There were 9 up-regulated proteins related to the release of esterified butyric acid,including D3Z0E6(Ketosaccharidase),P17225Q9D0A2(Polypyrimidine tract-binding protein),Q9DCM0(Persulfide dioxygenase),Q91VM9(Inorganic pyrophosphatase),Q0VD75(Alkaline phosphatase),O0870(Peroxidoreductase),Q3TB22(Fatty acid synthase),Q8VCT4(Carboxylesterase).Combined with bioinformatics functional identification,proteins related to the release of esterified butyric acid were significantly enriched in the pathways of sulfur metabolism and fatty acid biosynthesis,and also related to molecular function such as hydrolase activity(acting on ester bonds)and hydrolase activity.The results elucidated the mechanism of butyrate lotus seed starch targeting colon to produce butyric acid.