| Cassava is an important staple crop,which is rich in starch.The plant is drought-resistant and barren-resistant,making it important for sustainable agriculture.Cassava starch has a unique multi-scale structure,which leads to its special physicochemical properties.However,with high digestibility and high glycaemic index,cassava starch is not suitable for people with metabolic diseases such as obesity and diabetes.The multi-scale structure of cassava starch can be modified through many ways,which can decrease the digestibility of cassava starch.In recent years,many studies have applied phenolics in the modification of starch to obtain resistant starch(RS).RS may improve diabetes symptoms by lowering postprandial blood glucose level and reshaping gut microbiota.However,there is no literature proving that RS improves diabetes through changing gut microbiota through fecal microbiota transplantation.In this study,cassava starch and phenolic acids were used to modify starch.Three kinds of cassava starch phenolic acid esters were synthesized.Their digestibility and effects on type 2 diabetes as well as the mechanism were studied.It provides a scientific foundation for the development of cassava starch industry.The main results and conclusions of this study are as follows:1.Ferulic acid starch ester(FASE),p-coumaric acid starch ester(p-CASE)and sinapic acid starch ester(SASE)with low,medium and high degrees of substitution(DS)were synthesized by carbonyldiimidazole-mediated starch esterification and the physicochemical property changes were analyzed.The results showed that FT-IR and ~1H NMR confirmed the correct structure of the synthesized product without obvious impurities remaining.The reaction efficiency of cassava starch esters followed the order:ferulic acid>p-coumaric acid>sinapic acid,the DS order is consistent with the reaction efficiency,and FASE-3 had the highest DS which is 0.1404±0.0057.The granule structure of cassava starch was completely destroyed after esterification,the A-type crystal structure completely disappeared,and FASE emerged a new crystal structure(2θ=18.0°,20.0°,23.5°and 25.3°).The thermal stability of the esterified cassava starch was improved,but the solubility and swelling potential decreased due to the increase of hydrophobicity.The antioxidant activity of cassava starch esters was significantly improved and followed the order:SASE>FASE>p-CASE.2.Using in vitro hydrolysis curve,Englyst method,glycaemic index and in vitro simulateddigestion,the digestion pattern of phenolic acid starch esters was explored.The mechanism of reduced digestibility of phenolic acid starch esters were explored by molecular docking and particle size analysis.The results showed that phenolic acid starch esters had a lower end-point hydrolysis rate than native starch,and the end-point hydrolysis rate of the same phenolic acid starch esters increased with the increase of DS.Under the same synthesis conditions,the hydrolysis rate followed the order:SASE>FASE>p-CASE.The RS content order was consistent with the end-point hydrolysis rate,and SASE-3 possessed the highest RS content,reaching 39.0±1.2%.The content of slowly digestible starch changed with no regularity,while the content of readily digestible starch decreased significantly after esterification.All phenolic acid starch esters had significantly lower glycaemic index than native starch,and the lowest one was 47.1 from SASE.Molecular docking and particle size analysis showed that phenolic acid starch esters resisted digestion mainly through hydrophobic aggregation and steric hindrance.3.Extract the gut microbiota from the feces of healthy people and co-culture with SASE in vitro to explore the degradation of SASE by intestinal flora and the composition change of intestinal flora and its metabolites.The results showed that compared with the control(0.00±0.00 m L),SASE could significantly increase the gas production(10.67±0.06 m L)of the gut microbiota,indicating that the intestinal flora could utilize SASE.Compared with NCS,the gas production of SASE was significantly lower before 12 h(6.40±0.10 m L vs 10.20±0.00 m L),suggesting that SASE may only be degraded by specific taxa,and SASE is more likely to reach the distal colon.At the end of fermentation(24h),the SASE group had lower p H than NCS(5.45±0.03 vs 5.64±0.04)and higher total short-chain fatty acid content(115.5±1.0 mmol/L vs 101.7±1.4 mmol/L).After 12 h of glycolysis,85.4±1.5%of sinapic acid in SASE was released,and then the gas production and the content of short-chain fatty acids in the SASE group increased rapidly.Almost all sinapic acid was released after 24 h.SASE had no effect on the richness and evenness of the gut microbiota but the gut microbiota composition at phylum,genus,and speice level was changed greatly.4.ICR mice were applied to explore the acute oral toxicity,genotoxicity and subchronic toxicity of SASE,in order to clarify the safeness of SASE.The results showed that SASE did not cause the death of mice in the acute oral toxicity test,and the autopsy after the observation period did not find any toxicity of SASE.SASE show no obvious genotoxicity in bacterial reverse mutation toxicity,mouse bone marrow erythrocyte chromatin damage toxicity,and mouse sperm teratotoxicity analysis.During the 28-day oral toxicity test,SASE did not cause toxic reactions and death in mice,and no toxic reactions were found in the organs and blood of mice after the test.In conclusion,SASE has no acute,subchronic and genotoxicity,and is safe to eat.5.High fat diet and STZ injection were used to induce T2DM symptom in C57BL/6J mouse model,which was used to explore the effect of SASE on T2DM.Combining multi-omics methods such as microbiome,metabolome,and transcriptome,the mechanisms of SASE were preliminarily explored.The results showed that SASE intake for 8 weeks significantly decreased the fasting blood glucose,Hb A1c,AGEs,HOMA-IR,area under the curve of OGTT,and increased fasting insulin level,area under the curve of ITT in T2DM mice.The results of inflammatory factors showed that SASE significantly reduced the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors in T2DM.SASE also significantly reduced fecal p H,increased fecal water content,and promoted the synthesis of short-chain fatty acids by intestinal flora.SASE specifically up-regulated the relative abundance of specific strains of Actinobacteria,Dunaliella and Coriobacteriaceae＿UCG-002,as well as Faecalibaculum rodentium.Metabolome analysis showed that SASE up-regulated FOXO and AMPK signaling pathways.Liver transcriptome showed that SASE activated the gene expression in insulin sensitivity,glycogen synthesis pathway and downregulted the genes in gluconeogenesis pathway.6.Using fecal microbiota transplantation and in vitro co-culture to identify the role of gut microbiota in the improvement of T2DM by SASE.The expression of related proteins was analyzed by immunofluorescence and Western blot to find the liver metabolic pathways activated by short-chain fatty acids.The results showed that fecal microbiota transplantation in the SASE group could significantly improve the symptoms of diabetes in T2DM mice.In vitro co-culture found that Lactobacillus johnsonii could degrade SASE into sinapic acid and starch.Then Faecalibaculum rodentium and Dubosiella newyorkensis degraded starch into small molecules that can be used by Lactobacillus johnsonii.Immunofluorescence analysis showed that the expression of short-chain fatty acid receptor GPR43 in colon and liver were up-regulated,and the expression of GLUT2 in liver increased,thus the uptake of blood glucose was enhanced.Western blot results showed that short-chain fatty acids up-regulated the phosphorylation of AMPK,IRS1,AKT,GSK3βand FOXO1,up-regulated the expression of IRS1 and PI3K,down-regulated the expression of mature SREBP1c and PEPCK,activated the hepatic glycogen synthesis pathway,and promoted the Insulin sensitivity,promoted the accumulation of hepatic glycogen.In summary,this study clarified the digestion process of cassava starch phenolic acid esters,revealed the mechanism of SASE degradation by specific gut microbiota species and regulation of intestinal flora,pointed out that SASE modulated gut microbiota to increase hepatic glycogen synthesis and insulin sensitivity,and ultimately improved T2DM.This study reduced the digestibility of cassava starch through esterification,which provides a scientific basis for the promotion and high-value utilization of cassava starch.This study also provides new method for targeted colon delivery and specific gut microbiota regulation. |
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