Construction Of Maize Starch-phenolic Acid Complex And Its Mechanism Of Regulating Blood Glucose Homeostasis

Phenolic acids can affect the activity of starch hydrolase and form complex with starch to reduce the digestion rate of starch and delay the generation of glucose,so as to control the blood sugar level in the body.However,the inhibitory mechanism of phenolic acid on key enzymes of starch hydrolysis is still incomplete,and the construction methods of starch polyphenol complex are limited at present,and there are problems such as low binding amount and unclear anti-enzymatic hydrolysis mechanism.At the same time,the probiotic mechanism of undigested starch in starch-phenolic acids complex entering the large intestine and the mechanism of regulating blood glucose homeostasis need to be further explored.In this study,maize starch and three typical phenolic acids（chlorogenic acid,caffeic acid and ferulic acid）were taken as research objects.Firstly,the effects of chlorogenic acid,caffeic acid and ferulic acid on enzyme activity of starch hydrolase（α-amylase andα-glucosidase）were investigated.The maize starch phenolic acid complex was constructed by physical mixing,water heat treatment and extrusion treatment,respectively,to increase the binding amount of phenolic acid and enhance its anti-enzymatic properties.Further analyze the probiotic effect of digestion residue of maize starch-phenolic acid complex constructed by extrusion treatment and build a diabetic animal model.Combine islet cell function,intestinal microecology and hypothalamic appetite regulation mechanism,systematically study the mechanism of maize starch-chlorogenic acid complex regulating blood glucose homeostasis in the body.It provides theoretical guidance and design ideas for the control of blood sugar by nutritious and healthy starch foods.（1）Inhibitory effect of phenolic acid onα-amylase andα-glucosidaseThe inhibitory mechanism ofα-amylase andα-glucosidase activity by caffeic acid,chlorogenic acid and ferulic acid was clarified through in vitro enzyme inhibition experiment combined with 3D molecular docking method.The results showed that the three phenolic acids had the ability to inhibitα-amylase andα-glucosidase activities.From the value of half maximal inhibitory concentration(IC₅₀),it can be seen that the inhibitory capacity of the three phenolic acids onα-amylase andα-glucosidase was as follows:chlorogenic acid>caffeic acid>ferulic acid.The three phenolic acids showed mixed competitive inhibition ofα-amylase and non-competitive inhibition ofα-glucosidase.The three phenolic acids increased theα-helix ratio and decreased theβ-sheet ratio in the secondary structure ofα-amylase andα-glucosidase,indicating that the three phenolic acids made the secondary structure ofα-amylase andα-glucosidase more tightly,and wrapped the active site of hydrolyzed starch,resulting in a decrease in the hydrolysis rate of starch.At the same time,fluorescence quenching and 3D molecular docking also confirmed that the three phenolic acids can affect the amino acid structure of the two starch hydrolases,and the interaction forces are mainly hydrogen bonding and hydrophobic interaction.（2）Construction and digestive characteristics of the maize starch-phenolic acid complexThe mechanism of resisting enzymolysis of maizestarch phenolic acid complex constructed by extrusion method was elucidated by using modern structural characterization techniques and animal experiments.It was found that compared with physical mixing method and hydrothermal treatment method,high temperature and high pressure under extrusion treatment can increase the binding amount of phenolic acid,promote more phenolic acid to combine with maize starch through hydrogen bonding to form V-type maize starch-phenolic acid complex,improve the long-range order of maize starch,increase the proportion of V-type zone,and effectively block the contact and migration of starch hydrolase and starch molecules.The content of rapidly digestible starch（RDS）and resistant starch（RS）in the maize starch-phenolic acid complex constructed by extruded method decreased.In addition,the weakly bound phenolic acids will be gradually released during digestion,play the role of enzyme inhibition,and delay the process of starch hydrolysis.Furthermore,when chlorogenic acid,caffeic acid and ferulic acid were added at 2.0（w/w）,the postdietary blood glucose value of the maize starch-phenolic acid complex constructed by extrusion method was significantly lower than that of the maize starch-phenolic acid mixture/complex constructed by hydrothermal treatment method and physical mixing method.According to the in vitro and in vivo experiments,the mechanism of anti-enzymatic hydrolysis of maize starch phenolic acid complex constructed by extrusion method is to promote the long-term order and increase the proportion of V-type zone,cooperate with the inhibition of starch hydrolase during the release of phenolic acid,thus reducing the content of RDS,increasing the content of RS,and further reducing the postprandial blood glucose.（3）Fermentation characteristics of the digestive residue of maize starch-phenolic acid complex constructed by extrusion method.The prebiotic properties of the extrusion-constructed maize starch-phenolic acid complex were analyzed by in vitro simulated fermentation and 16 S rRNA high-throughput sequencing.The results show that intestinal flora will first attack the undefined regions of residues of extruded maize starch（REMS）,residues of the extruded maize starch-caffeic acid complex（REMS-CA）and residues of the extruded maize starch-chlorogenic acid（REMS-CHA）.Increased crystallinity followed by decreased crystallinity in the exposed crystallinity,while intestinal flora tended to attack crystallinity in the extruded crystallinity of the digestive residue of the maizestarch ferulic acid complex（REMS-FRA）,showing a continuous decrease in crystallinity.At the same time,phenolic acid can undergo phase I decarboxylation and dehydroxylation reactions when there is complex digestion residue in the fermentation solution,while phenolic acid can undergo phase II sulfuric acid esterification reaction when there is no complex digestion residue.In addition,extruded maize starch-chlorogenic acid digestion residues had the highest total short-chain fatty acid levels,in which chlorogenic acid assisted complex digestion residues to promote butyric acid production.Furthermore,the digestive residue of the complex significantly promoted the proliferation of Bifidobacterium,unclassfied＿o＿Lactobacillales,Lactococcus and Weissella genus,indicating that these four groups have potential probiotic effects.Moreover,the function prediction of KEGG metabolic pathway indicates that REMS-CHA has the ability to regulate blood glucose homeostasis.（4）The maize starch-chlorogenic acid complex restores islet function through IRS1/PI3K/Akt/Fox O1 signaling pathwayCombining proteomics and in situ cell biology,the protective mechanism of extruded maize starch-2.0 chrologenic acid（EMS-2.0CHA）complex constructed with 2.0%chlorogenic acid addition（w/w）on blood glucose fluctuation and islet cells was elucidated from protein level.The results showed that the biochemical indexes,glucose tolerance,insulin resistance and pancreatic oxidative stress of diabetic rats were significantly improved after 6 weeks after EMS-2.0CHA diet intervention.In addition,in the HE staining,it was observed that the volume and number of rat islet cells in the EMS-2.0CHA group were enlarged,and the insulin expression and level of EMS-2.0CHA were significantly increased in the results of immunohistochemical analysis.At the same time,the apoptosis and cell repair pathways of islet cells were investigated by in situ end labeling methods TUNEL and Western-Blot.It was found that EMS-2.0CHA could effectively reduce the apoptosis rate of islet cells by interfering with the IRS1/PI3K/Akt/Fox O1 signaling pathway.（5）The maize starch-chlorogenic acid complex regulates blood glucose homeostasis through the"gut-brain-pancreas"axisThe effects of EMS-2.0CHA on intestinal microecology and hypothalamic appetite in rats were further investigated by 16 S rRNA high-throughput sequencing and proteomics.The results showed that EMS-2.0CHA could increase the levels of satiating orexin（glucagon-like peptide-1,cholecystokinin and peptide YY）,decrease the expression of orexin（neuropeptide）and orexin（opioid-melanocorticogenic）protein in hypothalamus,and reduce the intake of starch and fat.In addition,EMS-2.0CHA is beneficial to the production of SCFAs,promotes the expression of short-chain fatty acid receptors in pancreatic tissue and brain,affects the expression of orexin in hypothalamus,and enhances insulin sensitivity.Similar to in vitro fermentation,EMS-2.0CHA significantly promoted the abundance of Bifidobacterium and Lactobacillus,indicating that EMS-2.0CHA can still play a probiotic role in diabetic rats.In conclusion,EMS-2.0CHA may regulate blood glucose homeostasis through the"gut-brain-pancreas"axis.The above studies indicated that the maize starch-chlorogenic acid complex constructed by extrusion method could effectively regulate the anti-enzymatic hydrolysis of maize starch,and at the same time could effectively regulate the body’s blood glucose homeostasis through the"gut-brain-pancreas"axis.The results of this study could provide design ideas for the subsequent application of phenolic acid in the field of staple food processing.