Study On Inhibition Of Enzymatic Hydrolysis Efficiency By Amphiphilic Substances During Starch Digestion

Regulating the rate of amylase hydrolysis is important for both industrial applications and human health management.Previous studies have focused on the discovery of natural amylase inhibitors and the synthesis of artificial amylase inhibitors,but there is no clear understanding of mechanism of the influence of amphiphilic molecules on the rate of amylase digestion during digestion.In this study,Pluronic was used as the amphiphilic substance model to explore the change of amylase and substrate starch structure in the amphiphilic substance system and the influence of temperature,concentration and other factors on hydrolysis,so as to obtain the mechanism of amphiphilic substance on the inhibition of amylase hydrolysis.?1?Enzymatic hydrolysis of starch in an amphiphilic system of Pluronic modelPluronic was used as an amphipathic substance model to study its effect on amylolytic rate.Pluronic?PL82.5,PL50,PL40,PL10?was added in the amylase hydrolysis experiment.In the 4-hour amylase hydrolysis experiment,the amylase hydrolysis rate of the blank group was 98%,and the experiment of adding PL10 was carried out.The enzymatic hydrolysis rate of the group was reduced by 16%compared with the blank group,and the inhibition of amylase hydrolysis by Pluronic showed a regular change with the hydrophobicity and concentration of Pluronic,the hydrophobicity increased,and the enzymatic hydrolysis rate showed a downward trend.The binding mode and site of interaction between Pluronic and amylose were studied by salicylic acid fluorescent probe method and ultraviolet spectroscopy.The fluorescence intensity at 406 nm and the absorbance at 540 nm-660 nm were observed.The change determines that the Pluronic and amylose binding sites are hydrophobic regions of amylose in a hydrophobic interaction.The effects of Pluronic and starch on the structure of starch were investigated by Fourier transform infrared spectroscopy and Raman spectroscopy.The results showed that the infrared absorption peaks showed obvious regularity at 576 cm^-1,996 cm^-11 and 1147 cm^-1.Shift,the offset value increases with the added Pluronic hydrophobicity.?2?Effect of amphiphilic molecules on the structure of?-amylaseBy studying the endogenous fluorescence of?-amylase and its change in absorption peak at 250 nm,it was found that the interaction between Pluronic and?-amylase resulted in a change in the spatial structure of amylase,and this change was related to the degree of hydrophobicity of Pluronic related.The effect of Pluronic on the secondary structure of?-amylase was further studied by circular dichroism?CD?.The?-helical structure and?-sheet structure increased with increasing hydrophobicity,and the?-turn angle decreased with hydrophobicity.The content of the random coil structure did not change significantly.Fourier deconvolution was performed on the absorption peak of 1700 cm^-1-1600 cm^-1?C=O stretching vibration?in the amide I band by Fourier transform infrared spectroscopy.The secondary structure change was studied and the results were consistent with the CD results.Synchronous fluorescence spectroscopy was used to study the effects of Pluronic on the microenvironment of?-amylase tyrosine and tryptophan.It was found that the microenvironment of tyrosine was enhanced in polarity and the fluorescence intensity was decreased.?3?Verification of the mechanism of amylolytic hydrolysis of fatty acid system on amphiphilic substancesTo validate the results obtained in the Pluronic model system,we used fatty acids with different chain lengths to further investigate how molecules with different degrees of hydrophobicity affect the efficiency of amylase hydrolysis.It was found that the fatty acid system also showed inhibition of the amylase digestion rate.Through the salicylic acid fluorescence experiment and the color difference experiment,it was found that the hydrophobic fatty acid and amylose interaction sites are hydrophobic regions of amylose,and the action mode is hydrophobic interaction,and the degree of binding is affected by the hydrophobicity of fatty acids.The effect of fatty acid on starch structure was verified by infrared experiments.The infrared absorption peaks showed obvious regular shift at 576 cm^-1?996 cm^-11 and 1147 cm^-1,and the offset value increased with the added Pluronic hydrophobicity.It was found in the fluorescence spectrum of the interaction between fatty acids and?-amylase that the more hydrophobic fatty acids caused the greater the decrease in fluorescence intensity of?-amylase at 346 nm.The experimental results are in agreement with the results obtained in the Pluronic system.In summary,this study used different pro-hydrophobic Pluronic as an amphipathic substance model and found that it has an inhibitory effect on amylase hydrolysis.Through infrared spectroscopy,ultraviolet spectroscopy,Raman spectroscopy and fluorescent probe experiments,it was found that the binding of amphiphilic Pluronic to the hydrophobic part of amylose affects the structure of starch;Pluronic also makes the spatial structure and amino acid of?-amylase The microenvironment changes,and the combined effect of the two causes the enzymatic hydrolysis of starch to be inhibited.Amylase hydrolysis experiments in different hydrophilic and hydrophobic fatty acid systems further validated this conclusion.This study is the first to explore the mechanism of amylolytic enzymatic hydrolysis from amylase and substrate.It not only analyzes the interaction mechanism between amphiphilic molecules and starch and amylase at the molecular level,but also changes the system.Hydrophilicity provides a new idea for the controlled digestion of starch.