Study On Interaction Mechanism Of Mulberry Leaf Protein With Resveratrol And Chlorogenic Acid

Mulberry leaf protein(MLP),one of the primary active components of mulberry leaves,has a number of beneficial properties that may reduce cholesterol,prevent diabetes,lower blood sugar,prevent obesity,antibacterial and antioxidant.Mulberry leaves are currently primarily used in the plant extract industry for the extraction of alkaloids and flavonoids.A significant portion of the residues that are extracted are rich in protein,polysaccharides,and other components,but most of them are not properly utilized,resulting in a serious waste of resources.Mulberry leaves and polyphenols frequently coexist during the food processing process,and this interaction alters the spatial structure of proteins,which in turn alters their functional characteristics.Therefore,it is very important to research the mechanisms and interactions between them.The interaction between mulberry leaf protein and phenols was investigated using spectroscopy analysis in this work.Resveratrol(Res)and chlorogenic acid(Cla)were chosen as the representations of polyphenols,respectively.In this study,the UV-visible spectrum,fluorescence spectrum,and Fourier transform infrared spectrum were used to characterize the interaction between MLP,Res,and Cla,and analyses were done on the interaction force and mode of action.Res and Cla’s molecules were optimized and their molecular structure were examined using density functional theory,which was also utilized to determine the molecules’ frontier molecular orbitals,quantum chemical parameters,and molecular electrostatic potential.To determine the interaction forces and locations between MLP and Res and Cla,molecular docking was employed to mimic the interaction.SEM was used to examine how interactions affected the structure of MLP.Finally,mulberry leaf protein-polyphenol complex characteristics and free radical scavenging activity were investigated.The following are the key findings of the study:(1)The results of an amino acid analysis revealed that MLP included seven of the essential amino acids.The proportions of essential amino acids(EAA)to total amino acids(TAA)and essential amino acids(EAA)to non-essential amino acids(NEAA)in the MLP were 35.10%and 54.10%,respectively.Near the ideal protein guidelines established by the Food and Agriculture Organization and World Health Organization(FAO/WHO).The addition of Res and Cla altered the shape of the MLP molecules,and Res and Cla interacted with MLP to form the novel complexes MLP-Res and MLP-Cla,according to spectroscopy studies.Res and Cla had a binding site on MLP,and their fluorescence quenching modes on MLP belonged to static quenching.Van der Waals forces and hydrogen bonds are important in their interplay.An exothermic process occurs when Res and Cla are interacted with MLP.(2)According to research using the density functional theory,Cla created intramolecular hydrogen bonds between the phenol hydroxyl groups on C13 and C14,increasing the reactivity.Res and Cla,respectively,have HOMO,LUMO,and energy gap values of-5.21,-1.16,4.05 and-5.85,-1.74,4.11 eV.The reaction sites for Res are the phenol hydroxyl groups of C1,C3 and C12 and C7=C8.The reaction sites for Cla are the phenol hydroxyl groups of C13 and C14 and the carbonyl group of C7.Res has a smaller energy gap value than Cla,and its molecules are softer and more easily polarized.Cla had a greater electrophilic index than Res,meaning that Cla was more likely to interact with the mulberry leaf protein.The system in which Res interacts with MLP is more stable because Res’s η value is higher than Cla’s.On the C1,C3 and C12 hydroxyl groups are where the majority Res nucleophilic reaction region is.The phenol hydroxyl group of C13 and C14 is where the majority of Cla nucleophilic reaction is.The hydroxyl group of C16 and the C=O of C7 are where the Cla electrophilic reaction region is.(3)According to molecular docking experiments,the hydrophobic cavity of MLP is where Res and Cla are in their best binding conformation.Res formed ahydrophobic interaction with the MLP residues Gly-185,Asn-485,Gln-186,and Ile-181 and a hydrogen bond with Tyr-118,Asp-117,Tyr-192,and Tyr-192.The lengths of the bonds were 3.12 ?,2.69 ?,3.01 ?,and 2.87 ? correspondingly.The lowest binding energy was-5.79 kcal/mol.In the MLP,Cla interacts with the following amino acids:His-116,Tyr-118,Asp-117,Gln-186,Ile-181,Gly-115,Arg-112,Ile-111,Leu-121,and SER-122.Through hydrogen bonds,Cla interacts with Ser-113,Gly-185,Gly-114,Asn-485,and Arg-478 in MLP.The lengths of the bonds are,in order,2.79 ?,3.20 ?,2.99 ?,3.02 ?,and 2.87 ?.The lowest binding energy was-4.53 kcal/mol.The lowest binding energy between Res and MLP is lower than the lowest binding energy between Cla and MLP,thus MLP-Res is more stable than MLP-Cla.(4)According to SEM examination,the addition of Res and Cla altered the appearance of MLP’s morphology,and the surfaces of MLP-Res and MLP-Cla were rougher than those of MLP.The incorporation of Res and Cla increased the thermal stability of MLP,according to the thermal stability analysis.Thermal denaturation temperature and enthalpy of denaturation rose by 4.32℃ and 10.91 J·g-1,respectively,in comparison to MLP.Thermal denaturation temperature and enthalpy of denaturation rose by 2.81℃ and 9.97 J·g-1,respectively,in comparison to MLP.The outcomes of an in vitro digestion and absorption experiment demonstrated that during the entire digestion stage,the degree of hydrolysis was MLP>MLP-Cla>MLP-Res.In the stomach digesting stage,MLP,MLP-Cla,and MLP-Res had varying degrees of hydrolysis(25.43%,23.60%,and 21.13%,respectively).The degree of hydrolysis in the intestinal digestion stage was 55.06%,53.52%,and 44.38%.(5)In vitro free radical scavenging experiment showed:The DPPH free radicals’capacity for scavenging was as follows:Cla>MLP-Cla>Res>MLP-Res>MLP.The ABTS free radicals’ capacity for scavenging was as follows:Res>Cla>MLP-Res>MLP-Cla>MLP;The OH free radicals’ capacity for scavenging was as follows:Cla>MLP-Cla>MLP>Res>MLP-Res.The order of total antioxidant activity was MLP>MLP-Res>MLP-Cla>Cla.The outcomes demonstrated that the addition of Res and Cla altered MLP’s capacity to scavenge free radicals.