Enzymatic Hydrolysis Process Of Eggshell Membrane Peptides And Its Effect On Alopecia Model Mice

Eggshell membrane refers to the clear film linling eggshells,commonly known as"phoenix cloth".As a prominent country for poultry breeding,China has a large output of eggshell membrane waste.The membrane has high nutritional value as a high-quality keratin protein resource,though it has not yet been fully developed and utilized.In recent years,there have been relatively few studies on nutritional supplement for hair loss,which plagues more and more adults.The development of a safer and more effective nutritional supplement is particularly important for preventing hair loss and promoting hair growth.The purpose of this thesis is to explore the biological relationship between eggshell membrane peptides and hair loss prevention.In the study,which is the subject of this thesis,eggshell membrane was used as the raw material to optimize the enzymatic hydrolysis process.The eggshell membrane was analyzed before and after the enzymatic hydrolysis with a scanning electron microscope,and the molecular weight and amino acid content of the eggshell membrane were determined.The chemical reaction method used to determine the antioxidant capacity of eggshell membrane peptides and the relationship between its antioxidant capacity and hair loss are discussed below.In order to provide theoretical support for the application of eggshell membrane peptides in the field of diet therapy for people with hair loss,the effect of eggshell membrane peptides on alopecia model mice was studied through animal experiments.The results of the study are as follows:First,using high-protein eggshell membrane as raw material and hydrolysis as the measurement index,the alkaline protease(AP-200a)was selected as the best protease,and optimized and analyzed through single factor and response surface experiments to determine the best Enzymatic hydrolysis conditions as:material-liquid ratio 1:9.5(w/v),enzyme addition(E/S)2.4%,enzymatic hydrolysis time 5 hours.Under these conditions,the degree of hydrolysis of the eggshell membrane polypeptide is 27.15%.The characteristics of the eggshell membrane peptides were studied,and the morphological characteristics were observed through scanning electron microscopy.It was found that the enzymatic hydrolysis reaction can destroy the fiber network structure of the eggshell membrane,which solves the problem of the eggshell membrane being difficult to decompose and difficult to dissolve in water.Using high performance liquid chromatography,it was determined that peptides with a relative molecular mass below5000 Da accounted for 98.1%,of which small molecule peptides less than 1000 Da accounted for 90.9%.An automatic amino acid analyzer was used to analyze its amino acid composition and found that the total content of hydrolyzed amino acids was 68.46 g/100g,which was 3.53 times that of free amino acids,of which essential amino acids accounted for 33.83%,hydrophobic amino acids accounted for 36.06%,and aromatic amino acids accounted for 7.52%.Therefore,this kind of enzymatic hydrolysis method can effectively decompose the eggshell membrane,improve the utilization rate of the eggshell membrane,and more fully utilize the nutrients of the eggshell membrane.This study examined the in vitro antioxidant activity of eggshell membrane polypeptides.Taking Vc as a reference object,the scavenging ability of eggshell membrane polypeptides on DPPH·,ABTS⁺·,·OH,and O₂^-·were investigated.The results showed that the scavenging abilities of the envelope peptides on DPPH·and ABTS⁺·were 93.03±0.51%and 94.53±0.92%,respectively,which were close to the effect of Vc.Its ability to remove·OH and O₂^-·is 43.33±1.10%and 53.40±0.70%,which is half of the Vc effect.Through the experiment of scavenging ABTS free radicals,it was verified that the alkaline protease(AP-200a)played a very good role in catalyzing the reaction during the enzymolysis process,and showed almost no antioxidant activity.Therefore,the eggshell membrane polypeptide has a certain antioxidant capacity and can effectively remove free radicals in the body.Finally,C57BL/6J mice were used to model hair loss,and the effect of eggshell membrane polypeptides on alopecia model mice was studied.At 28 days after the administration,the mice were euthanized.The skin and hair of the same area of the depilatory area on the back of each group of mice were taken,and the weight of the hair was weighed.The back skin tissue of each group of mice was cut into paraffin sections,and the number of hair follicles was measured by HE Statistical analysis.The results showed that compared with the model group,the hair weight of mice in the positive control group and the high-dose group increased significantly,with a significant difference(P<0.01),and the middle-dose group had a significant difference(P<0.05).Compared with the model group,the average number of hair follicles in the positive control group,high-dose group and medium-dose group increased significantly,with a significant difference(P<0.01),and a significant difference in the low-dose group(P<0.05).The demonstrates that the eggshell membrane polypeptide has an effect on alopecia model mice and has the effect of promoting the hair growth of mice.