| Almond(Amygdalus communis L.)is a kind of nut food,which is widely used in the food industry.In addition to the use of almonds for direct consumption,another use is to extract almond oil.During the oil extraction process,a large amount of defatted almond cake will be produced.This cake is rich in protein and has a complete range of amino acids.Blood lipids,lowering blood pressure,improving immunity,anti-cancer and antibacterial and other biological activities.Diabetes has become one of the four chronic diseases that threaten human health together with cardiovascular and cerebrovascular diseases,cancer,and chronic respiratory diseases.α-glucosidase inhibitors are a class of effective drugs for the treatment of diabetes.However,most of the first-line α-glucosidase inhibitors are non-food-derived chemical drugs with more or less side effects.Enzyme inhibitors have become one of the hot spots in the academic circles.It would be a very meaningful work to quickly screen out highly active α-glucosidase inhibitory peptides using almond protein as a raw material.In this paper,the virtual enzymatic hydrolysis of almond protein was carried out by computer,and the obtained almond peptide was screened,and the main binding mode of α-glucosidase and almond peptide was clarified by molecular docking technology;Hep G2 cells were used as a model to study the effect of almond peptide on improving insulin resistance Mechanism.Finally,sodium alginate-bovine serum albumin was used to nano-embed the amygdalin peptide and prepare hypoglycemic oral liquid,optimize the preparation process of oral liquid and test its stability.The main research content of this paper is as follows:(1)Through the amino acid sequence analysis of almond protein in the uniprot database,the protein with high α-glucosidase inhibitory potential was screened out,and the almond protein was obtained by virtual enzymolysis using Peptide Cutter,Peptide Ranker,Innovagen and other online tools oligopeptides,and screen out four α-glucosidase inhibitory peptides PGGD(proline-glycine-glycine-aspartic acid)with good biological activity,water solubility and absorption,distribution,metabolism,excretion,etc.),PPK(Proline-Proline-Lysine),GR(Glycine-Arginine)and PR(Proline-Arginine).The oligopeptides PGGD,PPK,and PR with high binding ability to α-glucosidase were screened by molecular docking technology,and the mechanism of interaction between peptides and α-glucosidase was studied.In vitro tests were used to evaluate the inhibitory effect of amygdalin PR and PGGD on α-glucosidase,and their IC50 values were 19.79 ±0.006 μmol/L and 20.63 ± 0.05 μmol/L).α-glucosidase inhibitory peptides,the amino acid sequences of which are PR and PGGD respectively.(2)The effects of PR and PGGD,two amygdalin peptides,on alleviating insulin resistance in Hep G2 cells were determined and further screened,and the mechanism of amygdalin PR on glucose metabolism in IR Hep G2 cells was studied from three metabolic pathways: glycogen synthesis,glucose transport,and glucose uptake.The results showed that amygdalin PR was better than PGGD at alleviating insulin resistance in cells.And PR can increase the expression and transport of GLUT4,regulate the phosphorylation of the S9 site in GSK-3β,activate pathways such as IRS-1/PI3K/Akt and AMPK,increase the expression of GSK-3β,and improve the glucose absorption capacity.((3)Sodium alginate-bovine serum albumin(BSA)was used as the wall material to embed amygdalin peptide,and combined with single factor and orthogonal experiments,the optimal process for nanoparticle preparation and amygdalin peptide embedding was determined.The results showed that: p H 5.5,volume ratio of sodium alginate and bovine serum albumin 4:1,covalent compound concentration 4.0 mg/m L were the optimal conditions for the preparation of nanoparticles.Under this condition,the particle size is95.1±3.06 nm,and the absolute value of the potential is 10.55±0.96 mv.The optimal embedding conditions of the almond peptide nanoparticle complex are core-to-wall ratio1:6,p H value 4,and ultrasonic time 6 minutes.The results can be obtained through the in vitro simulated digestion test: this complex can guarantee the hypoglycemic effect of almonds to a certain extent.Functional stability of peptide PR in the gut.(4)Through single factor combined with orthogonal test,the preparation process of almond peptide hypoglycemic oral liquid was optimized with the optimization index of centrifuge stability rate,and its properties and colony number were tested under different temperature and light time conditions,and the final product was tested in vitro The simulated digestion test was used to test its hypoglycemic effect.The results showed that when the compound stabilizers pectin,xanthan gum and citrus fiber were all added in an amount of 0.06%,the stability of the oral solution was better.And its stability is affected by temperature and storage time.Through the measurement of various microbial indicators after the oral liquid was stored for 20 days,it was found that the total number of colonies,Escherichia coli and mold content of the product were all within the specified range and reached the national standard.Finally,after simulated digestion,its α-glucosidase inhibitory ability was determined,and the hypoglycemic effect of almond hypoglycemic peptide oral liquid(IC50 value was 2.29 ± 0.003 mg/m L was comparable to that of commercially available acarbose tablets(IC50 value was 2.05 ± 0.013 mg/m L). |
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