| Whey is a by-product of the production of cheese and casein.It will produce excessive pollution and cause serious environmental problems if the whey was discharged directly into the environment without treatments.However,at the same time,whey retained more nutrients in the raw milk that has high nutritional value,including proteins,peptides,lipids,lactose,minerals and vitamins.Therefore,whey has great development potential as a by-product of production.Whey protein,one of the main components of whey,has high nutritional value and functional characteristics,which is widely used in various foods and is an ideal source of bioactive peptides.In this paper,the effects of ultrahigh pressure pretreatment on whey protein structure were studied with whey protein as research object.The process of enzymatic hydrolysis of whey protein after ultrahigh pressure pretreatment was screened.The optimum enzyme species and enzymatic hydrolysis conditions for the preparation of peptides with antioxidant activity and DPP-? inhibitory activity were determined.The enzymatic hydrolysis process was optimized by response surface.Peptides with antioxidant activity and DPP-? inhibitory activity in enzymatic hydrolysates were isolated and purified.The main conclusions are as follows:(1)The whey protein was treatment with ultrahigh pressure under different pressure and time conditions,the determination of the electrophoresis characteristics,surface hydrophobicity,fourier transform infrared spectrum,sulphur content,endogenous fluorescence spectrum indicated that electrophoresis has not changed significantly,surface hydrophobicity has a significant increase,alpha helix content increased,the content of beta folding down,free sulfhydryl content increased significantly,endogenous fluorescence intensity change significantly after the ultrahigh pressure processing.Among all the treatment conditions,the ultrahigh pressure treatment under the 400 MPa for 30 min had the most significant changes on the secondary and tertiary structures of whey proteins.(2)The whey protein treated with ultrahigh pressure was used as substrate,the optimum temperature,time,enzyme dosage and p H of whey protein hydrolyzed by trypsin,pepsin,alkaline protease,papain and acid protease were determined by single factor experiment.The optimum condition of whey protein hydrolyzed by pepsin was under 40 ? for 90 min with the enzyme dosage of 1.5% in the environment of p H 2.5.While for acid protease,The optimum condition was under 55 ? for 60 min with the enzyme dosage of 1.0% in the environment of p H 2.5.(3)The single enzyme hydrolysis,one-step hydrolysis method and two-step hydrolysis method were used to hydrolyze whey protein,the different enzymatic hydrolysis process were screened with the antioxidant activity and DPP-? inhibitory activity as index.The results showed that the substrate by two-step hydrolysis method that adding pepsase to hydrolyze whey protein for 60 min and the acid protease was then added for 60 min possessed the highest antioxidant activity of 65.18% as well as the DPPIV inhibitory activity of 62.28%,respectively.In addition,the antioxidant activity of some proteases by single enzyme hydrolysis were even higher than that of one-step hydrolysis or two-step hydrolysis of this enzyme.(4)The optimization experiments of response surface were carried out with the scavenging rate of ABTS free radicals as response value for pepsin and acid protease respectively.The results showed that the scavenging rate of ABTS free radicals for pepsin reached 35.97 % under 41 ? with the enzyme dosage of 1.4% in the environment of p H 2.6.The scavenging rate of ABTS free radicals for acid protease reached 52.51% under 55 ? with the enzyme dosage of 1% in the environment of p H 2.4.(5)Whey protein was hydrolyzed by pepsin and acid protease in two steps after response surface optimization,the ABTS radical scavenging rate and DPP-? inhibition rate increased 2.61%,3.03% and reached to 67.79%,65.31% respectively.(6)The whey protease hydrolysates were separated by gel filtration chromatography and RP-HPLC,and the components with the highest ABTS radical scavenging and DPPIV inhibitory rates were collected.The ABTS radical scavenging and DPP-? inhibitory rates reached 82.96% and 68.83% respectively with the concentration of 1 mg/m L.(7)The amino acid sequence of the component was determined by LC-MS/MS,and 14 polypeptides were selected for synthesis,then the biological activity of synthetic peptides were detected.The results showed that DDQNPHSSN polypeptide had higher antioxidant activity and DPP-? inhibitory activity at the same time.When the concentration was 1 mg/m L,the ABTS radical scavenging rate,DPPH radical scavenging rate and reducing power were significant,which reached 91.42%,88.76% and 0.637%,respectively,and the DPP-? inhibitory activity reached 66.28%. |
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