The Study Of Bioactivities Of Tibet Wild Peach Kernel Protein Enzymatic Peptides And Peptide-Ferrous Complex

Peach kernel is the dry seed of Rosaceae Prunus persica(L.)Batch and Prunus davidiana(Carr.)Franch.Peach kernel presents various bioactive effects such as blood-activating,runchang purging and relieving cough and asthma.What is more,the protein content of dry peach kernel is high between 25%and 30%.In the Linzhi Region of Tibet,China,The resource of wild peach is abundant while a small amount of peach kernel is consumed as traditional Chinese medicinal materials and most of the peach kernel is returned to nature as waste,which cause the waste of protein.In my reaserches,I investigated the optimum condition of the enzymatic hydrolysis,next in vitro antioxidant and angiotensin converting enzyme(ACE)inhibitory capacities of hydrolytic peach kernel peptides were detected,finaly the peach kernel peptides chelating ferrous complexes were studied.All results can provide theoretical basis for the development and utilization of peach kernel peptides.(1)The study aims to optimize the enzymatic hydrolysis condition and detect in vitro antioxidant capacities of hydrolytic Peach kernel peptides.The degree of hydrolysis(DH)and extraction rate of peptide,as responses,were optimized by using Response Surface Methodology(RSM).The optimization results were substrate concentration of 3.5%,pH value 10.4,enzyme dosage 4200 U/g and hydrolysis time of 4.9 h.Under this optimum condition,the DH and extraction rate of peptide were 61.12%and 67.91%,respectively.The SDS-PAGE analysis showed that the molecular weight of peach kernel hydrolytic peptides was under 12 kDa.4 components with different molecular weight were separated from peach kernel protein hydrolysate(PKH)by ultra-filtration.The scavenging capacities of DPPH radical,ABTS radical and nitrite as well as total antioxidant capacity test were adopted to evaluate the antioxidant capacity of 4 components and PPH.Results suggested that there was a significant difference in antioxidant capacity between different molecular weight components(P<0.05).The smaller the molecular weight is,the stronger the antioxidant activity is.Finally,the highest antioxidant capacity peptide PKH4(MW<3 kDa)was analyzed by Reversed Phase High Performance Liquid Chromatography(RP-HPLC).The Reversed Phase High Performance Liquid Chromatogram result indicated that with the concentration of the organic phase increasing,most of the peak time of the PKH4(MW<3 kDa)with the strongest antioxidant activity presented in the interval of 20-30 min.(2)The peach kernel proteins were extracted and treated with alkaline proteinase to generate peach kernel protein hydrolysate(PKH)which showed ACE inhibition activity.Using ultrafiltration,the hydrolysate was separated into four fractions and their anti-ACE activities were investigated.Results showed that all PKHs had ACE inhibitory activity that is negatively related with the sizes of the PKHs.The lowest size fraction PKH4(MW<3 kDa)had the highest ACE inhibitory activity(IC50 value=0.082 mg/mL).Lineweaver-Burk plots revealed that both PKH3 and PKH4 were non-competitive ACE inhibitors.The Ki of PKH4(0.1089 mg/mL)was lower than Ki of PKH3(MW 3?5 kDa)(0.2166 mg/mL).Amino acid composition analysis showed that the PKH4 contained significantly(P<0.01)high level of hydrophobic amino acids(HAA),branched-chain amino acids(BCAA)and aromatic amino acids(AAA).Tibet wild peach kernel protein hydrolysates had anti-ACE activity and the ACE inhibition activities of peach kernel peptides are related to molecular weight size and amino acid composition and content.(3)Peach kernel peptides and ferrous chloride as raw materials.The chelating rate of iron salts and different molecular weight peach kernel peptides were studied,as well as the structural characterization and variations of PKH-?-Fe complex(PKH-?,MW<5 kDa)in vitro simulating digestion were studied.The low molecular peach kernel peptide component has highest chelating rate.The positions and peak value of UV absorption peaks of peach kernel peptides changed after chelated with ferrous ions,and the intensity of endogenous fluorescence decreased obviously.Fourier transfer infrared spectra suggested that the-COO-,N-H,O-H of peptide could participated in the chelation of peptide with ferrous iron.The results of SEM demonstrated that PKH-?-Fe has smooth spherical particle structure.During simulating gastric digestion,The ferrous iron release rate of PKH-?-Fe was significantly lower than that of ferrous sulfate and ferrous lactate(P<0.05),which can avoid the formation of a large amount of ferric hydroxide precipitation.After entering simulating intestinal digestion,part of PKH-?-Fe was in ionic or chelate state.The absorption in body of peptide-ferrous complexes could be better than ferrous sulfate and ferrous lactate.(4)The antibacterial activity of chelates formed by ferrous ions with different molecular weight peach kernel peptides and different plant peptides,and chelates of ferrous,zinc,calcium and magnesium with peach kernel peptide were compared,respectively.Then the structures of all chelates were characterized-by Fourier transform infrared spectroscopy.Study results suggested that the antibacterial activity of low molecular weight peach kernel peptide-ferrous chelate was stronger than that with high molecular weight.Peach kernel peptide-ferrous,peach kernel peptide-zinc had strong antibacterial activity.There were significant differences(P<0.05)in the antibacterial activity between ferrous-chelating peptides of peach kernel,soybean,corn and wheat.Among them,peach kernel peptide-ferrous and wheat peptide-ferrous had strongest antibacterial activity.The MICs of Escherichia coli of peach kernel peptide-ferrous and wheat peptide-ferrous both were 5 mg/mL,and the MICs of Staphylococcus aureus were 2.5 mg/mL and 5 mg/mL respectively.Fourier transform infrared spectrum illustrated that the-COO',N-H,C-O of peptides were involved in chelation reaction with ferrous ions to form the ferrous-chelating peptides.