| Wheat germ,as the main by-product of wheat flour processing,contains about30% protein,is a high quality source of vegetable protein.At present,it is mostly used as feed or discarded directly,causing a huge waste of protein resources.In this paper,we extracted protein from wheat germ by subcritical water,investigated its structure and functional properties,and compared with traditional alkali-solution method.Based on this,the method of bioactivity tracing was used to obtain antioxidant peptides from the hydrolysis of wheat germ protein,and the composition of these peptides was analyzed.Finally,Caco-2 cell model was used to evaluate the absorption and transport of antioxidant peptides.The present study may provide theoretical support for the development and exploitation from wheat germ.The main contents and results are as follows:1.Extraction of wheat germ protein by subcritical water and its structure and functional properties.(1)Single-factor and response surface methodology analysis experiments were applied to optimize the protein extraction from wheat germ protein by subcritical water.The results showed that under the optimum extraction condition(pH 9.40,130 ℃for 15 min with the solid-liquid ratio of 1:20),the yield of protein was 42.25%,which was increased by 14.12% compared with traditional alkali-solution method.(2)There were somewhaat different in the secondary structure of wheat germ protein obtained between the by subcritical water and alkali-soluble method,however the functional properties were basically same.Compared with traditional alkalisolution method,the α-helix was decreased,while β-sheet and random coil were increased significantly,the structure of the conjugates displayed more flexibility,and the surface hydrophobicity of the protein was enhanced.There were some holes in the surface of the protein and the protein was found aggregated;The amino acid analysis result revealed that the protein extracted by subcritical water(29.02%)had a higher relative concentration of hydrophobic amino acid than that of alkali-solution extraction(25.53%).2.Antioxidant peptides obtained by enzymatic hydrolysis of wheat germ protein.(1)Wheat germ protein was hydrolized by using alcalase,the degree of hydrolysis(DH)and the yield of peptides were 16.35% and 63.31%,respectively.The scavenging activity of the hydrolyzation against DPPH· was all dose dependent,andthe IC50 value was 4.16mg/m L.Wheat germ protein hydrolysates were ultrafiltrated with different molecular.Four peptides with different molecular weight were obtained,the proportion of them was as follows: UF-1(<1KDa)47.45%,UF-2(1-3KDa)10.54%,UF-3(3-5KDa)11.86%,UF-4(>5KDa)30.15%,of which the molecular weight below3 KDa accounted for 57.99%.The reults of antioxidant capacity showed that the polypeptide with the molecular weight below 1KDa had the strongest antioxidant capacity.(2)Seven components were obtained from UF-1(MD<1KDa)by gel filtration Sephadex G-15.The results of each component DPPH scavenging ability showed that components G-15-4 and G-15-3 had stronger antioxidant activity than that of others.The IC50 of the components of G-15-4 and G-15-3 were 1.17 mg/mL and 1.23 mg/mL,respectively.It could be speculated from molecular weight distribution that the molecular weight of components of G-15-4 and G-15-3 were MD(181-370Da),MD(370-515Da),respectively.According to the analysis of amino acids of the purified G-15-4 and G-15-3,the main amino acids composition of G-15-4 were Phe,Tyr,Glu,His and Pro,and the main amino acids of G-15-3 were His,Glu,Phe,Tyr and Leu.3.Absorption and transportation of the component of G-15-4.(1)When the cells were cultured for 21 days,the TEER value of Caco-2 cells monolayer exceeded 1000 Ω·cm2,the TEER value of Caco-2 cells monolayer exceeded 800 Ω·cm2 when it was in the transportation medium.The alkaline phosphatase activity of AP side of the cells was 3 times of the BL side.The ratio of alkaline phosphatase activity of AP side and BL side of the Caco-2 cell model was about 3:1.The apparent permeability coefficient(Papp)of fluorescent yellow is less than 1.0×10-6 cm/s.It indicated that the integrality of Caco-2 cell monolayers has been established and can be used as a model for the studying of absorption and transportation in vitro.(2)The component of G-15-4 had no toxicity to Caco-2 cell in the concentration of 0.125-2mg/mL at 24 h with the determination of MTT.So we made a choice of the concentrations of 0.25,0.50,0.75mg/m L of G-15-4 for the transmembrane transportation experiment..(3)The absorption of the component of G-15-4 on the Caco-2 cell model with concentration and time dependent manner,and the ratio of the Papp both sides of BL and AP was between 0.5 and 1,indicating that the transmembrane transportation wasmainly passive transport;The transmembrane transport of G-15-4 on the Caco-2 cell model was related to the temperature,but P-gp glycoprotein did not participate in its transportation.It still needs to continue the depth study to confirm which the vector was. |
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