| Triticum aestivum Linn.is one of the most widely cultivated plants in the world.Wheat germ is the life source of wheat,although the weight is 2% of a wheat kernel,it has high nutritional and medicinal values.The protein is rich in wheat germ,which accounts for 30% of defatted wheat germ.Four protein fractions in wheat germ were extracted using Osborne method,namely albumin,globulin,prolamine and glutelin.The immunomodulating activities of four fractions were evaluated by in vitro cell method,and the fraction with the highest immunomodulating activity were successfully selected.Meanwhile,globulin,with low immunomodulating activity but high proportion and nutrition,was hydrolyzed with proteases,and the alcalase hydrolysate(AH)with the highest immunomodulating activity was obtained.Through purification,characterization,an immunopeptide P3-1-1 with high purity was obtained.The immune mechanism of P3-1-1 was investigated;simultaneously,the antiinflammtory and anti-hemolytic activities of the immunomodulatory peptide were studied.The results are as follows:Glutelin had the best immunomodulatory activities among four fractions,but the proportion was lower than albumin and globulin in wheat germ,moreover,its solubility and digestibility were poor.Although immunomodulatory activities of albumin was less than that of glutelin,the proportion,solubility and digestibility were preceded to those of glutelin,which can be used as an excellent protein source.The proportion of globulin in wheat germ was less than that of albumin,but its immunomodulatory activities was unsatisfactory.After alcalase hydrolysis for 3h,the immunomodulatory activities in RAW 264.7 cells including phagocytic activity,secretion of produce nitric oxide(NO),interleukins-6(IL-6),tumor necrosis factors-?(TNF-?)and reactive oxygen species(ROS)were respectively increased by 72%,37.73%,87.72% and 127.25%.The degree of hydrolysis(DH)and hydrophobic value of AH were 19.07% and 5985.17,respectively.Besides the solubility,the functionality of AH was lower than that of globulin.AH was digested in vitro,at pepsin digestion stage,the immunomodulatory activities decreased to a large extent;after pancreatin digestion,the immunomodulatory activities increased,although they were still lower than those of AH,they also possessed high immunomodulatory activities.AH was purified with Diethylaminoethyl(DEAE)sepharose Fast Flow,and four fractions were obtained,thereinto P3 exhibited the highest immunomodulatory activites;P3 was further purified with Sephadex G-15,two fractions were obtained,and P3-1 exhibited the highest immunomodulatory activites.P3-1 was then purified with semi-preparative reverse phase high-performance liquid chromatography(RP-HPLC),only one peak P3-1-1 was acquired.The molecular weight of P3-1-1 was 655.97 Da,the amino acid sequence was NH2-Glu-CysPhe-Ser-Thr-Ala-COOH,and the hydrophobic amino acids were occupied 50%.P3-1-1 could specifically reacted with Toll like receptor 2 and 4 which were on the surface of RAW 264.7 cells,the proposed signal pathways to activate cells to produce NO,IL-6,IL-10 and TNF-? were IRAK/TRAF6/JNK/JUN and IRAK/TRAF6/IKK/NF-?B,and ROS might further activate P13K/Akt/MAPK/NF-?B pathway to produce TNF-?.Apart from immunomodulating acitivity,P3-1 significantly decreased the LPS-induced proinflammtory cytokines(NO,IL-6 and TNF-?)of RAW 264.7 cells(p<0.05).P3-1 also significantly decreased AAPH-induced hemolysis of human erythrocytes,significantly decreased the ROS production,and the expression of antioxidases(catalase [CAT],superoxide dismutase [SOD] and glutathione peroxidase [GSH-Px])(p<0.05).In this study,both albumin and AH could be used as good immunomodulators,and applied as functional food for preventive treatment of hypoimmunity diseases.This study provides theory evidence in synthetically application of wheat germ proteins,research ideas in studying the mechanism of immunomodulatory peptides,and new materials in functional food field. |
PDF Full Text Request