Studies On The Preparation And Antioxidant Activity Of Water-Soluble Proteins From Stress Germinated Millet

Millet,a plant of annual grass,Setaria italica,Graminea,is one of main foodstuff corp and widely cultivated in the world for its nutrition and function of health keeping.There are four kinds of millet in common:Foxtail millet or Setaria italica,Finger millet or Eleusine coracona,Little millet or Panicum sumatrense, Kodo millet or Paspalum scorbiculatum.It was reported that the ethanol extracts of millet had antioxidant activity in vitro for contenting phenols substance,and the millet protein had an antiatheromatous function,no details about the composition and antioxidant activity of water extracts from germinated millet.In this paper,the antioxidant activity of water soluble protein from stress millet is investigated for its nutrition and health keeping function being used and exploited,supplying more powerful base in science,a new way for producing functional food.This is important to the value in practice and is meaningful in science and technology.To the soluble proteins in millet,it is very low in terms of hydroxyl radicals inhibition by its water-soluble proteins,and the corresponding composition extracts from water-germinated millet is in the same case.The method of stress germination was employed and enhanced the inhibition more powerful.A fractional factorial design,central composite experimental design and response surface methodology were used to optimize the optimal growth-promoting medium,the maximum value of 58.16%was achieved.The response surface methodology was applied to research the Germination conditions of the samples of stress germinated preparations.The temperature,the time and the pH of stress medium were chosen as varying factors to optimize the key parameters affecting the hydroxyl radical inhibition.The temperature and the pH of stress medium were screened to investigate the affecting conditions.The optimal conditions were:germination temperature of 28℃,time of 52 h and pH 7.6 of stress medium.The highest inhibition(60.38%) was achieved with the optimal conditions.The purification method of water-soluble protein extrats from stress millet was studied by the ion exchange chromatography,Gel Filtration Chromatography and Hydrophobic Interaction Chromatography.And the high purified protein was obtained,so these methods were feasible in practice.Ten fractions was obtained by ion exchange chromatography,the hydroxyl radicals inhibition were tested and the results shown in this sequence:F₄＞F₅＞F₁＞F₃＞F₆＞F₂＞F₇＞F₈＞F₉＞F₁₀.Among of them, the three relative high inhibition fractions were assured by High Performance Liquid Chromatography,their molecular weight distribution are all continuous.Five fractions were gained when F4 and F5 was analyzed by Gel Filtration Chromatography distribution,their tested inhibition results shown in this sequence:P₂＞P₃＞P₁＞P₅＞P₄.Subsequently,total four fractions were available when P₂ and P₃ was analyzed by Hydrophobic Interaction Chromatography,and their assayed inhibition results shown in this sequence:B＞D＞C＞A.The relative molecular weight analysis determined that fraction of B is 11084,of D is 11136, respectively.Their purified degree were preponderate over 90%,which illustrated this array of methods were feasible to separate and purify the sample of stress protein.The change of the composition of water extracts from stress millet,the six kinds of enzyme and respiration intensity in stress and non-stress during germination,was investigated respectively.And the possibly produced mechanism of stress protein was discussed by the combination with the effect of stress on the millet physiology. In 72 hours,the change of soluble protein went up at first,then came down and subject to a stable level in the end;and compared with the non-stress in further,the stress germination tested targets,namely,the level of amylase,proteinase, polyphenols oxidase,catalase,superoxide dismutase,glutathione peroxidase and respiration intensity,showed more advanced than that of the former.It was found that stress is heavy affecting the millet physiology.The stress protein may be produced by reason of the stimulus in medium acting as both first message and second message,resulting to tyrosine phosphorylation,transcription and the translation of stress protein.The whole process is regulated by redox in cell physiological response.The antioxidant characterization was studied in the end.The antioxidant test in vitro validated that the power of deoxidization,antioxidantion in oleic acid system, potential of scavenging DPPH·and O₂^·- were very well and presented content-efficiency relations.And the experiments in vivo showed that aqueous extracts and stress protein from stress millet obviously enhanced the activity of SOD in serum and that of GSH-Px in whole blood after sub-urgent aging mice administrating the preparations.The content of MDA in serum was decreased in the same treatment process.These preparations had a marked effect of antibiooxidation and delaying aging.The content of MDA in serum was increased,moreover the activity of GSH-Px in whole blood and the activity of SOD in serum decreased significantly in sub-urgent aging mice modeled with D-DAL.All showed that D-DAL destroyed the balance of over-oxidantion/antioxidantion in body and accelerated aging,which throw a light on the aging model used in this study was built successfully.