胁迫粟水溶性蛋白的制备与抗氧化性研究
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摘要
粟(Millet)粟属禾本科(Gramineae)、狗尾草属(Setaria italica)草本植物,是世界上主要粮食作物之一。粟可分为小米(Foxtail millet,学名:Setaria italica)、龙爪稷(Finger millet,学名:Eleusine coracona)、小粟(Little millet,学名:Panicum sumatrense)、库都粟(Kodo millet,学名:Paspalum scorbiculatum)四种,在世界各地均有分布,有很好的营养和保健功能。已有报道粟的乙醇粗提物因含有多酚类物质而具有体内外抗氧化作用,粟米蛋白具有抗动脉粥样化的功能。但对其萌发水提取物的主要成分及其抗氧化作用尚未见详细的报道,本文研究其胁迫萌发水提取物中胁迫蛋白的抗氧化作用,为开发利用粟的营养价值和保健功能提供有力的科学依据,为研究开发保健食品提供可以借鉴的新方法,具有有重要的学术意义和实践价值。
粟的四种可溶性粟米蛋白中,其水溶性蛋白羟自由基清除活性极低,水萌发后的水提取物活性也很低,采用胁迫萌发的方法有力地提高了其活性。使用析因实验和中心组合实验方法优化得到了最佳的胁迫培养基组成,提取物的羟自由基清除作用达到使用优化得到的培养基萌发粟,得到水溶性蛋白的羟自由基清除作用达到58.16%。
在研究粟的胁迫萌发制备条件时,选择温度、时间和pH值为影响因素,使用响应面分析方法,找到了影响羟自由基抑制活性的关键参数,优化了温度和pH值两个关键参数,得到了最佳萌发条件。使用优化的胁迫培养基,在胁迫萌发温度28℃、萌发pH值7.6、胁迫萌发时间52h得到水溶性蛋白的羟自由基清除作用达到60.18%。
研究了胁迫萌发粟水溶性蛋白的分离纯化方法,分别使用了离子交换色谱法、凝胶过滤色谱法、反相色谱法和高效液相色谱法对其进行分离纯化,并得到了高纯度的胁迫蛋白。使用离子交换色谱分析了胁迫粟水提取物的分子量分布情况,分离得到了十个组分,鉴定其羟自由基清除活性发现其活性大小顺序为:F_4＞F_5＞F_1＞F_3＞F_6＞F_2＞F_7＞F_8＞F_9＞F_(10),确定三个较大活性组分含氮物的分子量分布全都是连续的;使用凝胶过滤色谱分析了F_4和F_5的组成情况,共得到五个组分,活性测定结果说明它们的活性大小顺序是:P_2＞P_3＞P_1＞P_5＞P_4;使用反相色谱分析了P_2和P_3的组成情况,共分离得到了四个主要组分,活性测定其大小顺序为B＞D＞C＞A;相对分子质量分析确定组分B的相对分子质量为11084,D为11136;二者纯度均达到90%以上。采用以上分离方法是行之有效的。
研究了胁迫萌发过程中水提取物的成分变化和粟胁迫萌发与无胁迫萌发的六种酶及活性强度变化情况,并结合胁迫对粟的生理影响对胁迫蛋白产生的可能机理进行了探讨。胁迫萌发72小时内,可溶性蛋白上升再下降最后趋于稳定;淀粉酶、蛋白酶、多酚氧化酶、过氧化氢酶、超氧化物歧化酶和谷光甘肽过氧化物酶和呼吸强度的胁迫萌发指标与相应无胁迫萌发的指标比较表明,萌发进程也比无胁迫萌发大大提前;胁迫萌发对粟的生理影响很大,胁迫蛋白的产生可能是由于培养基中的刺激物兼有第一信使和第二信使的作用,导致发生蛋白酪氨酸磷酸化作用和mRNA的形成,并翻译形成胁迫蛋白,整个过程受氧化还原作用的调控。
研究了粟胁迫蛋白的抗氧化性质。体外抗氧化实验证明,粟胁迫蛋白的还原能力,对亚油酸体系中抗氧化作用、清除DPPH·和O_2·~—的能力,均与其浓度呈量效关系;体内抗氧化作用实验证明,灌服胁迫粟水提取物和粟胁迫蛋白样品可以显著提高D-半乳糖导致亚急性衰老小鼠血清中SOD和全血中GSH-Px的活力,能够清除过剩自由基,显著降低小鼠血清中MDA含量,起延缓衰老的作用,所造衰老模型是成功的。胁迫粟水提取物效果比粟胁迫蛋白更显著。
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:F_4＞F_5＞F_1＞F_3＞F_6＞F_2＞F_7＞F_8＞F_9＞F_(10).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:P_2＞P_3＞P_1＞P_5＞P_4.Subsequently,total four fractions were available when P_2 and P_3 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 O_2~(·-) 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.
粟的四种可溶性粟米蛋白中,其水溶性蛋白羟自由基清除活性极低,水萌发后的水提取物活性也很低,采用胁迫萌发的方法有力地提高了其活性。使用析因实验和中心组合实验方法优化得到了最佳的胁迫培养基组成,提取物的羟自由基清除作用达到使用优化得到的培养基萌发粟,得到水溶性蛋白的羟自由基清除作用达到58.16%。
在研究粟的胁迫萌发制备条件时,选择温度、时间和pH值为影响因素,使用响应面分析方法,找到了影响羟自由基抑制活性的关键参数,优化了温度和pH值两个关键参数,得到了最佳萌发条件。使用优化的胁迫培养基,在胁迫萌发温度28℃、萌发pH值7.6、胁迫萌发时间52h得到水溶性蛋白的羟自由基清除作用达到60.18%。
研究了胁迫萌发粟水溶性蛋白的分离纯化方法,分别使用了离子交换色谱法、凝胶过滤色谱法、反相色谱法和高效液相色谱法对其进行分离纯化,并得到了高纯度的胁迫蛋白。使用离子交换色谱分析了胁迫粟水提取物的分子量分布情况,分离得到了十个组分,鉴定其羟自由基清除活性发现其活性大小顺序为:F_4＞F_5＞F_1＞F_3＞F_6＞F_2＞F_7＞F_8＞F_9＞F_(10),确定三个较大活性组分含氮物的分子量分布全都是连续的;使用凝胶过滤色谱分析了F_4和F_5的组成情况,共得到五个组分,活性测定结果说明它们的活性大小顺序是:P_2＞P_3＞P_1＞P_5＞P_4;使用反相色谱分析了P_2和P_3的组成情况,共分离得到了四个主要组分,活性测定其大小顺序为B＞D＞C＞A;相对分子质量分析确定组分B的相对分子质量为11084,D为11136;二者纯度均达到90%以上。采用以上分离方法是行之有效的。
研究了胁迫萌发过程中水提取物的成分变化和粟胁迫萌发与无胁迫萌发的六种酶及活性强度变化情况,并结合胁迫对粟的生理影响对胁迫蛋白产生的可能机理进行了探讨。胁迫萌发72小时内,可溶性蛋白上升再下降最后趋于稳定;淀粉酶、蛋白酶、多酚氧化酶、过氧化氢酶、超氧化物歧化酶和谷光甘肽过氧化物酶和呼吸强度的胁迫萌发指标与相应无胁迫萌发的指标比较表明,萌发进程也比无胁迫萌发大大提前;胁迫萌发对粟的生理影响很大,胁迫蛋白的产生可能是由于培养基中的刺激物兼有第一信使和第二信使的作用,导致发生蛋白酪氨酸磷酸化作用和mRNA的形成,并翻译形成胁迫蛋白,整个过程受氧化还原作用的调控。
研究了粟胁迫蛋白的抗氧化性质。体外抗氧化实验证明,粟胁迫蛋白的还原能力,对亚油酸体系中抗氧化作用、清除DPPH·和O_2·~—的能力,均与其浓度呈量效关系;体内抗氧化作用实验证明,灌服胁迫粟水提取物和粟胁迫蛋白样品可以显著提高D-半乳糖导致亚急性衰老小鼠血清中SOD和全血中GSH-Px的活力,能够清除过剩自由基,显著降低小鼠血清中MDA含量,起延缓衰老的作用,所造衰老模型是成功的。胁迫粟水提取物效果比粟胁迫蛋白更显著。
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:F_4＞F_5＞F_1＞F_3＞F_6＞F_2＞F_7＞F_8＞F_9＞F_(10).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:P_2＞P_3＞P_1＞P_5＞P_4.Subsequently,total four fractions were available when P_2 and P_3 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 O_2~(·-) 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.
引文
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