【作者】 郭红英；

【导师】 董英；

【作者基本信息】 江苏大学 ， 食品科学， 2009， 博士

【摘要】 小麦是我国的主要粮食作物之一，年产量在1亿t以上。作为小麦加工的副产物，目前我国麦胚年蕴藏量达200万t。麦胚是小麦籽粒的生命源泉，其中含有约30％的蛋白质，是一种优质的植物蛋白质资源。麦胚蛋白通过特异的蛋白酶水解可生成具有抗氧化活性的氨基酸序列，即抗氧化肽。已有研究表明，人类许多慢性疾病及衰老现象均和体内的自由基水平失衡有关，过量的自由基对机体产生氧化性损伤，当损伤不能及时修复并且累积到一定程度就会导致疾病的发生。而来源于食品的天然抗氧化剂则有助于消除过量的自由基，维持人体内的自由基平衡。因此，利用麦胚蛋白制备对抗自由基、延缓衰老的功能性食品或食品配料，既可为麦胚等富含蛋白质的农副产品资源的深度开发和利用提供理论依据，也为实现小麦资源的综合利用、提高其附加值开辟新的途径。本论文以脱脂麦胚为原料，对麦胚蛋白在稀碱和微细化处理条件下的提取工艺进行研究，并对麦胚蛋白的酶解工艺、抗氧化活性及作用机理进行研究，主要研究内容及结果如下：1．首先采用锤式粉碎与胶体粉碎相结合的方法对脱脂麦胚进行微细化处理，然后利用碱提、超滤浓缩法提取制备麦胚蛋白，并通过Box-Behnken中心组合设计（CCD）及响应面分析（RSM）建立预测麦胚蛋白提取率的二次多项数学模型，优化蛋白质的提取工艺，在麦胚粒度为23μm、pH值9．5、提取温度51℃的条件下，经微细化处理的蛋白提取率为68．6％，比未经微细化处理的（过80目筛）提高30．63％。2．采用SDS—PAGE法测定麦胚蛋白的分子量为11．6kDa-93．8kDa。功能特性研究结果表明，麦胚蛋白在pH4-5范围内溶解度最低，与大豆分离蛋白相比，其吸油性、乳化活性、发泡性较强，吸水性、乳化稳定性、泡沫稳定性相对较弱。3．以DH及酶解产物对DPPH·清除率为考察指标，从5种商品蛋白酶中确定了麦胚蛋白的最适水解酶为碱性蛋白酶Alcalase。预处理工艺研究结果表明，对麦胚蛋白进行高速剪切乳化5min，可将其水解度（DH）和肽得率（TCA-PSI）分别提高29．85％和12．53％。通过单因素试验确定了高速剪切乳化预处理（22000r／min，5min）条件下Alcalase水解麦胚蛋白的最适参数为：酶用量[E]／[S]为4000U／g、底物浓度为4％、pH为8．5、温度55℃、时间240min。在此条件下，体系水解度为18．61±0．23％，肽得率为71．02±0．45％，平均链长为5．37个氨基酸，平均分子量为555．3Da。4．为研究不同分子量分布的肽段的抗氧化活性，选用截留分子量为5kDa、3 kDa、1 kDa的改良纤维素平板膜对麦胚蛋白酶解物进行超滤分级，选择麦胚肽一级超滤的最佳操作参数为：压力为0．10MPa、料液浓度为2．0％-2．5％、温度为35℃。采用阴阳离子交换树脂脱盐法对超滤分级肽进行纯化，以肽回收率和脱盐率为考察指标，确定10 CV／h为最佳流速。经过分级和脱盐处理的4种精制麦胚肽除分子量最大的肽Ⅰ外，均具有显著的抗氧化活性。5．采用5种化学模型对3种精制麦胚肽的抗氧化活性进行研究，结果显示，肽IV的还原力最强，当浓度为1．7 mg．mL^-1时，其还原力与1．0 mg．mL^-1 GSH相当；3种麦胚肽对DPPH·的清除均呈量效关系，其IC₅₀分别为肽Ⅳ为0．578mg．mL^-1、肽Ⅲ为0．769 mg．mL^-1、肽Ⅱ为1．661 mg．mL^-1，其参考对照GSH为0．28 mg．mL^-1。在邻苯三酚体系中，肽Ⅳ对O₂^-·的清除能力最强，与同剂量的GSH无显著性差异（p>0．05）。相同浓度下，肽Ⅲ清除·OH的能力和对Fe²⁺的螯合能力显著强于肽Ⅳ，表明肽Ⅲ与肽Ⅳ的抗氧化机制有所不同。荧光分光光度法测定肽Ⅳ和Ⅲ在全脂奶粉体系中均具有较好的抗氧化活性，当添加量达到脂质含量的5％时，抗氧化效果皆优于1％的BHT，而稍次于1％的GSH。6．采用离体小鼠肝脏线粒体，以Ca²⁺和Fe²⁺—Vc为诱导剂，建立了亚细胞水平的高效氧化损伤模型。经麦胚肽保护后，可显著抑制线粒体ATPase活性下降和MDA的生成，并有效促使膜蛋白羰基含量下降，对线粒体肿胀和膜电位的降低也都有明显的抑制作用。通过透射电镜观察线粒体超微结构的变化，发现麦胚肽对缓解线粒体氧化应激损伤、保持线粒体的完整结构具有显著作用。7．采用强阴离子型交换色谱、凝胶过滤色谱和反相高效液相色谱对麦胚肽Ⅳ进行分离纯化，得到了两种均为单一性组分的高抗氧化活性肽SEC-5和RP-2。采用ESI-MS对2种麦胚肽进行分子量测定及结构表征，SEC-5的分子量为296．2Da，氨基酸序列为Phe-Met；RP-2的分子量为246．1 Da，氨基酸序列为Leu-Asp。2个抗氧化肽的氨基酸组成和序列信息具有已报道抗氧化肽的表征结构特点。更多还原

【Abstract】 As one of the main cereal crops, the annual output of wheat has been beyond 100,000,000 tons in China. The wheat germ, an kind of important by-product of flour milling, is produced with annual yield about 2,000,000 tons. It is the life source and the most nutritious part of wheat kernel, which contains about 30% protein and is a high quality source of vegetable protein. Some competent peptide fragments with antioxidant activity will be obtained through specific enzymatic hydrolysis. It is shown that chronic diseases and senility were related to the imbalance of free radicals in our body. Excessive free radicals could cause the organism being oxidized damage. If the damage could not be repaired in time and be accumulated to certain degree, it would result in some diseases. Some natural antioxidants from food were helpful to eliminate excessive radicals and keep the balance of free radicals. The research on investigating and developing functional food and/or food ingredients with anti-aging and antioxidant activity from wheat germ protein hydrolysates can not only provide the theoretical basis for further utilization of agricultural byproducts such as wheat germ rich in protein, but also achieve comprehensive utilization and improve added value of wheat.In this dissertation, serial researches have been carried out concerning the processing of wheat germ protein by micronization and alkali extraction, the preparation and the action mechanism of antioxidant hydrolysates from wheat germ proteins. The results are as follows:Defatted wheat germ was micronized useing both hammer mill and colloid mill to increase the extraction rate of protein, followed by alkali extraction and ultrafiltration, respectively. A quadratic mathematical model was statistically constructed by using of Box-Behnken central composite design （CCD） and response surface methodology （RSM）. Under the optimized processing parameters of 23μm particle sizes, pH 9.5 and 51℃, a maximum extraction rate, 68.6%, was achieved, which is 30.63% higher than that of non- micronized wheat germ （passing through 80 mesh sieve）.The SDS-PAGE result showed that the molecular mass of wheat germ proteins was in the range of 11.6 kDa～93.8 kDa. The results of function determined indicated that in many ways such as the oil absorption, emulsifying activity and foaming activity, wheat germ protein was superior to those of soybean protein isolate （SPI）, but it was not as good as SPI on the water absorption, emulsifying stability and foaming stability. In addition, the solubility of wheat germ protein was very poor when pH was at the range of 4 and 5.Five kinds of proteases were selected by comparing the degree of hydrolysis （DH） and the scavenging activity of wheat germ protein hydrolysates against DPPH radical. Alcalase showed the optimal protease activity. The pretreatment, shearing and emulsifying for 5 min, can significantly improve hydrolysis degree and the yield of peptides, up to 29.85% and 12.53%, respectivly. After pretreated by shearing and emulsifying at 22000r/min for 5min, the optimized hydrolysing conditions were determined through single factor tests, Alcalase dosage is [E]/[S] 4000U/g, substrate concentration [S]4%, pH value 8.5, temperature 55?, time 240min. Under these conditions, DH was 18.61±0.23%, TCA-PSI, 71.02±0.45%, PCL, 5.37 and average molecular weight, 555.3.To better understand the antioxidant activity of peptides with different molecular weight, wheat germ protein hydrolysates were ultrafiltrated consecutively by upgrade cellulose FP membrane with the molecular cut off of 5 kDa, 3 kDa and 1 kkDa. The optimum conditions of the first-degree ultrafiltration were: pressure 0.10 MPa, material concentration 2%～2.5% and temperature 35℃. Graded hydrolysates were desalted by cation ion- exchange resins and anion ion- exchange resins. By comparing the desalting rate and peptide recovery rate, the optimum hydrolyte flow rate was 10 CV/h. The antioxidant experiment results showed that refined wheat germ peptides possess of noticeable antioxidant activity, except for peptides I with the largest relative molecular mass.The antioxidant effect of 3 wheat germ peptides evaluated using five different chemical models showed that at the concentration of 1.7 mg.mL^-1, peptides IV had the strongest reducing power, the same reducing power to that of 1.0 mg.mL^-1 GSH. The scavenging activity of 3 peptides against DPPH·were all dose independent. Their IC₅₀ were: peptides IV 0.578 mg.mL^-1, peptides HI 0.769 mg.mL^, peptidesⅡ1.661 mg.mL^-1, and the IC₅₀ of the control GSH was 0.28 mg.mL^-1. In the system of pyrogallic acid, peptidesⅣhad no significantly scavenging activity against O₂^-·with GSH. At the same concentration, the scavenging ability against·OH and the chelating activity on Fe²⁺ of peptidesⅢwere stronger than that of peptidesⅡ, which indicated that the action mechanisms of peptidesⅢand peptidesⅡwere somewhat different. In powder food, peptides III and peptides II with good antioxidant activity, comparing with that of 1% BHT and 1% GSH, were superior to the former and inferior to the latter when the concentration was 5% lipid content.Taken isolated rat heart mitochondria as targets, an oxidative damage pattern with high performance induced by Ca²⁺ and Fe²⁺-Vc was established on a subcellsular level. Wheat germ peptides could inhibit oxidative stress induced increase of MDA formation and carbonyl content of mitochondria as well as the lose of ATPase activity of mitochondria. The swelling of mitochondria and the reduction of membrane potential of mitochondria induced by Ca²⁺ and Fe²⁺-Vc were also prevented significantly by wheat germ peptides. The transmission electron micrographs of mitochondria showed that wheat germ peptides could release the injury to mitochondria induced by oxidative stress and help to maintain the integrity structure of mitochondria.The two peptides, SEC-5 and RP-2, showing strong antioxidant activity, were isolated using consecutive chromatographic methods including strong anion ion-exchange chromatography, gel-filtration chromatography and RP-HPLC. The molecular mass and the amino acid sequence of the two purified peptides were determined using electrospray ionization-mass spectrometry. For SEC-5, the molecular mass was 296.2, the amino acid sequence was Phe-Met; for RP-2, they were 246.1 and Leu-Asp, repectively.The amino acid composition and/or sequence of the two peptides agreed with the reported characteristics of antioxidant peptides.更多还原