【作者】 曹向宇；

【导师】 刘长江；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 小麦是世界三大粮食作物之一。作为小麦加工的副产物,麦麸的产量巨大,但有效利用率很低,麦麸资源未能得到充分、合理、有效的利用。麦麸资源的开发,不但可以解决小麦深加工中大宗副产品的出路问题,而且可以提高产品的附加值。麦麸中含有丰富的蛋白,约占麦麸总重量的12～18%,是一种潜在的植物蛋白资源,而以麦麸蛋白为原料进行生物活性肽的研究目前尚未见报道。本文在利用麦麸蛋白酶解制备生物活性肽方面进行了一系列研究,成功制备麦麸多肽,并证明其具有抗氧化活性及增强免疫力和抗疲劳作用。这一研究为拓宽麦麸的应用范围,进一步开发麦麸蛋白开辟了新的空间,为麦麸多肽的工业化生产提供理论依据和工艺参数,为麦麸多肽更加广泛地应用于保健品和食品领域提供理论基础与实验依据。本文对麦麸多肽的制备、纯化及生物活性进行了系统研究。以碱法、盐法提取麦麸蛋白,确定最佳提取条件;以羟基自由基和超氧阴离子自由基的清除率为指标,正交试验方法确定酶法制备麦麸多肽的最佳条件;通过超滤、SeaphadexG25凝胶过滤、DEAE-32离子交换层析和反相高效液相色谱对麦麸多肽进行分离纯化,应用质谱（MS）测定分子量,并对麦麸多肽的稳定性进行研究;通过体内、体外试验综合评价麦麸多肽的抗氧化能力;通过淋巴细胞转化试验、半数溶血值(HC₅₀)的测定、吞噬指数和免疫器官指数的测定,研究麦麸多肽增强免疫力功能;通过负重游泳实验和肝糖原、血尿素氮含量测定,研究麦麸多肽的抗疲劳作用;通过急性毒理试验、Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠睾丸精母细胞染色体畸变试验及彗星试验对麦麸多肽进行毒理研究和安全性评价;采用超声波法和旋转薄膜.超声波法对多肽进行微胶囊包埋和脂质体制备研究,确定最佳制备工艺条件。主要研究结果如下:1.碱法提取麦麸蛋白最佳条件为:料液比为1:15,pH为11,温度50℃,时间为2.5h,蛋白质的提取率可达57.86%。盐法提取麦麸蛋白质最佳条件为:盐浓度为2%,温度40℃,时间为2h,料液比为1:15,蛋白质的提取率可达27.68%。由两种方法提取结果可知,碱法是提取麦麸蛋白的较好方法。2.以碱性蛋白酶与复合风味蛋白酶分步水解制备麦麸多肽,其最佳工艺条件为:碱性蛋白酶:pH8,酶活添加量为6000u/g料,时间3 h,温度50℃:复合风味蛋白酶:pH6,酶活添加量为6000u/g料,时间1 h,温度55℃,在此条件下对麦麸蛋白进行水解,对O₂^-·和·OH的清除率分别为81.60%和86.16%。正交试验结果表明:在设定的四个因素中,对自由基清除能力影响程度依次为:pH＞时间＞酶活添加量＞温度。3.双酶分步水解后的麦麸多肽进行超滤研究表明,分子量低于10KDa的麦麸多肽抗氧化活性最强;采用SephadexG-25层析柱对低分子量的麦麸多肽进行分离,获得3个分离峰,其中第3个峰抗氧化活性最强。采用DEAE-32纤维素柱分离经SephadexG-25纯化后的活性组分,获得4个分离峰,其中第4个峰抗氧化活性最强。采用RT-HPLC分离经DEAE-32纤维素柱分离纯化后的活性组分,条件为:色谱柱:SUPELLOSIL C₁₈柱（25cm×4.6mm）;检测波长:215 nm;柱温:35℃;流速:0.5 mL/min;上样量:10μL;沈脱条件为:（80%甲醇,20%水）,分离出2个主峰,其中第1个峰具有较高的抗氧化活性。将收集的活性组分进行醋酸纤维素薄膜电泳,电泳结果为单一条带,说明收集的活性麦麸多肽纯度较高,质谱（MS）测定其分子量为1326.2Da。制备的麦麸多肽在pH6.0～8.0范围内比较稳定,pH＜4.0及pH＞8.0时,麦麸多肽的稳定性下降。4.抗氧化试验表明:经Sephadex G-25分离后制备的麦麸多肽具有较高的清除超氧阴离子自由基（O₂^-·）和羟自由基（·OH）的能力;可明显抑制溶血反应的发生,有效保护红细胞结构的完整性;并具有抑制·OH诱导的线粒体肿胀的功能;体外实验证明麦麸多肽具有较好的抗氧化作用。通过体内试验证明麦麸多肽具有很好的抗DNA氧化功能;能够促进小鼠血清和肝脏SOD、GSH-Px值的升高,降低脂质过氧化物MDA值,具有较好的提高机体内源性抗氧化酶活性,清除自由基,抑制脂质过氧化损伤作用。5.免疫试验表明:麦麸多肽具有增强免疫力的功效,其主要是通过促进机体的细胞免疫水平,增强巨噬细胞的吞噬功能,提高脾脏指数来实现其对机体的免疫作用的。麦麸多肽能延长小鼠负重游泳时间,增加小鼠肝糖原储备量,显著降低小鼠剧烈运动后血尿素氮含量,具有较强的增强动物运动耐力、缓解动物体力疲劳的作用。6.毒性试验表明:麦麸多肽小鼠急性毒性试验,LD₅₀＞20g/kg,属无毒级。Ames试验、骨髓细胞微核试验、睾丸精母细胞染色体畸变试验、彗星试验结果均为阴性,表明麦麸多肽是一种无毒物质,初步证明麦麸多肽的食用安全性。7.以明胶为壁材包埋制备麦麸多肽微胶囊,对包封率的影响顺序为超声功率＞芯材:壁材＞超声温度＞超声时间。明胶包埋麦麸多肽的最佳工艺条件为:芯材:壁材为1:8,超声功率为60W,超声时间为70min,超声温度为50℃,在此条件下,包封率可以达到59.68%。以饱和卵磷脂、胆固醇制备的麦麸多肽脂质体,对包封率的影响顺序为:超声时间＞饱和卵磷脂与胆固醇物质的量比＞水化温度＞麦麸多肽量,最佳制备条件为饱和卵磷脂与胆固醇物质的量比为1:1,水化温度为55℃,超声波处理时间为3min,麦麸多肽量为2mL,此时包封率为88.16%。更多还原

【Abstract】 Wheat is one of the three major grain crops in the world.The yield of wheat bran, byproduct of wheat processing,is very huge.At present,the effective utilization of wheat bran is very low,which is not fully,reasonably and efficiently utilized.Wheat bran peptide not only can broaden the range of wheat bran application and resolve the problem of bulk byproduct in the deep processing of wheat,but also enhance product added value.Wheat bran contains an abundant amount of protein,which is about 12～18%of the total weight,so it is a potential resource of plant protein.The study of wheat bran peptide made from wheat bran protein has not been reported yet.A series of experiments were taken to prepare bioactive peptides using enzymatic preparation from wheat bran protein,and wheat bran peptides were obtained successfully.The wheat bran peptide has good antioxidation,immunity enhancement and antifatigue effect.These researches can broad application area,develop new ways for further develop of wheat bran protein,provide theoretical basis and process parameters to industrial production of peptide and provide theoretical foundation and experimental basis for the more widely used of wheat bran peptide in health products and food.In this paper,preparation,purification and biological activity of wheat bran peptide was systematic studied.The wheat bran protein extraction was used by alkali method and salt method,the optimum extraction conditions were determined.The scavenging activity of hydroxyl radical and superoxide anion radical taken as index,the best conditions of enzymatic preparation of wheat bran peptide determined by orthogonal test method.The antioxidant capacity of wheat bran peptide was comprehensive evaluated by in vivo and vitro test.The function of immunoenhancement of wheat bran peptide were evaluated by lymphocyte transformation test,determination of half hemolysis value,phagocytic index,immune organ index.The antifatigue effect of wheat bran peptide was estimated by loaned swimming test and determination of liver glycogen and blood urea nitrogen.The toxicological and safety evaluation of wheat bran peptide were tested by acute toxicity test,Ames test,mouse bone marrow micronucleus test,the chromosome aberration test of mouse primary spermatocyte, comet assay.Wheat bran peptide was isolated and purified by ultrafiltration,SeaphadexG25, DEAE-32 and reversed-phase high performance liquid chromatography.The molecular weight is measured by mass spectrometry（MS）.The stability of wheat bran peptide was studied. Microcapsule and liposome of wheat bran peptide were prepared by ultrasonic method and rotary film-ultrasonic method,the optimum preparation conditions were determined.The main results are as followed.1.The optimum extraction conditions of wheat bran protein by alkali method are as follows:solid-liquid ratio of 1:15,pH 11,temperature 50℃,time 2.5h,protein extraction rate is 57.86%.The optimum extraction conditions of wheat bran protein by salt method are as follows:concentration of 2%,temperature 40℃,time of 2h,solid-liquid ratio 1:15,protein extraction rate is 27.68%.Optimized by the two methods,alkali method is a better way.2.The best conditions of enzymatic preparation of wheat bran peptide prepared by double-enzyme fractional hydrolysis with Alkaline protease and Flavourzyme were as follows: pH8,enzyme activity 6000u/g material,time 3 h,temperature 50℃with Alkaline protease, pH6,enzyme activity 6000u/g material,time 3 h,temperature 55℃with Flavourzyme,under these conditions for hydrolysis of wheat bran protein,and clearance rate of O₂-^·and·OH were 81.60%and 86.16%.Orthogonal test results show that the influences of the effect of free radical scavenging capacity are:pH＞Time＞addition of enzyme activity＞temperature.3.Ultrafiltration were carried out to separate wheat bran peptide,less than 10KDa molecular weight is the strongest antioxidant activity,SephadexG-25 column chromatography was used to separate low molecular weight of wheat bran peptide,three separate peaks were got,of which the third peak with the strongest antioxidant activity.DEAE-32 cellulose column was used to separate the active component purified by the SephadexG-25,four separate peaks were got,of which the fourth peak with the strongest antioxidant activity. RT-HPLC was used to separate the active component purified by the DEAE-32 cellulose column,conditions are as follows:Column:SUPELLOSIL C₁₈ column（25cm×4.6mm）, detection wavelength:215 nm,column temperature:35℃,velocity:0.5 ml/min,sample loaded volume:10μL,elution conditions are as follows:（80%methanol,20%water）,two peaks were isolated,of which the first has higher antioxidant activity.The active component was collected for cellulose acetate membrane electrophoresis,a single band was obtained.The molecular weight is 1326.2Da analysised by mass spectrometry（MS）.Wheat bran peptide has high purity.Preparation of Wheat bran peptide in the range of pH6.0～8.0 is more stable,pH＜4.0 and pH＞8.0,the stability of wheat bran peptide is decreased.4.The wheat bran peptide extracted by Sephadex G-25 has a higher scavenging activity of hydroxyl radical and superoxide anion radical,can inhibit hemolysis significantly,prevent the integrity of red blood cells effectively,inhibit mitochondria swelling induced by·OH, wheat bran peptide has good antioxidation in vitro.Wheat bran peptide has antioxidation of DNA in vivo,can promote mice serum and liver SOD,SOD,GSH-Px values and reduce the lipid peroxide MDA value,with better improvement of the body endogenous antioxidant enzyme activity,scavenging free radicals,inhibit lipid peroxidation injury.5.Immunological tests show that wheat bran peptides have enhanced the effectiveness of the immune system,through the promotion of the level of cell-mediated immunity mainly, and enhance of macrophage phagocytic function,increase of spleen index to achieve its role of the immune.Wheat bran peptide can extend mice swimming time,increase liver glycogen storage quantity in mice,reduce blood urea nitrogen significantly,enhance exercise tolerance and alleviate physical fatigue of animals.6.Toxicity test show that wheat bran peptide in mice acute toxicity test,LD₅₀＞20g/kg,is non-toxic class.Results of Ames test,mouse bone marrow micronucleus test,the chromosome aberration test of mouse primary spermatocyte and comet assay were negative. Wheat bran peptide is a non-toxic material.The present studies thus provide preliminary proof of safety for wheat bran peptide.7.Wheat bran peptide microcapsule was prepared with gelatin embedded as wall material,the influence order of entrapment efficiency is ultrasonic power＞core material:wall material＞ultrasonic temperature＞ultrasonic time.The best conditions of wheat bran peptide embedded with gelatin are as follows:core material:wall material for the 1:8,ultrasonic power of 60W,ultrasonic time of 70min,ultrasonic temperature 50℃,in these conditions,the rate of entrapment efficiency can be achieved 59.68%.Wheat bran peptide liposome was prepared with satured phospholipids and cholesterol, the influence order of entrapment efficiency is ultrasonic time＞amount of substance ratio of lecithin and cholesterol＞hydration temperature＞amount of wheat bran peptide,The best conditions of wheat bran peptide embedded with satured phospholipids and cholesterol are as follows:molar ratio of lecithin:cholesterol for the 1:1,the hydration temperature of 55℃, ultrasonic processing time for 3min,wheat bran polypeptide of 2mL,in these conditions,the rate of entrapment efficiency can be achieved 88.16%.更多还原