【作者】 夏明；

【导师】 应铁进；

【作者基本信息】 浙江大学 ， 食品科学， 2011， 博士

【摘要】 生物活性肽是指对生命活动具有调节作用的多肽,通常以多肽链序列的形式存在于蛋白质之中。利用天然的蛋白质作为原料,使用蛋白酶水解制取各种生物活性肽,是生物活性肽制备技术中最具可行性的一种。使用酶解法制取生物活性肽,有间歇式水解和酶膜生物反应器中连续水解两种手段,前者自动化程度低,操作简便,后者可以连续运行,生产效率高。抗菌肽是生物活性肽家族中的一员,它是生物体抵抗外来微生物侵袭的重要手段,通过破坏细胞膜结构、诱导细胞凋亡、抑制细胞呼吸和DNA合成等机制来达到抑菌作用。从制备抗菌肽的天然蛋白质的来源上讲,乳蛋白是制备抗菌肽的重要原料。乳蛋白中的乳酪蛋白、乳清蛋白和乳铁蛋白,它们的水解产物对微生物的影响存在差异。本论文的研究,主要分为以下几个部分：第一是探索了乳蛋白的分离与检测技术,优化分离工艺,提出了新的乳铁蛋白的检测方法；第二是在间歇式反应条件下,对不同类型的乳蛋白进行酶解,优化酶解反应条件,测定水解产物的抗菌特性；第三是尝试在酶膜反应器中连续水解乳蛋白制备抗菌肽。论文首先以脱脂乳粉作为原料,通过控制等电点的手段制备酪蛋白,得率可以达到7.8%。除去酪蛋白后的乳清,在中性条件下经过10K的超滤膜处理,可以脱去其中的乳糖和盐分,成为高纯度的乳清分离蛋白。论文提出了一种借助高效液相色谱测定乳制品中乳铁蛋白的方法。先通过额外加入的亚铁离子使乳铁蛋白的构型更加稳定。然后用60%乙醇沉淀蛋白除去糖分,再用醋酸盐缓冲液提取乳铁蛋白并分离掉酪蛋白,最后用高效液相色谱检测。采用LiChrosorb RP-C18色谱柱,在0.1%三氟乙酸协助下,甲醇与水线性梯度洗脱。乳铁蛋白的线性范围是0.4～2mg/mL (R2= 0.9980),平均回收率95.4%。可以用于一般乳制品中乳铁蛋白的测定。此外,一种借助乳铁蛋白铁结合能力测定奶粉中乳铁蛋白的方法也被提出并与HPLC的检测结果进行比较。先通过额外加入的亚铁离子使乳铁蛋白中的铁达到饱和以获得稳定的铁含量,然后对蛋白反复醇沉和水溶以除去游离铁,再用原子吸收分光光度计测定铁含量,依据铁与蛋白含量的相互关系计算出乳铁蛋白含量。该法快速准确,加样回收率超过96%,检测范围为3.5-100mg/g。该法可以用于婴儿配方奶粉中乳铁蛋白的测定。以牛初乳作为原料,经等电点沉淀和加压过滤除去酪蛋白,随后在pH 7.5条件下加入乙醇至50%,可以使乳清中的乳铁蛋白沉淀并获得粗制品,粗制品经低压液相色谱纯化以及切向流超滤脱盐后可以获得高纯度的乳铁蛋白,该乳铁蛋白制品经HPLC和HPEC分析,杂质含量均已低于检测限度。在乳铁蛋白水解工艺的优化过程中,使用平行试验法设计实验,借助Mathematic7.01软件实施曲面回归分析初步得出,当以蛋白质水解程度最大为目标时,胃蛋白酶水解乳铁蛋白反应体系适宜的条件是：底物浓度2%,温度37℃,pH 2.2-2.3,酶用量7U/g。当水解反应进行到60min时,所得水解产物抑菌活力最强。在乳清蛋白水解工艺的优化过程中,使用SPSS 18.0软件设计正交实验方案,并进行单变量一般线性模型的方差分析。使用碱性蛋白酶水解乳清蛋白适宜的工艺参数为：酶底比5%,pH8.0,温度45℃。水解产物不对病原微生物产生抑制作用,但可以促进益生菌的生长。将胰蛋白酶和中性蛋白酶以1：1的比例复合使用来水解酪蛋白。使用Design Expert软件的Box-Behken模块中的响应面分析法设计实验方案并作回归分析,发现在pH7.4,温度44℃,底物浓度32g/L,酶底比2.5%下,水解90min后,水解度可以达到15%,此时水解多肽产物抵抗病原微生物的活力达到最强,该水解多肽同时又能促进益生菌的生长。本文使用了自制酶膜生物反应器连续水解乳铁蛋白制备乳铁素。使用截留分子量为10k的聚醚砜超滤膜作为试验用膜堆。发现只要控制好底物流加速率并在线添加酶活,酶膜生物反应器就可以实现稳定产出。在酶膜生物反应器中乳铁蛋白的转化率和乳铁素的得率较间歇式反应有明显提高。更多还原

【Abstract】 Bioactive peptides are the peptides that can regulate the metabolism, and exist in the protein in the form of polypeptide chains’sequences. The hydrolysis of natural protein by protease is the most feasible approach to attain diverse bioactive peptides. The hydrolysis method includes two types, namely batch-type hydrolysis and continuous hydrolysis utilizing the enzymatic membranes bioreactors (EMB). The former is easy and simple to control and has low automaticity; while the latter can be operated continuously and has efficient output.Antibacterial peptide which is one of the important categories of bioactive peptides offers vital protection to organism to resist microbial attack. The antibacterial peptide restrains the microorganism through various mechanisms, such as destroying the structure of cell membrane, inducing the cell apoptosis and inhibiting the cell respiration and DNA synthesis. Lactoprotein, as one kind of the natural protein, is an important material in the production of antibacterial peptide. The hydrolysates of the casein, whey protein and lactoferrin which are the ingredients of lactoprotein have different influences on the microorganism.This study compasses three parts. Firstly, the study optimized the separation technique of lactoprotein, and suggested new detection methods for some of the proteins. Secondly, using the batch-type reaction, different kinds of lactoprotein were hydrolyzed for the purposes of the optimization of hydrolysis parameters and the analysis of hydrolysates’antibacterial activities. Thirdly, antibacterial peptide was continuously produced by a designed enzymatic membrane bioreactor.The casein was separated from defatted milk powder and reach 7.8% yield through the isoelectric point precipitation. The whey was processed with the ultrafiltration membrane (10K) in the neutral pH condition, in order to erase the lactose and salinity and produce high purified whey protein isolate.In this study, a method to determine the lactoferrin in milk products by HPLC was identified. In a bid to obtain a configuration of lactoferrin that can be precipitated easily, the lactoferrin in milk was saturated by additional ferrous ion. Subsequently, the milk was precipitated by 60% thanol to eliminate the sugar, and dissolved in acetic acid buffer to remove casein protein and extract lactoferrin. The remaining product was then measured by HPLC, using LiChrosorb RP-C18 as separation column, linear gradient elution by methanol and water with the assistance of 0.1%TFA. The linear range of the lactoferrin detection is between 0.4 to 2mg/mL (R2= 0.9980), and the average recovery rate was 95.4%. This method is useful in detecting the lactoferrin in the milk products.In addition, an alternative method to determine the lactoferrin in milk powder, based on iron binding function of lactoferrin, was proposed and compared with the HPLC method. First, ferrous ion was added to lactoferrin to achieve a saturated iron ion binding. And then, the protein was precipitated by the ethanol and dissolved in water repeatedly in order to eliminate the excessive iron ion. The amount of iron in lactoferrin was measured by atomic absorption spectrophotometer. Through the correlation between the amounts of iron and protein, the amount of lactoferrin can be calculated. This method has advantages in terms of accuracy and speediness. The lactoferrin recovery rate is above 96%. The lactoferrin detection ranges from 3.5 to 100 mg/g. This method can be useful in the detection of lactoferrin in the infantile milk powder.The colostrums were preprocessed with the isoelectric point precipitation and pressure filtration to remove the caseins. In the condition of pH 7.5, the ethanol was mixed into the whey up to 50% of the mixture. The addition of the ethanol assists the precipitation of lactoferrin in the whey and to be harvested as crude product. The crude product was further purified with low pressure liquid chromatography and desalted with tangential flow ultra filter to yield high purity lactoferrin. The HPLC and HPEC analyses of lactoferrin indicated that the impurity content of lactoferrin is below the range of detection.The parallel test was employed to design the experiments for the optimization of lactoferrin hydrolysis. The calculation of response surface regression performed with software Mathematic 7.01 showed that the optimum parameters of lactoferrin hydrolysis by the pepsin are substrate concentration 2%, temperature 37℃, pH 2.2-2.3, and dosage of enzyme 7U/g, when the aim of experiment is to maximize the degree of proteolysis.The orthometric experiments aimed at optimization of whey protein hydrolysis were designed by the software SPSS 18.0. The optimized parameters attained from the orthometric experiments were further used to formulate a univariate general linear model. The data of parameters estimated by the univariate general linear model were compared with the real data using the variance analysis. The results revealed that the optimum parameters for the whey protein hydrolysis by the alcalase include the ratio of enzyme to substrate 5%, pH 8.0 and temperature 45℃. The hydrolysate does not inhibit the pathogenic microorganism, but facilitate the growth of probiotics.The casein was hydrolyzed by the mixture of trypsin and neutral proteinase (ratio 1:1). The response surface analysis in the Box-Behken function was used to design the experiments, employing the software Design Expert. The results of experiments were then analyzed with regression analysis, which revealed that the degree of hydrolysis can reach 15%, with pH 7.4, substrate density 32g/L, ratio of enzyme to substrate 2.5%, and 90 minutes of hydrolysis. When the degree of hydrolysis reaches 15%, the antibacterial activity of polypeptide product is strongest. The polypeptide can also boost the growth of probiotics.In this study, lactoferrin was continuously hydrolyzed to generate lactoferrcin by a designed EMB apparatus. The membrane used in the experiment was the polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 10k. The study found that the EMB can have stable output, if the supplement rate of substrate is well controlled and the enzyme was added on line. The production of lactoferrcin and the conversion rate of lactoferrin were higher in the EMB reactor than in the batch reactor.更多还原