小麦胚芽蛋白质谱识别及释放行为研究

发布时间：2019-01-07 20:08

【摘要】：小麦胚芽蛋白是以脱脂小麦胚芽为原料经过碱溶酸沉等方式制备而成的蛋白质产品,在食品、化工等领域具有重要的应用前景。传统的基于碱溶酸沉技术的提取工艺存在用水量高、提取率低和乳清废水排放量大等一系列问题。此外,资源利用率和产品附加值较低。本文旨在以麦粕中蛋白质为研究对象研究一种新型的碱溶酸沉提取小麦胚芽蛋白的方法,并在基于质谱的蛋白质种类识别的基础上,研究了不同的提取条件对小麦胚芽中各种种类的蛋白质的释放行为造成的不同影响。通过小麦胚芽中试放大实验可将所得产品应用到实际生产中,并通过改变提取温度、溶剂的pH值及提取时间等因素可进一步提高蛋白得率及蛋白含量,主要结论如下:1.基于HPLC-MS的蛋白质识别方法研究,利用HPLC对小麦胚芽蛋白提取液进行了分离,收集了分离出的主要蛋白质组分,采用不同的蛋白酶进行酶解的技术并结合了高效液相色谱串联质谱技术对高效液相分离出各种种类的蛋白质进行了一一识别。结果表明不同种类蛋白质在色谱柱中保留时间的不同,总计识别出多种蛋白质,其中丰度较高的蛋白质有17种。2.脱脂小麦胚芽蛋白质释放行为研究,在蛋白质组分识别的基础上,对麦粕中每一种蛋白质的溶出过程进行了检测,并对比了所有种类的蛋白质在提取过程中的释放速率以及释放量的显著变化。根据实验的结果可知,低分子量的蛋白释放速率显著快于高分子量蛋白质的释放速率,而且动态提取的过程要比静态提取的过程各组分蛋白的释放速率要高很多,动态提取过程中保留时间为18.78min的色谱峰中Chromosome3B蛋白释放速率最快,提取2次的蛋白释放率已达90%。3.基于小麦胚芽蛋白的酶解方法研究,考察了利用各种蛋白酶在不同的固液比、提取温度、溶液pH值的条件下水解脱脂小麦胚芽蛋白制备得到小麦胚芽肽。比较木瓜蛋白酶、枯草杆菌蛋白酶,以及混合蛋白酶水解脱脂小麦胚芽蛋白的进程曲线,结果显示复合蛋白酶的酶解效果最好,其较佳作用条件为:底物浓度11.11%,pH 7.0,温度45℃,酶与底物比1:200,时间为60 min。在此条件下,小麦胚芽蛋白的酶解效率最高为87%。此外,利用中试反应釜,管式离心机,喷雾干燥机,冷冻干燥机等设备可将小麦胚芽蛋白的制备应用到企业生产,可制备出蛋白含量较高的小麦胚芽蛋白粉。本研究将利用碱溶酸沉技术应用于小麦胚芽蛋白的提取,并考察了不同提取条件对蛋白收率及释放速率的影响,同时对小麦胚芽蛋白的分离鉴定进行优化,对建立一种新型制备小麦胚芽蛋白的提取工艺具有重要的参考价值。
[Abstract]:Wheat germ protein is a protein product prepared from defatted wheat germ by alkaline acid precipitation. It has important application prospect in food, chemical industry and other fields. A series of problems such as high water consumption, low extraction rate and large discharge of whey wastewater exist in the traditional extraction process based on alkali soluble acid precipitation technology. In addition, resource utilization and product added value are low. The aim of this paper is to study a new method of extracting wheat germ protein from wheat meal by alkaline acid precipitation, and based on the recognition of protein species based on mass spectrometry. The effects of different extraction conditions on the release behavior of various kinds of proteins in wheat germ were studied. The obtained product can be applied to actual production through the pilot scale experiment of wheat germ, and the protein yield and protein content can be further improved by changing the extraction temperature, the pH value of solvent and the extraction time. The main conclusions are as follows: 1. Based on the study of protein recognition method based on HPLC-MS, wheat germ protein extract was separated by HPLC, and the main protein components were collected. Different protease hydrolysis techniques and high performance liquid chromatography-tandem mass spectrometry (HPLC / MS) were used to identify all kinds of proteins isolated by high performance liquid chromatography (HPLC). The results showed that the retention time of different kinds of proteins in the chromatographic column was different, and a total of many proteins were identified, of which 17 were found to have higher abundance. The protein release behavior of defatted wheat germ was studied. Based on the recognition of protein components, the dissolution process of each protein in wheat meal was determined. The release rate and release amount of all kinds of proteins were compared. According to the experimental results, the release rate of low molecular weight protein was significantly faster than that of high molecular weight protein, and the dynamic extraction process was much higher than that of static extraction process. The release rate of Chromosome3B protein was the fastest in the chromatographic peak with retention time of 18.78min during dynamic extraction, and the release rate of Chromosome3B protein reached 90.3in two times extraction. Based on the method of enzymatic hydrolysis of wheat germ protein, wheat germ peptide was prepared by hydrolysis of degreased wheat germ protein under different solid-liquid ratio, extraction temperature and pH value of solution. Compared with papain, Bacillus subtilis protease and mixed protease, the results showed that the enzymatic hydrolysis effect of complex protease was the best, and the optimum conditions were as follows: substrate concentration 11.11%. PH 7.0, temperature 45 鈩，

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