本文選題：小麥面筋蛋白　切入點：酶法水解　出處：《江南大學》2017年博士論文　論文類型：學位論文

【摘要】：小麥面筋蛋白作為小麥淀粉加工過程中的副產(chǎn)物,主要用于面制品和飼料工業(yè),是一種物美價廉、食用安全的植物蛋白。小麥面筋蛋白復雜的分子組成和結(jié)構(gòu)特性,且含有高比例的疏水性氨基酸,造成該蛋白的水溶性較差,極大地限制了其在食品工業(yè)中的應用。采用蛋白酶酶解小麥面筋蛋白制備生物活性肽的研究與開發(fā),將有助于小麥產(chǎn)業(yè)鏈向高附加值產(chǎn)品延伸。但傳統(tǒng)酶解工藝不夠成熟,尚存在苦味較重、小肽含量較低等問題,制約了產(chǎn)品的開發(fā)利用。本文以小麥面筋蛋白為原料,圍繞酶解技術(shù)在制備低苦味肽粉中的應用及其脫苦原理進行研究。首先分析了小麥面筋蛋白酶解產(chǎn)物的苦味特性,設(shè)計內(nèi)切酶和端解酶連續(xù)水解,制備小麥面筋蛋白低苦味肽粉,并采用脫酰胺改性改善小麥面筋蛋白酶解產(chǎn)物的風味。為針對性優(yōu)化小麥面筋蛋白-Proteax酶水解體系,探討了小麥面筋蛋白酶解產(chǎn)物中苦味肽的釋放機制,最終確定了分步酶解小麥面筋蛋白制備低苦味小肽的工藝。內(nèi)切酶和端解酶連續(xù)水解,比單一的內(nèi)切酶和端解酶有更強的水解能力,因此連續(xù)水解可以更有效的制備小肽。采用中性蛋白酶、木瓜蛋白酶、風味蛋白酶、堿性蛋白酶、胰蛋白酶、復合蛋白酶和Proteax酶酶解小麥面筋蛋白,通過滋味稀釋分析評估單酶酶解產(chǎn)物的苦味強度,構(gòu)建內(nèi)切酶和端解酶連續(xù)水解方案。對比不同方案中酶解產(chǎn)物的肽氮含量、氨基酸組成、分子量分布和氨基酸序列對感官特性和電子舌分析的影響。研究結(jié)果表明連續(xù)水解制備的酶解產(chǎn)物,在苦味肽含量上的差異不大。氨基酸序列和分子量分布影響多肽的苦味閾值,決定酶解產(chǎn)物的苦味強度。與其它方案制備的酶解產(chǎn)物相比,Pro-m酶解產(chǎn)物具有最高的肽氮含量(61.54%)、小肽含量(53.26%)和最低的苦味值(1.33),并且其可溶性氮含量也高達82.82%。因此,確定以小麥面筋蛋白-Proteax酶水解體系為基本框架,開展后續(xù)工作。采用Glutaminase SD-C100S、Glutaminase和鹽酸(HCl)對Pro-m酶解產(chǎn)物進行脫酰胺改性,促使酶解產(chǎn)物中谷氨酰胺轉(zhuǎn)化為谷氨酸,發(fā)揮鮮味肽和游離谷氨酸增鮮抑苦的特性,改善酶解液的風味。對比分析脫酰胺改性酶解產(chǎn)物的風味特征、分子量分布和氨基酸組成,探討脫酰胺改性酶解產(chǎn)物中呈鮮物質(zhì)的主體成分以及其對苦味的抑制效果。研究結(jié)果表明,脫酰胺度增大,鮮味物質(zhì)的含量上升,引起酶解產(chǎn)物鮮味增強、苦味降低。谷氨酸鈉親水性強,比鮮味肽有更強的唾液溶解性,能夠更好的抑制苦味信號的傳導。鮮味肽比谷氨酸鈉有更久的受體作用時間,增強了鮮味的持續(xù)時間,會表現(xiàn)出一種更為柔和、協(xié)調(diào)的濃厚感。為針對性優(yōu)化小麥面筋蛋白-Proteax酶水解體系,研究了小麥面筋蛋白酶解產(chǎn)物中苦味肽的釋放機制。采用異丁醇萃取苦味釋放特征發(fā)生明顯變化的酶解產(chǎn)物,有效萃取酶解產(chǎn)物中苦味極強的苦味肽。系統(tǒng)地闡述了Proteax酶水解過程中苦味肽氨基酸序列、分子量分布、氨基酸組成的變化規(guī)律,并利用偏最小二乘回歸法分析了異丁醇萃取物、感官屬性、分子量分布與氨基酸組成之間的相關(guān)性。研究表明,相對于氨基酸序列的變化,苦味肽的氨基酸組成、分子量分布對苦味肽的苦味強度有一個更顯著的影響。酶解反應在0-120 min時,苦味氨基酸在苦味肽(分子量處于180-500 Da、500-1000 Da和1000-3000 Da區(qū)間)中所占的比例逐漸增大。酶解反應在120-300 min時,苦味肽中苦味氨基酸的含量不再提高。萃取物中高分子量苦味肽的含量降低,尤其是苦味最強,相對分子量處于500-1000 Da區(qū)間的苦味肽。因此,小麥面筋蛋白-Proteax酶水解體系中苦味肽的苦味強度呈先升高后降低的趨勢。采用Proteax酶、堿性蛋白酶、復合蛋白酶和胰蛋白酶分別水解小麥面筋蛋白,生成可溶性酶解產(chǎn)物和水不溶性聚集體。以水不溶性聚集體為原料,重復上述操作,收集聚集體酶解產(chǎn)物。對可溶性酶解產(chǎn)物和聚集體酶解產(chǎn)物進行苦味評定,發(fā)現(xiàn)采用同一種蛋白酶,在水解度大小一致的前提下,聚集體酶解產(chǎn)物比可溶性酶解產(chǎn)物產(chǎn)生更高的苦味強度。根據(jù)上述結(jié)論提出分步酶解技術(shù),優(yōu)化了小麥面筋蛋白-Proteax酶水解體系。即采用Proteax酶水解小麥面筋蛋白35 min,離心去除沉淀,加入Glutaminase SD-C100S,采用Design-Expert 8.0.6軟件優(yōu)化復合酶法水解工藝。酶解工藝以低苦味值為目標,復合酶法的最佳工藝參數(shù)為:酶解時間280.79 min,酶解溫度53.09°C,pH6.94,苦味預測值為0.41。對結(jié)果進行驗證,調(diào)整酶解參數(shù)為酶解時間280 min,酶解溫度53°C,pH6.9。制備出苦味值僅為0.43的小麥面筋蛋白低苦味肽粉。
[Abstract]:Wheat gluten as a by-product of wheat starch processing, mainly used for flour products and feed industry, is a kind of high quality and inexpensive, safe to eat the vegetable protein. Molecular wheat gluten protein complex composition and structure characteristics of hydrophobic amino acids and contains a high proportion of the protein, resulting in poor water solubility, which greatly limits its application in the food industry. The research and development of protease preparation of bioactive peptide of wheat gluten system solutions, will contribute to the wheat industry chain to high value-added product extension. But the traditional enzymolysis technology is not mature, there are still bitter heavy, small peptide content is low, restricted the development and utilization of products. In this paper, wheat gluten was used as raw material, around the low bitter peptide powder in the system and the principle of debittering enzyme technology. First analysis of wheat gluten hydrolysate. Bitter taste characteristics, design enzymes and exopeptidase continuous hydrolysis, preparation of wheat gluten powder with low bitter peptide, deamidation of wheat gluten hydrolysate flavor. For the optimization of enzymatic hydrolysis of wheat gluten -Proteax system, discusses the release mechanism of bitter peptide products of wheat gluten protein by enzymatic hydrolysis finally, determine the process by hydrolysis of Wheat gluten to produce low bitter peptide. Exopeptidase enzymes and continuous hydrolysis, enzyme hydrolysis ability has better solution than the single enzyme and end, therefore continuous hydrolysis preparation of small peptides can be more effective. Using neutral protease, papain, flavourzyme, alkaline protease, trypsin, wheat gluten, composite protease and Proteax enzyme, through analysis and evaluation bitter taste dilution strength products of single enzyme hydrolysis, enzyme and construction of exopeptidase hydrolysis continuous square Comparison of different schemes in the case. The amino acid hydrolysate peptide nitrogen content, composition, influence analysis of the sensory characteristics of electronic tongue and the molecular weight distribution and amino acid sequence. The results showed that the product of continuous hydrolysis preparation of enzyme solution, differences in the content of the bitter peptides. Little amino acid sequence and molecular weight distribution of bitter threshold polypeptide the decision of bitterness intensity hydrolysates. With other solutions prepared hydrolysates compared to Pro-m hydrolysate peptide has the highest nitrogen content (61.54%), the content of small peptide (53.26%) and the lowest value (1.33), and the bitter taste of the soluble nitrogen content as high as 82.82%. so determined to wheat gluten protein -Proteax enzyme hydrolysis system as the basic framework, to carry out follow-up work. By Glutaminase SD-C100S, Glutaminase and hydrochloric acid (HCl) of Pro-m hydrolysis products were deamidation of glutamine, prompted the enzymolysis products into the valley Ammonia acid, umami peptide and free glutamate play by fresh suppression and bitter characteristics, improve hydrolysate flavor. Comparative analysis of flavor characteristics of the deamidation of hydrolysates, molecular weight distribution and amino acid composition of a main component of fresh matter and the bitter taste of the inhibitory effect of deamidation of hydrolysates. The results show that the deamidation degree increases, the rising content of umami substances, causing hydrolysate flavor enhancement, reduce the bitter taste. Glutamate strong hydrophilicity, saliva solubility stronger than umami peptides, can inhibit the bitter better signal conduction. Umami peptides receptors longer than the role of glutamate increased duration of flavor, will show a more gentle, strong sense of coordination. In order to optimize the enzymatic hydrolysis of wheat gluten -Proteax system of wheat gluten protein enzymolysis bitter peptide product of release The isobutanol extraction mechanism. Bitter product release features changed by enzymatic hydrolysis, bitter peptide product bitter strong effective extraction enzyme. Systematically bitter peptide amino acid sequence of Proteax hydrolysis process, molecular weight distribution, variation of amino acid composition, and analysis of isobutanol extract, using partial least squares regression of sensory attributes correlation weight distribution and amino acids composition. The results show that, compared to the changes in the amino acid sequence, amino acid composition of bitter peptides, the molecular weight distribution of bitterness intensity of bitter peptides have a more significant effect. The enzymatic reaction in 0-120 min, in the bitter bitter amino acid peptide (molecular weight at 180-500 Da, 500-1000 Da and 1000-3000 Da interval) in the proportion increased gradually. The enzymatic reaction at 120-300 min, the content of amino acid in the bitterness of bitter peptides and improving extraction. 鐗╀腑楂樺垎瀛愰噺鑻﹀懗鑲界殑鍚噺闄嶄綆,灝ゅ叾鏄嫤鍛蟲渶寮，

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