【作者】 ULUKO HANKIE；

【导师】 吕加平；

【作者基本信息】 中国农业科学院 ， 农产品质量与食物安全， 2014， 博士

【摘要】 乳蛋白浓缩物(MPC)是一种新型乳制品,由于其具有高蛋白、低糖、低脂肪的特点,MPC需求量逐渐增加。有研究表明MPC用在奶酪、冰淇淋和酸奶上能改进产品的物理化学特性。然而,同其它乳蛋白如乳清蛋白和酪蛋白钠的浓缩物相比较时,MPC的功能特性相对较差。这可以通过一定的技术手段,增强浓缩物中的天然蛋白质基团暴露率加以改进。本研究利用微波(microwave,MW)和超声(ultrasound,US)技术将MPC进行预处理,以期提高MPC的水解率及功能特性。首先,利用微波或者超声对MPC做预处理,然后利用5种蛋白酶(碱性蛋白酶,胰蛋白酶,中性蛋白酶,碱性蛋白酶和风味蛋白酶)将其水解。微波及超声处理能够显著提高MPC水解产物的稳定性及水解程度,但不能降低水解物的苦味。水解物的血管紧张素转换酶(ACE)抑制活性有所提高,5分钟的超声波预处理后的中性蛋白酶水解物ICso值为0.23mgmL-1。利用液相色谱法分析MPC经中性蛋白酶酶解后产物中肽的水解状况。研究发现,在1-5分钟超声处理样品中出现的小分子肽不存在于对照组样品和8分钟超声处理的样品中。这可能由于随着预处理时间增长,ACE抑制活性的增加,这些小肽在8分钟预处理时被分解。同ACE抑菌活性相关的蛋白通过鉴定,其序列为：p-酪蛋白上WMHHQPHQPLPPT, AVPYPQRDMP, LSQSKVLPVPQ, AMAPKHKEMPFPKYP,αs1-酪蛋白上IKHQGLPQEVLNEN,LLRLKKYKVPQ.其他的活性肽通过芯片消化技术进行鉴定。采用响应曲面法得出用于生产出具有最高ACE抑制活性的中性蛋白酶水解物的最佳条件是超声预处理时间为4.11min,酶解时间2.32h及酶与底物的比例2.33%。在此条件下,理论上的ACE抑制活性是0.044mgmL-1。使用此处理方式进行中试规模验证实验。成功地从牛奶蛋白浓缩物中生产出ACE抑制物和抗氧化肽。生产出了四种分离组分(F1,>8kDa；F2,3.5-8kDa；F3,0.2-3.5kDa；and F4,<0.2kDa),研究表明,组分3是理想的ACE抑制齐(IC50:0.096±0.010mgmL-1)而组分2是很好的抗氧化肽。通过经济成本分析,此方法用于生产中可取得良好的经济效益。更多还原

【Abstract】 Milk protein concentrate (MPC) is a relatively new milk product with increasing demand and usage as an ingredient because of its high protein and low sugar/fat content. It has found applications in cheese, ice cream and yogurts with reported improvements in physicochemical characteristics. However, when compared to other milk proteins such as whey protein and sodium caseinate concentrates, the functional characteristics of MPC are inferior. This can be improved by the application of technologies that enhances the exposure of the native proteins in the concentrate. It is in this context that microwave (MW) and ultrasound (US) technologies were investigated in this study as pretreatments in order to enhance the subsequent enzyme hydrolysis and functional characteristics of milk protein concentrate.Initially, MPC was pretreated either by MW or US before enzymatic hydrolysis with5enzymes (Alcalase, Trypsin, Neutrase, Alkaline Protease and Flavourzyme). Pretreatments increased the degree of hydrolysis and stabilized the solubility of the hydrolysates but could not significantly reduce bitterness of the hydrolysates The angiotensin converting enzyme (ACE) inhibitory activity of the hydrolysates were improved with5-min ultrasound pretreated-Neutrase hydrolysates giving IC50value of0.23mgmL-1. Then, the peptide profiles of the Neutrase-derived hydrolysates of MPC were analysed by liquid chromatography. The profiles indicated that new small peptides in US pretreated samples (1-5min) which were not present in the control samples and8min pretreated samples, could be responsible for increased ACE inhibitory activity. These small peptides were digested in the8min pretreated samples. The6peptides responsible for ACE inhibitory activity were identified as WMHQPHQPLPPT, AVPYPQRDMP, LSQSKVLPVPQ, AMAPKHKEMPFPKYP from β-casein and IKHQGLPQEVLNEN, LLRLKKYKVPQ from αs1-casein using MALDI-TOF-LC-MS2. Other possible bioactive peptides were predicted using the in silico digestion.Using response surface methodology, the optimum conditions for producing Neutrase hydrolysates with the highest ACE inhibition were US pretreatment time, hydrolysis time and enzyme-to-substrate ratio of4.11min,2.32h and2.33%, respectively. Under these optimal conditions, the theoretical ACE inhibition was0.044mgmL-’. Using the near-optimal conditions, a batch mode, pilot-scale process, was successful in the production of ACE inhibitory and antioxidative peptides from milk protein concentrate (MPC). Four fractions were produced (F1,>8kDa; F2,3.5-8kDa; F3,0.2-3.5kDa; and F4,<0.2kDa) and analyses showed that F3was ideal for ACE inhibition (IC50=0.096±0.010mgmL-1) while F2was good for antioxidative peptides. Economic analysis showed that it was feasible to produce these peptides at a profit.更多还原