玉米黄粉蛋白酶解过程的研究及产物分析

利用中性蛋白酶和复合风味蛋白酶对玉米黄粉蛋白进行酶解，研究了双酶法水解玉米黄粉蛋白的过程，并对水解产物进行分析。结果表明，双酶法水解玉米黄粉蛋白的最佳酶解条件为：pH＝6.7，T＝47℃，S%=0．9%，S／E复＝30，E复／E中＝8，水解时间40min，试验指标水解度(DH)％可达到32.82％，酸溶性多肽得率(YASP)％可达到87.46%，氮溶指数(NSI)％接近37.43％。经分析测定，水解产物中氨基酸含量4.84mg／g Pr，多肽含量18l.86mg／g Pr;多肽分子量主要分布于1089—1728Dal范围内。     

不同蛋白酶和水解条件对酪蛋白酶解产物性质的影响

采用碱性蛋白酶、木瓜蛋白酶、菠萝蛋白酶和胰蛋白酶分别对酪蛋白进行酶解,发现碱性蛋白酶水解酪蛋白的水解程度最大。当酪蛋白经不同的酶水解至相同水解度(DH)时,酶解物的分子量分布存在显著差异。随后选定碱性蛋白酶以考察水解条件对酪蛋白酶解产物性质的影响,采用p H-stat法控制水解过程p H恒定(8.5±0.02),制备DH8%和12%的酶解产物,分别测定其DPPH自由基清除率,TCA可溶性氮含量,采用SE-HPLC测定分子量分布。结果表明,不同酶解条件下,DH 8%和DH 12%水解产物的DPPH自由基清除率分别为32.76%~34.02%和39.87%~41.02,DH 8%和DH 12%水解产物的TCA可溶性氮含量值分别为58.98%~60.43%和69.69%~70.34%。方差分析显示水解度相同,不同产物的DPPH自由基清除率无显著性差异,TCA可溶性氮含量的结果亦如此(p0.05)。HPLC的结果进一步证明尽管水解条件不同,水解至相同DH时,水解产物的分子量分布一致。     

小麦蛋白酶解制备活性多肽的研究

分别采用胃蛋白酶、酸性蛋白酶、木瓜蛋白酶、胰蛋白酶水解小麦蛋白,实验结果表明,胃蛋白酶作用效果最好。以水解度（DH）和得率为指标,对底物浓度、胃蛋白酶浓度、反应温度、反应时间和反应体系pH值等影响因素进行了单因素试验,在此基础上通过正交试验最终确定了酶解小麦蛋白的最佳条件为：底物浓度10％、E／S7％、温度35℃、时间3h、pH2．0,在此条件下蛋白质水解度为4．62％、产物得率为50．64％。利用SDS．聚丙烯酰胺凝胶电泳分析小麦蛋白的水解产物,实验结果表明,所有水解产物的分子量均小于7．0kD,其中大多数处于4．1～1．4kD,说明小麦蛋白已被水解成多肽。     

胞壁蛋白酶（CEP）酶解对酪蛋白结构及功能特性的影响

通过纳米粒度分析、傅立叶红外光谱（FTIR）、乳化性、乳化稳定性、蛋白溶解性、抗氧化性及ACE抑制率的测定,分析探讨酪蛋白及其不同水解度（DH 2.4%、4.5%、7.1%、8.3%）的嗜酸乳杆菌胞壁蛋白酶（CEP）酶解产物的结构及功能特性。FTIR分析表明CEP酶解改变了酪蛋白各种构象所占的比例,酪蛋白二级结构发生了不同程度的变化;纳米粒度分析表明酪蛋白颗粒大小随水解的加深先减小后增大,其水解物颗粒在DH 4.5%时最小,乳化稳定性最大;酪蛋白的乳化性随水解的加深先增大后减小,DH 7.1%时增至最大,与其溶解性的变化趋势一致;此外酪蛋白的酶解物具有一定的ACE抑制活性及抗氧化性,且DPPH清除能力在一定范围内随水解度及浓度的增大而增大,当DH为8.3%,浓度为5mg/mL时,DPPH清除能力增大至35.00%。因此CEP酶解可有效改善酪蛋白的结构及功能特性,为乳源性功能多肽的开发提供理论依据。     

不同水解度下的大豆分离蛋白的结构表征

利用中性蛋白酶水解大豆分离蛋白(SPI),并通过荧光探针法、SGS-PAGE凝胶电泳及扫描电镜技术对不同水解度(DH)下的SPI的疏水性进行分析,且对其二级结构和微观结构变化进行表征。结果表明:随着水解度的增加,SPI的表面疏水性呈增长趋势。通过不同DH下制备的SPI的电泳分析,随着DH的增大,SPI被逐渐水解形成更多的低分子量多肽,且不同DH下的SPI的多肽与亚基分布相似,随着DH的增大,SPI的低分子量多肽含量先增大后降低,新增的聚集肽含量增多。不同DH下的SPI,皆呈单个球状蛋白分布,分布较为松散,每个球状蛋白上皆有亚基分布,大部分球蛋白球体表面呈塌陷状态,随SPI的DH增大,SPI的球蛋白表面塌陷越明显,且亚基未被解离。     

Lactobacillus helveticus 9的酪蛋白水解作用研究

基于酪蛋白水解度和多肽含量数据,研究了两株瑞士乳杆菌(L.helveticus 9和L.helveticus 6)与酪蛋白共培养后的蛋白水解效果,菌株L helveticus 9蛋白水解活性显著高于菌株L.helveticus 6.在不同发酵时间下,菌株L.helveticus 9对酪蛋白的水解作用随时问延长而逐渐递增,培养48h时酪蛋白的水解度可达17.3%,多肽含量达到0.62mg/ml:SDS-PAGE分析和Sephadex G-25层析鉴定表明,酪蛋白水解产物多肽含量随培养时间延长而增加.显然,具有较高蛋白水解活力的菌株L.helveticus 9可以用于从酪蛋白中来制备潜在的生物活性肽.     

酶法制备不同水解度高温豆粕水解产物的理化特性研究

采用Alcalase酶和木瓜蛋白酶分别对高温大豆粕进行酶解,通过控制酶解反应得到水解度为5%、10%和15%的6种水解产物,研究两种酶对不同水解度的水解产物理化特性的影响。结果表明,Alcalase酶和木瓜蛋白酶均可产生6种不同分子量范围的水解产物,但各部分比例具有显著差异(P0.5),其平均分子量随水解度的增加逐渐减少,Alcalase酶的水解产物中小于2562 Da小分子量肽所占比例更高。豆粕蛋白的疏水基团在酶解反应中发生暴露与断裂的数量差,导致其表面疏水性随水解度增加呈现先下降再上升的变化,即水解度为10%的表面疏水性最低。zeta电势的绝对值随水解度不断上升,分子间的斥力增大,相同水解度下两种酶对zeta电势的影响并不显著。此外,在pH值为3、5、7和9时,水解产物的溶解性随着水解度的增加而逐渐增高,乳化活性和乳化稳定性则逐渐降低。     

不同条件下水解酪蛋白所得到的抗菌肽抑菌效果比较

本文采用胰蛋白酶水解酪蛋白制备抗菌肽，针对不同底物浓度下，改变酶与底物浓度比，水解产物的抑菌活性随着水解度的变化情况进行对比。确定了在45℃时该反应的最适底物浓度是9％（g/ml)；最佳酶与底物浓度比为1：120。同时，通过在不同水解时间取点，发现水解所得到产物的抑菌强度随水解度的增大而增大。     

碱性蛋白酶水解大豆蛋白过程的拟合及水解产物性质的研究

采用Alcalase碱性蛋白酶在50、60℃下以不同酶底比(1∶100、2∶100和3∶100)分别制备水解度(DH)为5%、10%和15%的大豆蛋白水解产物。考察水解反应动力学以及水解产物的DPPH自由基清除率和三氯乙酸可溶性氮含量,并且采用HPLC表征水解产物相对分子质量变化。结果表明:碱性蛋白酶在所考察的条件下水解大豆蛋白,符合指数形式动力学方程;水解产物水解度从5%增加到15%,DPPH自由基清除率和三氯乙酸可溶性氮含量分别由17.07%、16.28%增加到31.84%、64.85%;大豆蛋白经碱性蛋白酶水解至相同水解度时,在同一温度下,随着酶底比的增加,水解产物的DPPH自由基清除率和三氯乙酸可溶性氮含量增加,水解产物中相对分子质量大于10 k D的肽段比例逐渐减小,小于0.5 k D的肽段比例逐渐增大。同一种酶水解同一种蛋白质,虽然水解反应动力学均符合指数形式动力学方程,但是当大豆蛋白被水解至相同水解度时,不同条件下制备的水解产物的性质与结构均有显著性差异。     

剪切乳化预处理对玉米蛋白酶解反应的促进作用

研究了剪切乳化预处理对黑曲霉酸性蛋白酶水解玉米蛋白反应的影响。实验结果表明，在酸性蛋白酶与玉米蛋白粉的质量比为10％，底物浓度为80g／L，反应温度为55℃，水解24h的条件下，当玉米蛋白粉未经高速剪切乳化预处理时，蛋白质溶出率为62．79％，所得的水解产物中分子量小于1000u的寡肽占45％；而当玉米蛋白粉经7000r／min高速剪切乳化预处理1h后，蛋白质溶出率提高到88．47％，所得的水解产物中分子质量小于1000u的寡肽占78％。利用高效液相色谱法测定了玉米蛋白粉酶解产物的氨基酸组成，发现高速剪切乳化预处理能增加水解产物中支链氨基酸的含量，有利于生产高F值寡肽。     

鱼鳞明胶酶解制备超氧阴离子清除活性肽的研究

研究了Alcalase2.4L水解鱼鳞明胶的工艺条件及产物的超氧阴离子清除活性。以水解度为指标,在单因素实验的基础上,通过响应面实验优化得到最佳酶解条件为温度54.5℃,pH7.7,底物浓度1.8%(W/V),加酶量1%(W/W),在此条件下水解2h的水解度为13.02%。对酶解产物进行体外超氧阴离子清除活性分析,结果表明,不同水解度的酶解产物表现出不同的抗氧化活性。在优化条件下水解4h,水解产物的超氧阴离子清除活性最高,IC50为0.36mg/mL,此时水解度为14.11%。     

ENZYMATIC HYDROLYSIS OF WHEY PROTEINS BY TWO DIFFERENT PROTEASES AND THEIR EFFECT ON THE FUNCTIONAL PROPERTIES OF RESULTING PROTEIN HYDROLYSATES

Whey protein concentrate (WPC 80) was hydrolyzed by Alcalase 2.4 L and Protamex to 5, 10, 15 and 20% degree of hydrolysis (DH). WPC 80 and its hydrolysates were analyzed, compared and used for measuring some functional properties. All hydrolysates were different from WPC 80 in protein, moisture and ash content. Free amino groups and protein solubility increased with increasing DH. The peptides produced by hydrolysis had smaller molecular sizes, and their average molecular weight decreased as the DH increased. Except hydrolysates generated by Alcalase 2.4 L at 5% DH, all others showed poor emulsifying and foaming properties compared with unhydrolyzed WPC 80. Gelation properties of WPC 80 and its hydrolysates were different. The global amino acid compositions did not differ significantly between the different hydrolysates, and they were very close among WPC 80 and its hydrolysates except for Methionine, Glycine, Histidine and Valine.     

Influence of Hydrolysis Degree on the Functional Properties of Salmon Byproducts Hydrolysates

ABSTRACT: Protein hydrolysates from salmon heads were obtained by enzymatic treatment with Alcalase^(r) 2.4L. Response surface methodology (RSM) allowed optimization of temperature, enzyme/substrate, and pH leading to various hydrolysates (11.5% to 17.3% hydrolysis degree [DH]) and protein recovery ranging from 47% to 70%. Size exclusion chromatography of hydrolysates showed that small peptides increased only at high DH. The nitrogen solubility index (NSI) of hydrolysates was higher than 75% over a wide range of pH values, whereas hydrolysates with high DH had the best solubility. Emulsifying capacity, emulsion stability, and fat absorption capacity were better when DH was low.     

Functionality of Bacillus proteinase hydrolysates of sodium caseinate

Sodium caseinate (NaCN) was digested with Protamex, a Bacillus proteinase, at 40°C and pH 7.0 to degree of hydrolysis (DH) values of 2.7%, 5.3% and 13.3%. The solubility, emulsifying, foaming and viscosity properties of the hydrolysates were investigated between pH 2.0 and 10.0. Foam expansion of >1300% was observed for the 5.3% DH hydrolysate at pH 4.0, compared to 670% for unheated NaCN. Significantly improved foam expansion properties (P<0.005) were observed over the entire pH range examined for the 13.3% DH hydrolysate compared to unheated or heat-treated NaCN. Hydrolysis resulted in significantly improved solubility (P<0.005) around the isoelectric point and significant improvements in the emulsifying activity and stability (P<0.005) at alkaline pH compared to unheated NaCN. Hydrolysis with Protamex increased the apparent viscosity of NaCN at the isoelectric point. Reversed-phase HPLC profiles showed that high DH samples contained high levels of hydrophilic peptides.     

Characteristics of Bellamya purificata snail foot protein and enzymatic hydrolysates

The foot muscle protein of Bellamya purificata (mud snail, named Luosi in Chinese) was investigated. Its conformation change and increase in solubility were researched during enzymatic hydrolysis. The protein conformation was looser following an increase in pH (from 10 to 12), while the β-sheet was the main conformation at pH 12. Blending, ultrasonic extraction, ultradispersing and alkaline treatment increased the solubility of the foot muscle protein. The effects of several proteases on its hydrolysis were compared and Proleather FG-F was chosen. The relative molecular mass distribution, the free amino acids (FAA) content and the angiotensin-I converting enzyme (ACE) inhibitory activity of the hydrolysates were quantitatively analyzed and compared. In the Proleather FG-F hydrolysates, the percentage of the peptides with molecular weight between 150 and 2000 Da were 84.65%, much more than that in the Alcalase 2.4L hydrolysates (68.44%). Proleather FG-F released much less FAA (5.80%), than Alcalase 2.4L (17.01%). The IC₅₀ of the Proleather FG-F hydrolysate was 0.69 mg/ml, whereas for the Alcalase 2.4L hydrolysate the value was 3.30 mg/ml. Finally, response surface methodology (RSM) was used to optimize the factors (pH, enzyme: substrate ratio- E/S- and temperature) affecting Proleather FG-F hydrolysis.     

Functional Properties and Bitterness of Sodium Caseinate Hydrolysates Prepared with a Bacillus Proteinase

The functional and physicochemical characteristics, and bitterness were evaluated on sodium caseinate hydrolysates generated with a commercial Bacillus proteinase complex. At low degrees of hydrolysis (0.5 and 1.0% DH) the hydrolysates compared to the unhydrolyzed substrate had increased emulsion activity at pH 2 and 4. However, higher DH resulted in lower emulsion activities. At pH 8 and 10, the low DH hydrolysates displayed increased foam expansion and decreased foam drainage compared to the starting substrate. The Bacillus proteinase hydrolysates at higher DH had similar bitterness values to tryptic hydrolysates at equivalent DH. However, the Bacillus hydrolysate at 0.5% DH was less bitter (p<0.05) than the tryptic hydrolysate at the same DH.     

Alcalase 2.4L催化牛骨胶原蛋白水解反应及其动力学研究

对碱性蛋白酶Alcalase 2.4L水解牛骨胶原蛋白的工艺以及动力学特征进行了研究.探讨底物浓度、酶量、温度、pH、时间等因素对胶原蛋白水解度的影响,研究确定了碱性蛋白酶Alcalase 2.4L水解牛骨胶原蛋白的最佳水解条件为:在pH8.5,60℃,底物浓度为80g/L,加酶量为40μL/g条件下水解3h,水解度可达13.5%,优于其他蛋白酶.根据所建立的pH-stal酶解体系确定了Km为1.6362mol/L,Vmax为0.3444mol/L·h,为更有效地利用胶原蛋白资源奠定了理论基础.     

Functional Properties of Veal Bone Hydrolysates

Meat hydrolysates were prepared from crushed veal bone residues, using a commercial preparation of Bacillus subtilis (55°C, pH 7.0). Influence of degree of hydrolysis (DH = 4.0; 5.0; 5.6; 6.2, 8.2%) on functional properties of freeze-dried hydrolysates was studied as compared to sodium caseinate and gelatin, for possible use as natural stock bases for soups, sauces and ready-cooked dishes. Limited proteolysis was controlled using pH-stat and the trinitrobenzene sulfonic acid (TNBS)-methods with strong correlation. Partial hydrolysis improved protein solubility more than 74% over a wide pH range. Emulsifying capacity (EC), activity (EA) and stability (ES) were determined by conductivity measurements. EC and EA increased with protein concentration ranging from 0.025 to 0.25%, while EC decreased with DH. Correlations were high between EA, EC, DH, and adsorption rate.     

Nutritional composition and antioxidant properties of protein hydrolysates prepared from echinoderm byproducts

Protein hydrolysates were prepared from echinoderm byproducts, including viscera (SCV) of Atlantic sea cucumber ( Cucumaria frondosa ) and digestive tract (UDT) and non-commercial grade gonads (UGN) of green sea urchin ( Strongylocentrotus droebachiensis ). Enzymatic hydrolysis was performed on defatted materials using Alcalase^® 2.4L (0.75% w/w) and reaction was carried out overnight (∼16 h, 55 °C, pH 8.0). Freeze-dried hydrolysates were analysed for their nutritional composition, nitrogen solubility index and antioxidant activity. Degree of hydrolysis was low, with values of 5.6%, 4.6% and 7.0% for SCV, UDT and UGN, respectively. Hydrolysates showed high protein content (∼55%), high proportion of essential amino acids (∼35% of total amino acids) and good solubility (nitrogen solubility index ≈ 68%). They contained variable concentrations of major and trace elements with a predominance of Na and K. Hydrolysates showed apparent antioxidant activities in both ORAC assay (267–421 μmol TE g⁻¹) and inhibition of lipid oxidation test (54–57%). Antioxidant activities were thought to be associated with the presence of antioxidant peptides in hydrolysates. Our results showed that hydrolysates from Atlantic sea cucumber and green sea urchin byproducts might serve as alternative sources of dietary proteins, with good nutritional composition, high solubility and interesting protection against oxidative stress.