大豆蛋白酶解肽的分子量分布及抑制ACE活性关系研究

研究不同蛋白酶作用的酶解肽表现在血管紧张素转化酶(ACE)活性抑制差异,酶解产物的水解度、分子量分布与ACE抑制率的相互关系。用胰蛋白酶、胃蛋白酶、中性蛋白酶、木瓜蛋白酶、碱性蛋白酶等五种蛋白酶对大豆分离蛋白酶解,进行了多肽增量、水解度、超滤膜分离及其ACE抑制率对比等实验。结果表明,碱性蛋白酶的多肽增量最大,胃蛋白酶次之,依次为木瓜蛋白酶和中性蛋白酶,胰蛋白酶则出现反常;水解度随着酶解的时间而增加,碱性蛋白酶的最大水解度可达到21%,依次为胃蛋白酶、木瓜蛋白酶和中性蛋白酶,最低为胰蛋白酶仅为9%左右;与此对应的碱性蛋白酶的酶解物的ACE活性抑制率为最高(44.9%),胃蛋白酶次之(43.5%)。分子量范围在1000Da以下组分对ACE的抑制效果最高,碱性蛋白酶作用获得的小分子肽组成为71.25%,胃蛋白酶的为69.35%,但其对应的ACE抑制率却为64.57%和78.49%。中性蛋白酶、木瓜蛋白酶和胰蛋白酶作用获得的小分子肽的ACE抑制率分别为45.7%、47.3%和29.6%。胃蛋白酶的降压肽制备效果为最好,其次为碱性蛋白酶、木瓜蛋白酶、中性蛋白酶和胰蛋白酶。     

几种酶对鲢鱼蛋白水解条件的比较

通过比较胰蛋白酶、碱性蛋白酶、中性蛋白酶、胃蛋白酶以及木瓜蛋白酶在不同条件下对鲢鱼蛋白进行酶解发现，胰蛋白酶对鲢鱼蛋白的酶解效果最好，在最佳条件下水解度可达到35％左右。     

核桃多肽制备酶解关键技术研究

以核桃仁为原料,以水解度和对α-淀粉酶抑制率为评价指标,正交设计研究核桃蛋白酶解中相关的单酶水解、多酶水解、酶添加顺序、复合酶最佳配方等关键因子。结果表明,单酶对核桃蛋白的水解度大小依次为:碱性蛋白酶中性蛋白酶≈酸性蛋白酶胃蛋白酶胰蛋白酶,而酶解产物对α-淀粉酶抑制率大小依次为:酸性蛋白酶中性蛋白酶碱性蛋白酶胃蛋白酶胰蛋白酶,并发现依次添加单酶比同时添加的效果更好。综合考虑,先加中性蛋白酶再加碱性蛋白酶的添加方式最佳,可使核桃蛋白水解度达到40%左右,同时还保证酶解产物对α-淀粉酶抑制率较大,可达到85.9%。     

核桃分离蛋白酶解产物结构与功能的变化

核桃蛋白是优质植物蛋白,但其溶解度较低,限制了其在食品中的应用。为拓宽核桃蛋白的应用范围,采用不同的蛋白酶（碱性蛋白酶、胃蛋白酶、胰蛋白酶、木瓜蛋白酶、中性蛋白酶）对核桃分离蛋白进行酶解,然后分析不同蛋白酶酶解产物的水解度、二级结构和功能性（溶解性、吸水性、吸油性、乳化特性和起泡特性）。结果表明：碱性蛋白酶酶解产物的水解度最高,为22.16%,其次是木瓜蛋白酶的,为20.06%,而胰蛋白酶的最低,为13.95%;FTIR结果显示这5种蛋白酶酶解产物的二级结构中均以β-折叠及β-转角为主;在pH 7.0时,与核桃分离蛋白的吸水性（2.36 g/g）和吸油性（4.65 g/g）相比,5种蛋白酶酶解产物的吸水性和吸油性分别提高了11.58～17.15 g/g和7.58～15.44 g/g;与核桃分离蛋白相比,在不同pH（2～12）下,5种蛋白酶酶解产物的溶解度显著提高;在不同的pH和NaCl浓度下,5种蛋白酶酶解产物的乳化特性和起泡特性也不同。从提高蛋白功能性角度考虑,碱性蛋白酶为核桃分离蛋白的最佳酶解用酶。     

山羊乳酪蛋白酶解条件优化及其抗菌活性研究

为制备山羊乳酪蛋白抗菌肽,以新鲜无抗山羊乳为原料,采用碱性蛋白酶、中性蛋白酶、木瓜蛋白酶、胰蛋白酶和胃蛋白酶在最适条件下酶解山羊乳酪蛋白,以沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑菌圈直径为指标,筛选最适水解酶,并通过单因素实验和正交实验确定水解酶水解山羊乳酪蛋白的最适条件。结果表明:木瓜蛋白酶是酶解山羊乳酪蛋白的最佳蛋白酶,其酶解物对沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑制作用最强;确定了木瓜蛋白酶水解山羊乳酪蛋白的最佳工艺。     

罗非鱼鳞明胶的制备及其酶解产物活性研究

以罗非鱼鱼鳞为原料,通过热水提取鱼鳞中的明胶,利用多种蛋白酶对明胶进行酶解,测定其不同酶解产物的抗氧化性、血管紧张素I转换酶(ACE)抑制活性、α-葡萄糖苷酶(AG)抑制活性等生物活性。实验结果表明:100℃热水或120℃热水提取明胶1.5h的得率分别为20.2%和37.9%。6种蛋白酶(木瓜蛋白酶、胰蛋白酶、胃蛋白酶、酸性蛋白酶、中性蛋白酶、碱性蛋白酶)水解明胶后,胰蛋白酶水解液和碱性蛋白酶水解液的羟自由基清除力为76.7%和76%,高于Vc的65.6%;除中性蛋白酶对DPPH自由基没有清除力以外,其余5种蛋白酶均对DPPH自由基有清除力,但是低于Vc;胃蛋白酶水解液对α-葡萄糖苷酶的活性抑制作用最强,为67.1%,而中性蛋白酶、碱性蛋白酶和胰蛋白酶水解液对α-葡萄糖苷酶没有抑制作用。6种蛋白酶的水解产物均有的ACE抑制活性,没有抗凝血活性。     

黄鲫(Setipinna taty)蛋白酶解液的抑菌活性及稳定性研究

以黄鲫为原料测定其氨基酸组成,比较风味蛋白酶、胰蛋白酶、胃蛋白酶、碱性蛋白酶和木瓜蛋白酶水解黄鲫蛋白所得酶解液对大肠杆菌的抑菌作用,并对抑菌效果最强的蛋白酶酶解液进行抑菌稳定性研究。结果表明：黄鲫蛋白必需氨基酸含量丰富,其中胃蛋白酶的水解液对大肠杆菌抑菌作用强,相对分子质量主要分布在3000～1000;黄鲫胃蛋白酶酶解液对热稳定,酸性低pH值可增强其抑菌效果,可耐受胰蛋白酶和β-内酰胺酶处理,该酶解液有作为天然抗菌剂应用的前景。     

猪血红蛋白酶解制备ACE抑制肽的研究

本实验选用碱性蛋白酶、胰蛋白酶、胃蛋白酶、风味蛋白酶、中性蛋白酶和木瓜蛋白酶等六种商业蛋白酶在各自最适反应条件下分别水解猪血红蛋白12h,研究其水解产物对血管紧张素转换酶抑制率和蛋白水解度的影响。结果显示:采用胃蛋白酶酶解获得的产物ACE抑制率最高。胃蛋白酶的酶解条件为底物5%(质量分数),酶与底物浓度比E:S=3%,温度37℃,pH2.0,水解4h后其ACE抑制率为81.10%,水解度为6.64%。     

酶解花生蛋白制备血管紧张素转化酶抑制肽

采用碱性蛋白酶、胃蛋白酶、胰蛋白酶、复合风味酶（固／液）、木瓜蛋白酶水解花生蛋白制备血管紧张素转化酶（ACE）抑制肽,通过体外检测法测定其ACE抑制率。结果表明,碱性蛋白酶水解物的ACE抑制率最大。根据Box—Behnken的中心组合实验设计原理对碱性蛋白酶酶解工艺进行优化,结果表明：当温度为53．7℃,底物浓度为7．72％,酶与底物质量比4．18％,pH=8．0,水解时间为120min时,其ACE抑制率可达72．78％。     

复合酶解可溶性蛋膜蛋白制备多肽的工艺优化

为研究蛋膜蛋白的利用,采用碱性蛋白酶、木瓜蛋白酶、胰蛋白酶、中性蛋白酶及胃蛋白酶进行单一酶解筛选实验,然后进行复合酶解实验。采用二次正交旋转组合设计,以水解度、氮收率为指标,研究酶制剂种类、加酶方式、复合酶比例、总加酶量、酶解时间、pH 值及温度对制备多肽工艺的影响。综合考虑水解度和氮收率因素,最终确定复合酶解可溶性蛋膜蛋白制备多肽的最佳工艺条件为：总加酶量16000U/g,并以碱性蛋白酶与木瓜蛋白酶的酶活力配比为8:2 先后加入;酶解时间为碱性蛋白酶2h、木瓜蛋白酶1h;pH 值为碱性蛋白酶9.0、木瓜蛋白酶5.5;酶解温度为50℃。该条件下制备的蛋膜蛋白酶解产物水解度和氮收率分别为46.12%、85.56%。     

Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes

Eleven peptides, selected on the basis of physicochemical characteristics and their theoretical release from β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) by trypsin or chymotrypsin, were chemically synthesised to evaluate their immunomodulating properties. Murine splenocyte proliferation in the absence and presence of mitogen and different peptide concentrations were measured after 72–96 h incubations. β-Lg f78–83 had no effect on proliferation; β-Lg f15–20, f55–60, f84–91, f92–105, f139–148, f142–148 and α-La f10–16 stimulated proliferation to different extents; β-Lg f1–8, f102–105 and α-La f104–108 showed a cytotoxic effect. Regression analysis revealed the relationship of positive charge, hydrophobicity and length to the stimulatory proliferative effect. β-Lg f15–20, f55–60 and f139–148 also induced various inhibiting and/or stimulating effects on cytokine secretion. The results confirm that peptides releasable by digestive enzymes from α-La and β-Lg have the potential to influence the specific immune response through the modulation of splenocyte proliferation and cytokine secretion.     

Coacervate formation of α-lactalbumin–chitosan and β-lactoglobulin–chitosan complexes

α-Lactalbumin (α-La) and β-lactoglobulin (β-Lg) are important whey proteins with isoelectric points of pH 4.80 and 5.34, respectively, and evidence negative charge over a range of pHs. Chitosan exhibits a cationic property under pH 6.5. In an effort to determine the physicochemical properties of mixtures of 0.5% α-La and 0.1% chitosan, and 0.5% β-Lg and 0.1% chitosan, optical structure, turbidity, electrophoresis, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were assessed in a pH range of 2.0–8.0. The results demonstrated that α-La, β-Lg, and chitosan precipitated at pH values of approximately 5.0, 5.0, and 7.0, respectively. The mixtures of α-La and chitosan as well as β-Lg and chitosan coacervated at a pH range of 6.0–6.5. The turbidity of α-La and α-La–chitosan achieved a maximum at pH 5.0, whereas those of β-Lg and β-Lg–chitosan achieved maximum values at a pH of 6.5. The electrophoregram showed a large band with high molecular weight in increasing pH values from 5.0 to 6.0, which suggested that α-La and β-Lg form polymers with chitosan. The denaturation temperature and enthalpy of α-La were shown to increase, whereas those of β-Lg were reduced. The SEM images demonstrated that the α-La was characterized by uneven and associated cluster morphology, whereas that of β-Lg was even, globular, and harbored dense particles, whereas the chitosan evidenced a flat morphology. Our assessment of the complex demonstrated that α-La and β-Lg attached to the surface of the chitosan. The α-La–chitosan and β-Lg–chitosan complexes evidenced opposite charges at a pH range of 5.0–6.0, and formed coacervates. It appears, therefore, that the α-La–chitosan and β-Lg–chitosan coacervates might be applied as a delivery system for foods, nutraceuticals, cosmetics, and drugs.     

Fractionation of α-Lactalbumin and β-Lactoglobulin from Whey Protein Isolate Using Selective Thermal Aggregation, an Optimized Membrane Separation Procedure and Resolubilization Techniques at Pilot Plant Scale

An optimized fractionation method in the pilot scale for production of isolated α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) was developed. The method comprises following steps: (1) selective thermal precipitation of α-La, (2) aging of the formed particles, (3) separation of native β-Lg from the precipitate via microfiltration and ultrafiltration, (4) purification of β-Lg, (5) resolubilization of the precipitate, and (6) purification of α-La. The native status of the isolated fractions was confirmed by reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and differential scanning calorimetry (DSC). Protein fractions with a purity of 91.3% for α-La and 97.2% for β-Lg were produced. These values were based on the native protein detectable in RP-HPLC. High overall yields for α-La between 60.7% and 80.4% and for β-Lg between 80.2% and 97.3%, depending on membrane operation parameters, were achieved. The method offers potential for pilot plant scale and possibly industrial application to produce pure native fractions of α-La and/or β-Lg.     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

Thermal Properties of Yak α-Lactalbumin and β-Lactoglobulin: a DSC Study

A differential scanning calorimetry (DSC) method was used to investigate the denaturation temperature of yak α-lactalbumin (α-La), β-lactoglobulin (β-Lg), and a mixture of two proteins and the thermal properties of α-La and β-Lg in the presence of glucose, lactose, sucrose, NaCl, CaCl₂, and at various pH (4.0–10.0). The denaturation temperature (T _d) of α-La increased from 52.1 °C in the absence of β-Lg to 53.9 °C in the presence of β-Lg, while the T _d of β-Lg decreased from 81.4 °C in the absence of α-La to 79.9 °C in the presence of α-La. α-La was thermal stable in the range of pH 4.0–10.0, while β-Lg was more thermal stable in acidic pH than in alkaline pH. Sugars, Na⁺, and Ca²⁺ influenced the stabilization of the two proteins against thermal denaturation with greatly influenced for β-Lg. α-La kept reversibility in the presence of sugars, NaCl, CaCl₂, and over a wide pH range (4.0–10.0), with most of the reversibility values being greater than 90%. In contrast, β-Lg was completely irreversible whether in its native state or in the presence of the additives.     

Comparison of the gel-forming ability and gel properties of α-lactalbumin,lysozyme and myoglobin in the presence of β-lactoglobulin under high pressure

α-Lactalbumin (α-La) and lysozyme (LZM) each contain four disulfide bonds but no free SH group, whereas myoglobin (Mb) possesses no disulfide bond or free SH group. In this work, the pressure-induced gelation of α-La, LZM and Mb in the absence and in the presence of β-lactoglobulin (β-Lg) was studied. Solutions of α-La, LZM and Mb (1–24%, w/v) did not form a gel when subjected to a pressure of 800 MPa and circular dichroism analysis revealed that both α-La and LZM are pressure-resistant proteins. In the presence of β-Lg (5%, w/v), however, a pressure-induced gel formed for α-La and LZM (each 15%, w/v) but not for Mb (15%, w/v). One- and two-dimensional SDS-PAGE demonstrated the disulfide cross-linking of proteins was responsible for the gelation. Although α-La and LZM are homologous and have the same disulfide bond arrangement, the texture and appearance of the gels formed from α-La/β-Lg and LZM/β-Lg were markedly different even when induced under the same experimental conditions. Microscopic analysis indicated that phase separation occurs during the gelation of LZM/β-Lg but not during the gelation of α-La/β-Lg. NMR relaxation measurement revealed that the association of water molecules with the protein matrix in the α-La/β-Lg gel is tighter compared to that in the LZM/β-Lg gel. These results indicate that the gel-forming ability of a globular protein under high pressure is related to the primary structure of the protein, and that the gel properties depend on the cross-linking reaction and on the phase behavior of protein dispersion under high pressure.     

芝麻蛋白制备金属螯合肽的酶解工艺研究

分别采用木瓜蛋白酶、胰蛋白酶和碱性蛋白酶Alcalase对芝麻蛋白进行水解,结果表明胰蛋白酶为制备金属螯合肽的最佳酶。通过优化胰蛋白酶酶解工艺条件发现最佳条件为:底物质量浓度5%(g/100mL),酶添加浓度20u/g(底物),水解时间5h,得到的酶解产物金属螯合率最强,与Fe2+的螯合率为90.9%,与Zn2+的螯合率为93.5%。通过考察水解度对酶解产物金属螯合率及抗氧化能力的影响,结果显示水解度在18.9%到22.4%之间的酶解产物金属螯合率强,且水解度在一定范围内金属螯合率和抗氧化能力均与水解度呈正相关性。     

Heat-Induced Gelation of Individual Whey Proteins A Dynamic Rheological Study

Heat-induced gelation of the bovine whey proteins [serum albumin (BSA), β-lactoglobulin (β-Lg) and α-lactalbumin (α-La)] has been studied individually and in mixture at different conditions by a dynamic rheological method. Values in the shear stiffness modulus (/G*/) appeared on heating at low protein concentration for BSA (～2%) and at intermediate concentration for β-Lg (～ 5%). α-La did not form a heat-induced gel of concentrations up to 20% (w/v). The ratio of viscous to elastic properties (loss factor) at maximum possible measuring temperature was below 0.07 for the BSA gels and 0.1–0.3 for the β-Lg gels. The temperature of gelation was highly dependent on pH. In mixture one protein could not be exchange for another without changing the gelation behavior of the mixture.     

热处理对α-乳白蛋白变性及其与其他乳蛋白相互作用影响的研究进展

本文综述了热处理导致的α-乳白蛋白（α-lactalbumin,α-La）变性及其与其他乳蛋白成分之间的相互作用和影响因素。α-La的热稳定性受其分子内结合的Ca2＋影响,变性后无法自聚,但可以与β-乳球蛋白（β-lactoglobulin,β-Lg）和血清白蛋白（serum albumin,SA）形成聚集体。经高温短时（high temperature short time,HTST）巴氏杀菌处理生成的α-La/β-Lg聚集体可用于生产低黏度、低浊度和高溶解性的蛋白饮料,α-La/SA聚集体具有良好的凝胶结构。α-La/β-Lg聚集体可与酪蛋白胶束表面的κ-酪蛋白结合,生成的聚合物有利于缩短生产发酵乳的时间,改善酸乳凝胶结构。α-乳白蛋白还能与免疫球蛋白G结合,采用HTST、超巴氏杀菌和超高温灭菌处理可降低乳品的致敏性。在实际生产中可根据需要利用上述反应,选择合适的热处理方式。     

Hydrolysis of bovine β-Lactoglobulin by various proteases and identification of selected peptides

β-Lactoglobulin (β-Lg) was incubated with a number of proteases to seek conditions under which relatively large fragments were produced in good yield. The proteases used were bromelain, papain, pepsin, trypsin, endoproteinase Arg-C, aminopeptidase and carboxypeptidase Y. Bromelain-catalysed hydrolysis led to rapid hydrolysis of β-Lg and the formation of a large number of small peptides. Pepsin hydrolysed only a fraction of the β-Lg molecules, and the few released fragments were degraded into small peptides. Both papain and trypsin degraded β-Lg with the formation of medium-sized peptides (1-5 kDa), of which seven were identified. Endoproteinase Arg-C did not hydrolyse native β-Lg, whereas heat-treated β-Lg was degraded and six peptides with molecular masses around 2 kDa were identified. None of the exopeptidases catalysed complete removal of a substantial part of β-Lg. A few conditions under which relatively large and easily purifiable fragments were produced from β-Lg were identified.