酶解对乳清蛋白抗原性影响的研究

研究了酶解对乳清蛋白抗原性的影响。选择了7种常见蛋白酶在同一水解模式下水解乳清蛋白，用竞争ELISA法测定水解物的残留抗原性，从而间接测定其过敏性变化。结果表明，酶解能有效降低乳蛋白抗原性，但水解物仍能与特异抗体反应，保留一部分抗原性。不同酶对乳清蛋白过敏原的影响不同，酶的特异性对乳清蛋白水解物的抗原性有较大的影响，碱性蛋白酶降低乳蛋白抗原性的效果最佳，对抗β-乳球蛋白（β-LG）和抗α-乳白蛋白（α-LA）抗体的抗原性分别降低了50．02％和99．72％。     

马克思克鲁维酵母(Kluyveromyces marxianus)对发酵乳蛋白、质构的影响

在以保加利亚乳杆菌与嗜热链球菌为发酵剂的基础上,添加一定比例的马克思克鲁维酵母进行共同发酵,研究其对发酵乳发酵及后熟过程中pH、蛋白含量、各乳蛋白相对含量及质构的影响并研究其相关性,为开发添加马克思克鲁维酵母的新产品提供数据参考。结果表明,酵母能促进发酵乳凝乳,降低凝乳时间、显著降低发酵乳pH;酵母的添加降低了蛋白含量、增加了发酵乳中蛋白的水解情况,并对发酵与后熟过程中蛋白的代谢产生影响,其中对κ-酪蛋白影响显著,除此之外,添加酵母后增加了发酵乳硬度、稠度、黏性等,相关性分析表明,pH、蛋白含量、κ-酪蛋白、β-乳清蛋白、α-乳白蛋白与发酵乳质构具有显著相关性,且添加酵母后相关性增加。     

碱性蛋白酶水解乳清分离蛋白工艺优化

目的 降低乳清分离蛋白中的致敏蛋白含量, 制备低致敏性乳制品。方法 利用碱性蛋白酶水解乳清分离蛋白, 研究酶添加量、初始pH、酶解时间以及温度对乳清分离蛋白水解度的影响。在单因素的实验基础上, 采用Box-Behnken实验设计方法进行四因素三水平的响应面优化实验。结果 在P<0.05的水平下, 4个因素对乳清分离蛋白的水解度都有显著影响。最优的水解工艺为: 酶添加量6.4%、初始pH 11、酶解时间4 h、温度60 ℃。乳清分离蛋白在此条件下水解后, 水解度达到21.11%。酶解液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)分析显示, 经过这一优化工艺水解, 10 kDa以上的蛋白基本全部被降解。高效液相色谱法(high performance liquid chromatography, HPLC)分析酶解产物的多肽及蛋白质分子量分布, 结果显示酶解产物的分子量大都分布在3.5 kDa及以下。采用间接竞争酶联免疫吸附法原理测定2种标志性致敏蛋白(β-乳球蛋白和α-乳白蛋白)的残余抗原性, 发现2种致敏蛋白的残余抗原性也有不同程度的降低。结论 通过碱性蛋白酶水解后, 乳清分离蛋白中具有致敏性的大分子蛋白转变为小分子的肽类, 从而降低了致敏性。     

胰蛋白酶水解对乳蛋白抗原性影响的研究

以胰蛋白酶为水解用酶,利用小鼠动物模型从体外和体内两个方面研究了水解作用对牛乳蛋白抗原性的影响。结果表明,胰蛋白酶水解的最适条件为50℃,E/S为0.6%下水解1.5 h。此水解条件下乳蛋白的总致敏性降低最多,分别为α-乳白蛋白(α-LA)抗原性降低率为80.16%,β-乳球蛋白(β-LG)抗原性降低率为51.94%,酪蛋白(CN)抗原性降低率为73.26%。动物实验表明:酶解组小鼠过敏症状较未水解的牛乳蛋白组相比明显减轻。与乳蛋白相比,酶解物显著抑制特异性IgE的产生,IgE质量浓度下降了42.36%。血浆组胺实验表明,酶解物降低血浆中组胺的释放,组胺质量浓度比乳蛋白组下降了32.77%。     

酶解作用对牛乳乳清蛋白抗原性影响的研究

为了探明胰蛋白酶水解作用对乳清蛋白致敏性或者抗原性的影响,利用小鼠动物模型从体外和体内两个方面研究了水解作用对乳清蛋白致敏性的影响.结果表明,酶解物中β-乳球蛋白抗原性降低率为53.92％,α-乳白蛋白抗原性降低率为82.31％.酶解组小鼠过敏症状较未水解的乳清分离蛋白(WPI)组相比明显减轻.与WPI组相比,酶解物显著抑制特异性IgE的产生,IgE质量浓度下降了40.55％.血浆组胺实验表明,酶解物降低血浆中组胺的释放,组胺质量浓度比WPI组下降了28.72％.     

乳清蛋白-低聚异麦芽糖模拟胃液消化过程中抗原性的变化

目的 研究乳清蛋白-低聚异麦芽糖和乳清蛋白在模拟胃液消化过程中抗原性及游离氨基酸的变化。方法 乳清蛋白(WPI)-低聚异麦芽糖制备后, 对WPI-低聚异麦芽糖及WPI模拟胃液消化过程中的抗原性变化和游离氨基酸含量进行分析。结果 糖基化后乳清蛋白中精氨酸、酪氨酸、胱氨酸和赖氨酸的含量显著降低。经过模拟胃液消化, 乳清蛋白和乳清蛋白-低聚异麦芽糖中α-乳白蛋白的抗原性降低到1 μg/mL以下, 乳清蛋白中β-乳球蛋白抗原性降低到42.83 μg/mL, 乳清蛋白-低聚异麦芽糖中β-乳球蛋白抗原性降低到15.66 μg/mL。结论 经过模拟胃消化, 乳清蛋白-低聚异麦芽糖中α-乳白蛋白和β-乳球蛋白的抗原性比乳清蛋白中α-乳白蛋白和β-乳球蛋白的抗原性低; 在模拟胃液消化过程中, 乳清蛋白-低聚异麦芽糖比乳清蛋白更容易受到胃蛋白酶酶解。     

羊乳清蛋白部分和深度水解工艺、水解物致敏性及肽谱分析

利用羊乳清蛋白制备低致敏性配料是目前乳品工业的研究热点。乳清蛋白是乳中主要的蛋白质之一,也是引起婴幼儿过敏反应的主要成分,将蛋白质水解为小分子肽是降低其致敏性的有效方法。以山羊乳清蛋白为原料,研究了部分水解乳清蛋白和深度水解乳清蛋白的水解工艺和水解物特性(水解度、分子质量分布和β-乳球蛋白抗原性),并利用液相色谱串联质谱法(LC-MS/MS) 比较了部分水解和深度水解工艺中过敏表位酶切位点的差异。研究结果表明,中性蛋白酶和碱性蛋白酶对羊乳清蛋白水解效果较好,其中碱性蛋白酶的水解度最高,达21.26%。经电泳分析,单酶水解后的产物中仍存在大分子多肽链,深度水解工艺需要复合酶水解。在酶底比为4000U/g时,使用碱性蛋白酶在pH值为10.0、温度为55℃条件下水解羊乳清蛋白1.0h,部分水解产物的水解度为12.31%,分子质量在5kDa以下的多肽占95.18%,β-乳球蛋白抗原性下降率为9.40%。中性蛋白酶和碱性蛋白酶的质量比为1∶1,酶底比为6000U/g,在pH值为8.5、温度为50℃条件下,水解羊乳清蛋白3.0h,深度水解产物的水解度为35.58%,分子质量低于3kDa的多肽为97.26%,β-乳球蛋白抗原性下降率为40.97%。部分水解和深度水解均能破坏β-乳球蛋白的大部分过敏表位,但相较于部分水解,深度水解能更大程度地降低乳清蛋白的致敏性。研究旨在为低致敏性羊水解乳清蛋白的生产提供一定的理论参考。     

发酵生产低致敏乳源蛋白基料的研究

利用益生菌发酵,从而降低牛乳蛋白抗原性是目前研究的一个新领域,对于功能性蛋白基料开发具有十分重要的意义。实验中筛选出1株乳杆菌作为发酵菌种,用于生产低致敏乳源蛋白基料。研究其水解乳蛋白的能力及发酵产物中抗原降解的情况。研究发现,发酵乳中α-乳白蛋白(α-LA),β-乳球蛋白(β-LG),as-酪蛋白(αS-CN),β-酪蛋白(β-CN)和牛血清白蛋白(BSA)的抗原降解率分别为31.8%,20.54%,4.08%,18.67%和25.47%。确认菌种为瑞士乳杆菌。可被用于生产低致敏乳源蛋白基料。     

牛乳蛋白过敏原改性的研究

对牛乳中的主要过敏原酪蛋白、β-乳球蛋白（β-LG）及α-乳白蛋白（α-LA）进行了介绍。重点论述了热处理、蛋白水解、糖基化作用和乳酸发酵等技术对乳蛋白过敏改性的研究现状，提出了牛乳中过敏蛋白原改性的研究方向。     

水牛奶乳清蛋白的热稳定性

以水牛奶为材料,利用native-PAGE和SDS-PAGE,通过不同温度和加热条件下总乳清蛋白和单体蛋白质量浓度的变化,研究水牛奶乳清蛋白的热稳定性。结果表明:水牛奶乳清蛋白在native-PAGE条件下只有β-LG一条条带,在SDS-PAGE条件下得到4条单体乳清蛋白条带;随着提取温度和时间的增加,水牛奶单体乳清蛋白和总乳清蛋白质量浓度均呈下降趋势,说明水牛奶乳清蛋白热稳定性较差;4种乳清单体蛋白中,α-LA的热稳定性最好,IG的热稳定性最差,热稳定性顺序为:α-LAβ-LGBSAIG。     

黑木耳多糖-乳清蛋白复合物的制备及其抗原性的研究

蛋白质和多糖在控制条件下通过美拉德反应会发生一定程度的共价复合,能显示更优越的性能。采用黑木耳多糖作为糖基供体,用糖基化的手段与牛乳中乳清蛋白结合形成木耳多糖-乳清蛋白复合物,并在现有的条件下探索不同质量比与不同反应时间对糖基化进程的影响,采用间接竞争ELISA法测定复合物中β-乳球蛋白和α-乳白蛋白抗原性的影响。结果表明,乳清蛋白与黑木耳多糖质量比为1：1,反应时间为24h,是糖基化反应最佳条件并且能有效减低乳清蛋白抗原性,其中β-乳球蛋白抗原性降低率为75.7%,α-乳白蛋白抗原性降低率为25%。     

Stabilizing vitamin D3 using the molten globule state of α-lactalbumin

α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D₃ (vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA–vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.     

Effects of Maillard reaction conditions on the antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with maltose

The effects of Maillard reaction conditions (weight ratio of protein to sugar, temperature and time) on the antigenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in conjugates of whey protein isolate (WPI) with maltose were investigated. Response surface methodology was used to establish models to predict the antigenicity of α-LA and β-LG and find an optimal reaction condition under which the antigenicity of α-LA and β-LG reduces to minimum value. Conjugating WPI with maltose was an effective way to reduce the antigenicity of α-LA and β-LG. The antigenicity of α-LA decreased from 32.25 μg mL⁻¹ to 10.91 μg mL⁻¹. And the antigenicity of β-LG decreased from 272.4 μg mL⁻¹ to 38.17 μg mL⁻¹. Temperature had the greatest effect on the antigenicity of α-LA, while weight ratio of WPI to maltose was the most significant factor on the antigenicity of β-LG.     

超高压处理对乳清分离蛋白结构及致敏蛋白含量的影响

以乳清分离蛋白为研究对象,通过测定圆二色性光谱、巯基含量以及内源性荧光光谱等研究了不同超高压水平(100、200、400和600 MPa)对其二级、三级结构的影响,并采用水解度测定、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及2个标志性致敏蛋白(β-乳球蛋白和α-乳白蛋白)含量的检测来解析超高压对乳清分离蛋白致敏性的影响。结果表明,超高压处理能够使乳清分离蛋白的α-螺旋和β-转角部分转化为β-折叠和无规则卷曲,可以增强乳清分离蛋白的巯基含量,在400 MPa时,表面巯基含量提高了104.82%,也造成了乳清分离蛋白内源性荧光强度的显著变化以及最大吸收波长的红移,电泳图谱以及水解度未显示出明显差异。通过酶联免疫吸附实验原理检测致敏蛋白含量发现,超高压可以使α-乳白蛋白含量显著减少,但是,400 MPa的超高压处理却使β-乳球蛋白含量增加。综上表明,超高压处理能够显著改变乳清分离蛋白的二级、三级结构,暴露出结构内部的疏水基团,并对致敏蛋白产生影响。     

Major whey proteins in ovine and caprine acid wheys from indigenous greek breeds

Acid whey filtrates from the bulk milk of different indigenous greek ovine and caprine breeds were investigated for the quantitative and qualitative characteristics of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG). For comparison reasons acid wheys from bovine milk and from Saanen and Alpine caprine breeds were included. The main characteristic of ovine acid wheys was the low α-LA percentage. The β-LG/α-LA ratio of ovine acid wheys ranged from 3.91 in Chios breed to 6.65 in Boutsiko breed. It was higher than the estimate for bovine acid wheys which ranged from 3.09 to 3.37. The chromatographic and isoelectric focusing profiles of ovine β-LG and α-LA from the different breeds were also variable. The β-LG percentage of caprine acid wheys was lower compared to ovine and bovine acid wheys. Their β-LG/α-LA ratios ranged from 2.02 in Saanen breed to 3.04 in the indigenous breed Skopelos.     

Effect of residual chymotryptic activity in a trypsin preparation on peptide aggregation in a β-lactoglobulin hydrolysate

Peptide composition and peptide aggregation in β-lactoglobulin (β-LG) hydrolysate were studied as related to residual chymotryptic activity in a commercial trypsin (CT) preparation. Residual chymotryptic activity produced smaller and more hydrophobic peptides in tryptic hydrolysate of β-LG, which enhanced peptide aggregation, mainly at acidic pH. The contribution of the chymotryptic peptide β-LG 15–20 to this aggregation process appeared to be very important, but other peptides (i.e., β-LG 41/43−60, 1–8 and 61−69/70+149−162) and residual α-LA may also decrease peptide solubility. When using CT mixtures in the preparation of whey protein hydrolysates, the impact of residual chymotryptic activity should not be neglected because of its influence on peptide–peptide interactions and on the resulting solubility of the hydrolyzed product.     

Short communication: Effect of kefir grains on proteolysis of major milk proteins

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of κ-, α-, and β-caseins (CN), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20°C was monitored. Significant proteolysis of α-LA and κ-, α-, and β-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for α-LA and α- and β-CN. α-Lactalbumin and β-CN were more easily hydrolyzed than α-CN. No significant reduction was observed with respect to β-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of α-LA and β-LG: α-LA was hydrolyzed between 60 and 90% after 12 h (for 6 and 12 g of kefir) and no significant β-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of α-LA and κ-, α-, and β-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. β-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase β-LG digestibility in whey-based or whey-containing foods.     

Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins

Native-PAGE (polyacrylamide gel electrophoresis) was used for the simultaneous qualitative and quantitative analysis of whey proteins of raw, commercial and laboratory heat-treated bovine milks. Four whey protein bands, including β-lactoglobulin variants (β-LG A and B), could be distinctively separated in the gel. The results showed that levels of the major whey proteins were reduced by less than 23% in the pasteurized milks and by more than 85% in the UHT milks as compared with raw milk. The α-lactalbumin (α-LA) exhibited the strongest heat-tolerance: about 32% of it remained in its native state after the milk was heated at 100 °C for 10 min. About 42% of β-LG A and 53% of β-LG B were lost after the milk was heated at 75 °C for 30 min. Blood serum albumin (BSA) was lost almost completely when the milk at pH 5.0 was heated at a temperature of 75 °C or higher. The β-LGA and β-LGB were much more stable at low pH than in neutral conditions.     

Proteolytic pattern, antigenicity, and serum immunoglobulin E binding of beta-lactoglobulin hydrolysates obtained by pepsin and high-pressure treatments

This study evaluates the use of high pressure to enhance pepsin hydrolysis of β-lactoglobulin (β-LG). The protein was subjected to high pressure before and during the proteolytic process. Analysis of remnant β-LG, identification of the peptides produced, and evaluation of antigenicity (binding to commercial antibodies) and binding to IgE of allergic patients’ sera were conducted in the hydrolysates. The results showed that the application of high pressure before the enzyme treatment slightly improved the proteolytic process but did not reduce the antigenicity or IgE binding of the hydrolysates. The application of high pressure during the enzymatic treatment enhanced the production of large intermediate fragments that were further proteolysed to smaller fragments as proteolysis proceeded for longer periods. At 400 MPa, all the intact protein was removed in minutes, simultaneously decreasing its antigenicity and serum IgE binding properties. However, for considerable reduction of the antigenicity and IgE binding of β-LG, extending the incubation time with the enzyme was needed to reduce the amount of potentially allergenic intermediate peptides. Changes of β-LG under pressure at acidic pH are discussed.     

Identification and characterization of yak α-lactalbumin and β-lactoglobulin

α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak β-LG had 2 variants, β-LG A (P02754) and β-LG E (L8J1Z0). Both domestic yak and wild yak milk contained β-LG E, but it was absent in bovine milk. The amino acid at site 158 of β-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and β-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and β-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.