超高压对乳清分离蛋白结构和抗氧化活性的影响

为了研究超高压处理对乳清分离蛋白结构的影响,该研究对乳清分离蛋白进行了不同条件的超高压处理,之后测定表面疏水性、傅里叶变换红外光谱、自由巯基含量和内源荧光光谱分析乳清分离蛋白的结构变化。与未经处理的乳清分离蛋白相比,200 MPa及以上压力显著提高了乳清分离蛋白的表面疏水性,在400 MPa-30min时达到最大值。超高压处理使乳清分离蛋白的α-螺旋、β-折叠含量发生明显变化,证明了其对乳清分离蛋白二级结构的影响。超高压处理增加了蛋白自由巯基含量,在400 MPa-30 min时增加49%,并且超高压处理也引起了乳清分离蛋白内源荧光强度的显著变化。在所有的超高压处理条件中,400 MPa-30 min对乳清分离蛋白结构的影响最为显著,并显示出了最高的抗氧化活性。研究表明,超高压处理能显著改变乳清分离蛋白的二、三级结构,暴露出疏水基团等活性基团,从而对抗氧化活性产生影响。     

连续式微滤膜分离乳清蛋白的研究

文章研究了跨膜压力对连续式微滤膜分离技术工艺参数、分离效果及组分组成的影响。以脱脂乳为原料,使用0.1μm陶瓷微滤膜三级连续在线洗滤工艺分离乳清蛋白和酪蛋白。实验使用0.08、0.11、0.14 MPa 3个梯度,在50℃,3.5倍浓缩的条件下连续生产240 min。计算跨膜压力并且检测截留液和透过液中的α-乳白蛋白（α-La）,β-乳球蛋白（β-LG）含量及钾、钙、钠、镁等金属离子的含量。结果表明一级膜通量下降是导致整体膜通量下降的主要因素,经过240 min实验通量下降约17.2%。研究了不同跨膜压力下的膜通量变化情况,膜通量与跨膜压力呈正相关关系,水洗恢复率与跨膜压力呈负相关关系。随着实验时间的延长,膜表面形成不可逆的污堵层,乳清蛋白分离率下降,透过液中乳清蛋白含量下降,150 min后α-乳白蛋白浓度下降37%,β-乳球蛋白浓度下降36.5%。乳清蛋白中2种主要蛋白质比例会随着跨膜压力变化而变化,随着跨膜压力的升高β-乳球蛋白含量会逐渐升高。三级连续膜过滤后,乳清蛋白最高分离率90%左右（α-乳白蛋白为90.4%,β-乳球蛋白为92.7%）。乳中蛋白质的形态和功能受金属离子影响...     

SDS-PAGE测定乳基婴幼儿配方食品中乳清蛋白方法的研究

采用SDS-PAGE对乳基婴幼儿配方食品内乳清蛋白进行了分离和定量分析,优化了试验条件,简化了数据的处理方法。结果表明采用该法对乳粉内6种重要蛋白(α-酪蛋白、β-酪蛋白、κ-酪蛋白、β-乳球蛋白、α-乳白蛋白和血清白蛋白)能实现较好地分离,依据乳清蛋白和酪蛋白的比值可以推算出乳清蛋白在乳粉内的含量。对乳清蛋白含量不同的乳粉及市售婴儿粉的测定表明,SDS-PAGE法是一种操作简便、准确快速测定乳粉内乳清蛋白含量的方法。     

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。     

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。     

超声波协同马克思克鲁维酵母Z17胞内粗酶处理降低乳清蛋白的致敏性

以超声预处理过的乳清蛋白为酶解底物,采用OPA法、ELISA分析等手段,探究马克思克鲁维酵母Z17粗酶水解乳清蛋白、降低乳清蛋白致敏性【以α-乳白蛋白(α-LA)和β-乳球蛋白(β-LG)为抗原性表征】的最优超声预处理-酶解条件。结果表明:乳清蛋白水解度受初始pH值和酶解温度的影响显著,α-LA、β-LG抗原性受初始pH值的影响显著,超声间歇时间和超声功率的交互作用对α-LA、β-LG抗原性影响显著。采用响应面法获得马克思克鲁维酵母Z17转化乳清蛋白的最优酶解条件是:超声间歇时间16 s,超声功率400 W,初始pH 6.16,酶解温度18.48℃,预测α-LA抗原性、β-LG抗原性的降低率达到最大值,分别为65.56%和57.96%。     

糖基化反应条件对乳清蛋白——麦芽糖复合物抗原性的影响

研究了将麦芽糖通过糖基化引入到乳清蛋白制备乳清蛋白-麦芽糖,用间接竞争ELISA法测定不同反应时间不同质量比的乳清蛋白-麦芽糖中α-乳白蛋白和β-乳球蛋白的抗原性的变化。结果表明,糖基化能有效降低α-乳白蛋白和β-乳球蛋白的抗原性,α-乳白蛋白的抗原性可以从26.2 mg/L降低到14.4 mg/L,β-乳球蛋白的抗原性可以从95.1 mg/L降低到22.4 mg/L。反应时间对不同质量比的乳清蛋白-麦芽糖中α-乳白蛋白和β-乳球蛋白的抗原性有较大影响。蛋白与糖的质量比为1～8时,反应相同时间的乳清蛋白-麦芽糖中α-乳白蛋白和β-乳球蛋白的抗原性随蛋白与糖的质量比的下降而下降。     

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％.     

德式乳杆菌保加利亚亚种的乳清蛋白利用能力比较

为了探究德式乳杆菌保加利亚亚种对乳清蛋白的利用能力,研究了8株德式乳杆菌保加利亚亚种菌株在乳清蛋白培养体系中的生长和产酸情况,发酵过程中氨肽酶Pep N、Pep C和Pep X活力变化及乳清蛋白主要组分β-乳球蛋白、α-乳白蛋白和牛血清白蛋白的利用情况,探讨了不同菌株氨肽酶活力变化与乳清蛋白水解能力之间的相关性。结果表明,在乳清蛋白培养体系中,最适宜菌株生长的初始p H是5. 5,该条件下菌株在3 h内快速生长到达稳定期。在发酵过程(12 h)中,3种氨肽酶活力均表现出先升高后降低的变化趋势,但不同菌株的酶活力之间存在显著差异性。8株菌株均表现出对α-乳白蛋白较强的水解能力(26. 93%～31. 33%),但对于β-乳球蛋白的水解能力存在菌株差异性,β-乳球蛋白的变异体A和B的水解率分别是10. 56%～22. 82%和9. 04%～23. 83%。菌株的氨肽酶活力与β-乳球蛋白的水解能力之间存在一定相关性,DQHXNS8L6和DQHXNS15M2在3 h内即能达到最高的氨肽酶活力并有效水解β-乳球蛋白,具有发酵乳清蛋白饮料的应用潜力。     

丙二醛氧化对乳清蛋白结构及其抗氧化活性的影响研究

研究丙二醛(MDA)氧化对乳清蛋白羰基、巯基和二聚酪氨酸等氧化产物的影响,比较氧化前后乳清蛋白DPPH和ABTS自由基清除能力、脂质过氧化抑制能力及还原力,分析MDA对乳清蛋白体外抗氧化活性的影响,采用SDS-聚丙烯酰胺凝胶电泳、红外光谱和圆二色谱等方法,研究MDA对乳清蛋白分子聚集状况及二级结构的影响。结果表明MDA对乳清蛋白的氧化程度与其浓度相关。300 mmol/L MDA氧化的乳清蛋白羰基含量较氧化前提高了3.2倍,总巯基含量较氧化前下降了34.18%。但二聚酪氨酸含量显著下降(P0.01),说明MDA作用下,乳清蛋白中酪氨酸残基未氧化生成二聚酪氨酸。MDA氧化提高了乳清蛋白的DPPH和ABTS自由基清除力2～2.2倍;但降低了其脂质过氧化抑制能力及还原力(P0.01)。高浓度MDA氧化乳清蛋白,导致β-乳球蛋白、α-乳白蛋白及BSA形成二聚体、三聚体及多聚体,同时促使乳清蛋白二级结构中无规则卷曲含量上升。说明MDA氧化改变了乳清蛋白空间结构及体外抗氧化活性。     

Antigenicity and conformational changes of β-lactoglobulin by dynamic high pressure microfluidization combining with glycation treatment

The combined effect of previous dynamic high-pressure microfluidization treatment (40, 80, 120, and 160 MPa) and subsequent glycation with galacto-oligosaccharides (GOS) on the antigenicity of β-lactoglobulin (β-LG) was investigated. The antigenicity of β-LG-GOS decreased at relatively low pressure (≤120 MPa). Surface sulfhydryl group content of β-LG-GOS increased and surface hydrophobicity of β-LG-GOS decreased. Additionally, protein unfolding in β-LG-GOS samples was reflected by quenching of fluorescence intensity, the red-shift of fluorescence spectra, decreased UV absorption, and circular dichroism analysis, indicating tertiary and secondary structural changes of β-LG. The conformational changes may contribute to the alteration of antigenicity.     

二次杀菌对贮藏期间酱卤羊肚肌原纤维蛋白氧化、特性及结构的影响

为了探明超高压处理和热处理对酱卤羊肚肌原纤维蛋白氧化、特性及结构的影响。本实验以未经二次杀菌处理的样品为空白（CN）,对酱卤羊肚分别进行400 MPa、15 min超高压处理（ultra-high pressure treatment,UHPT）和85 ℃水浴40 min热处理（heat treatment,HT）,真空包装后4 ℃条件下贮藏,通过测定肌原纤维蛋白羰基含量、总巯基含量、蛋白组成、溶解度和浊度,分析两种二次杀菌方式对蛋白氧化及特性的影响;同时采用光谱法（紫外吸收光谱、内源荧光光谱和拉曼光谱）对蛋白结构进行分析。结果表明：贮藏初期经过超高压和热处理后的肌原纤维蛋白羰基含量均上升更快,并且UHPT组整体高于HT组。贮藏期间各组总巯基含量呈先下降后上升的趋势,并且相比CN组,HT组和UHPT组在贮藏前期下降速率更快,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果表明,二次杀菌后,主要受到影响的蛋白为肌动蛋白和原肌球蛋白。超高压和热处理均会显著提高蛋白浊度（P＜0.05）,但超高压处理的影响较小。光谱分析表明,两种二次杀菌方式均使色氨酸残基向非极性环境转移,并且α-螺旋相对含量降低、无规卷曲相对含量增加、折叠比上升,且随贮藏时间的延长,结构产生了有序到无序的转变。相关性分析表明：贮藏时间与总巯基含量、溶解度、α-螺旋相对含量具有显著相关性,并且蛋白氧化与蛋白特性和结构之间存在一定的相关性。超高压作为二次杀菌方式在尽可能保证贮藏期间酱卤羊肚的品质的同时具有更大的优势。     

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.     

超高压辅助中华管鞭虾脱壳及对其肌肉品质的影响

为研究超高压处理对虾仁品质的影响,采取不同的压力(100~500 MPa)处理中华管鞭虾,通过测定虾仁肌原纤维蛋白盐溶性、巯基含量、表面疏水性、Ca~(2+)-ATPase活性及虾仁色泽、质构、肌肉超微结构,研究超高压处理在辅助脱去虾壳的同时对虾仁肌肉及其蛋白生化特性的影响。结果表明,与对照组相比,200、300 MPa处理中华管鞭虾,在保持虾仁结构完整性及其良好品质的同时能显著提高脱壳效果,400、500 MPa处理后虾肉发生熟化;200 MPa处理组虾肉硬度、弹性和咀嚼性比对照组分别提高35.37%、7.46%和24.93%,肌原纤维蛋白除表面疏水性上升外,其盐溶性、总巯基含量和Ca~(2+)-ATPase活性无明显变化;而300、400、500 MPa处理后肌原纤维蛋白盐溶性、巯基含量及Ca~(2+)-ATPase活性下降明显。结合脱壳效果和对虾仁肌肉及其肌原纤维蛋白生化特性的影响,选择200 MPa处理中华管鞭虾,可有效提高脱壳效果,还能保持虾仁良好品质。实验结果可为冻虾仁生产中利用超高压辅助中华管鞭虾脱壳技术的应用提供支持。     

Front-face fluorescence spectroscopy combined with chemometrics to detect high proteinaceous matter in milk and whey ultrafiltration permeate

Proteinaceous matter can leak into the permeate stream during ultrafiltration (UF) of milk and whey and lead to financial losses. Although manufacturers can measure protein content in the finished permeate powders, there is currently no rapid monitoring tool during UF to identify protein leak. This study applied front-face fluorescence spectroscopy (FFFS) and chemometrics to identify the fluorophore of interest associated with the protein leak, develop predictive models to quantify true protein content, and classify the types of protein leak in permeate streams. Crude protein (CP), nonprotein nitrogen (NPN), true protein (TP), tryptone-equivalent peptide (TEP), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents were measured for 37 lots of whey permeate and 29 lots of milk permeate from commercial manufacturers. Whey permeate contained more TEP than did milk permeate, whereas milk permeate contained more α-LA and β-LG than did whey permeate. The types of protein leak were thus identified for predictive model development. Based on excitation-emission matrix (EEM) of high- and low-TP permeates, tryptophan excitation spectra were collected for predictive model development, measuring TP content in permeate. With external validation, a useful model for quality control purposes was developed, with a root mean square error of prediction of 0.22% (dry basis) and a residual prediction deviation of 2.8. Moreover, classification models were developed using partial least square discriminant analysis. These classification methods can detect high TP level, high TEP level, and presence of α-LA or β-LG with 83.3%, 84.8%, and 98.5% cross-validated accuracy, respectively. This method showed that FFFS and chemometrics can rapidly detect protein leaks and identify the types of protein leak in UF permeate. Implementation of this method in UF processing plants can reduce financial loss from protein leaks and maintain high-quality permeate production.     

Efficiency of removal of whey protein from sweet whey using polymeric microfiltration membranes

Our objective was to measure whey protein removal percentage from separated sweet whey using spiral-wound (SW) polymeric microfiltration (MF) membranes using a 3-stage, 3× process at 50°C and to compare the performance of polymeric membranes with ceramic membranes. Pasteurized, separated Cheddar cheese whey (1,080 kg) was microfiltered using a polymeric 0.3-μm polyvinylidene (PVDF) fluoride SW membrane and a 3×, 3-stage MF process. Cheese making and whey processing were replicated 3 times. There was no detectable level of lactoferrin and no intact α- or β-casein detected in the MF permeate from the 0.3-μm SW PVDF membranes used in this study. We found BSA and IgG in both the retentate and permeate. The β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) partitioned between retentate and permeate, but β-LG passage through the membrane was retarded more than α-LA because the ratio of β-LG to α-LA was higher in the MF retentate than either in the sweet whey feed or the MF permeate. About 69% of the crude protein present in the pasteurized separated sweet whey was removed using a 3×, 3-stage, 0.3-μm SW PVDF MF process at 50°C compared with 0.1-μm ceramic graded permeability MF that removed about 85% of crude protein from sweet whey. The polymeric SW membranes used in this study achieve approximately 20% lower yield of whey protein isolate (WPI) and a 50% higher yield of whey protein phospholipid concentrate (WPPC) under the same MF processing conditions as ceramic MF membranes used in the comparison study. Total gross revenue from the sale of WPI plus WPPC produced with polymeric versus ceramic membranes is influenced by both the absolute market price for each product and the ratio of market price of these 2 products. The combination of the market price of WPPC versus WPI and the influence of difference in yield of WPPC and WPI produced with polymeric versus ceramic membranes yielded a price ratio of WPPC versus WPI of 0.556 as the cross over point that determined which membrane type achieves higher total gross revenue return from production of these 2 products from separated sweet whey. A complete economic engineering study comparison of the WPI and WPPC manufacturing costs for polymeric versus ceramic MF membranes is needed to determine the effect of membrane material selection on long-term processing costs, which will affect net revenue and profit when the same quantity of sweet whey is processed under various market price conditions.     

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.     

Reaction Kinetics of the Denaturation of Whey Proteins in Milk

The kinetics of the neat-induced irreversible denaturation of β-lacto-globulins (β-LG) A and B and of α-lactalbumin (α-LA) in milk were examined over a wide temperature/time range (70-150°C, 2-5400 sec). Denaturation of β-LG was best described with an apparent reaction order of 1.5 (α-LA; first order). The abrupt changes in the temperature dependence of the rate constants (β-LG at 90°C, α-LA at 80°C) were interpreted in terms of the different activation energies and entropies occurring in the two temperature ranges. By using the kinetic parameters for calculating lines of equal degrees of denaturation in a plot of log-time versus 1/absolute temperature it was possible to predict the effect of different heat treatments on the denaturation of individual proteins.