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。     

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。     

糖基化反应对大豆蛋白-乳糖复合物抗原性及结构的影响

本文将乳糖通过糖基化反应引入到大豆分离蛋白(SPI)上制备大豆分离蛋白-乳糖复合物,采用间接竞争ELISA法测定不同温度、不同质量比、不同反应时间下大豆分离蛋白-乳糖复合物中β-伴大豆球蛋白的抗原性变化,并对糖基化产物进行了结构特性的研究。结果表明,糖基化能有效降低β-伴大豆球蛋白的抗原性,其抗原性从93.79%降到37.75%。糖基化改性后,大豆蛋白中游离氨基含量降低,在反应60 h时,游离氨基含量下降最大;傅里叶红外光谱结果表明,与原大豆分离蛋白相比,大豆分离蛋白-乳糖糖基化产物的α-螺旋、β-转角、无规卷曲的含量下降,而β-折叠的含量增加;SDS-PAGE电泳及PAS染色结果表明,随着糖基化反应的程度增加,SPI谱带逐渐的减弱,说明SPI与乳糖分子发生了共价连接。     

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％。     

大豆蛋白-木糖复合物的抗原性、致敏性及结构特性研究

大豆是营养丰富的植物蛋白资源,但也是八大过敏原之一;糖基化修饰是一种降低食物过敏原的有效方法。本文将木糖通过糖基化的方法引入大豆分离蛋白制备大豆分离蛋白-木糖复合物,采用间接竞争ELISA法,测定在一定温度、一定质量比、不同反应时间条件下,大豆分离蛋白-木糖复合物中大豆球蛋白的抗原性和过敏原性的变化,并且对糖基化产物进行了结构特性的研究。结果表明,糖基化能有效降低大豆球蛋白的抗原性和过敏原性,其抗原性从83.01%降到67.43%;过敏原性从46.32%降低到29.48%;两者在反应10 h时免疫活性都较低。通过三硝基苯磺酸(TNBS)法、SDS-PAGE电泳证明了糖基化反应的发生;傅里叶红外光谱结果表明,与大豆分离蛋白相比,SPI-木糖糖基化产物的α-螺旋、β-转角的含量下降,而无规卷曲、β-折叠的含量增加。     

糖基化改性对大豆蛋白抗原性及结构特性的影响研究

本文以大豆分离蛋白和葡聚糖为原料,在干热条件下进行美拉德反应,制取不同时间下的糖基化复合物。以β-伴大豆球蛋白和大豆球蛋白抗原抑制率为指标,采用间接竞争ELISA方法测定糖基化产物的抗原性,在反应6 d时,糖基化产物中β-伴大豆球蛋白和大豆球蛋白的抗原性分别降低了31.94%和21.26%。糖基化产物颜色加深,且游离氨基含量降低,说明大豆蛋白与糖发生了不同程度的反应。红外光谱中糖链的引入,使蛋白质分子展开,β-转角和无规则卷曲结构含量的降低,影响了β-伴大豆球蛋白α亚基的抗原表位,从而可能使大豆蛋白的抗原性降低。糖基化反应影响抗原性的关键作用在于蛋白与糖结合部位对蛋白质结构的变化。     

糖基化改性对大豆蛋白抗原性及结构特性的影响

以大豆分离蛋白和葡聚糖为原料,在干热条件下进行美拉德反应,制取不同时间下的糖基化复合物。以β-伴大豆球蛋白和大豆球蛋白抗原抑制率为指标,采用间接竞争ELISA方法测定糖基化产物的抗原性,在反应6 d时,糖基化产物中β-伴大豆球蛋白和大豆球蛋白的抗原性分别降低了36.90%和18.12%。糖基化产物颜色加深,且游离氨基含量降低,说明大豆蛋白与糖发生了不同程度的反应。红外光谱中糖链的引入,使蛋白质分子展开,β-转角和无规则卷曲结构含量降低,影响了β-伴大豆球蛋白α亚基的抗原表位,从而可能使大豆蛋白的抗原性降低。糖基化反应影响抗原性的关键作用在于蛋白与糖结合部位对蛋白质结构变化的影响。     

高压脉冲电场结合糖基化对β-乳球蛋白抗原性与结构的影响

β-乳球蛋白(β-lactoglobulin,β-Lg)是一种营养丰富的牛乳清蛋白,但也是导致牛乳过敏的主要过敏原;本文以β-Lg为研究对象,通过酶联免疫吸附法、电泳、圆二色谱及荧光光谱等方法,在蛋白与糖的质量比为1:2、修饰温度60℃、反应时间3 h、反应环境p H 7.4和不同高压脉冲电场(pulsed electric fields,PEF)强度反应条件下,对β-Lg-半乳糖复合物中β-Lg的抗原性变化及反应产物的结构特性进行研究。结果表明:经过PEF结合糖基化处理后,β-Lg抗原性显著降低,且PEF预处理会促进β-Lg抗原性降低,在电场强度25 k V/cm下预处理90μs后进行糖基化,β-Lg抗原性降低最多,降低了约72.9%;其分子量增大;α-螺旋和β-转角结构减少,而β-折叠和无规则卷曲结构逐渐增多;表面疏水性和内源性荧光强度均降低;自由巯基含量先升高后下降,这为制备低致敏性β-Lg提供了一种新的方法。     

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%。     

乳清蛋白-海藻酸钠的制备及其特性的测定

将海藻酸钠通过糖基化反应引入乳清蛋白,制备乳清蛋白-海藻酸钠复合物,探讨干法条件下反应时间、反应温度、海藻酸钠与乳清蛋白质量配比对复合物接枝度和溶解度的影响,确定了最佳糖基化条件为反应时间6.3 d,反应温度56.1℃,海藻酸钠与乳清蛋白质量配比4.1∶1。在此条件下,乳清蛋白-海藻酸钠共价复合物的接枝度为75.89%、溶解度为32.56%与理论预测值基本相符。     

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.     

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.     

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.     

Interactions of milk proteins during heat and high hydrostatic pressure treatments — A Review

Pressure treatment of β-lactoglobulin (β-LG), whey protein concentrate (WPC), whey protein isolate and skim milk has been explored by many groups using a wide range of techniques. In general terms, heat treatment and pressure treatment have similar effects: denaturing and aggregating the whey proteins and diminishing the number of viable microorganisms. However, there are significant differences between the effects of the two treatments on protein unfolding and the subsequent thiol-catalysed disulfide-bond interchanges that lead to different structures and product characteristics. Application of a range of techniques has given insight into the subtle differences between the pathways from native proteins to the final product mix. This review covers some of the techniques used and their strengths, and the probable pathways from native protein to the final products. β-LG is one of the most pressure-sensitive proteins and α-lactalbumin (α-LA) is one of the most pressure resistant. In a heated WPC system, bovine serum albumin is very sensitive and β-LG is more resistant. In a heated milk system, β-LG reacts with κ-casein (κ-CN) and not with α_S2-CN, but, in pressure-treated milk, β-LG forms adducts with either κ-CN or α_S2-CN. In both treatments, the role of β-LG is central to the ongoing reactions, involving α-LA and κ-CN in heated systems but involving κ-CN, α_S2-CN and α-LA in pressurized systems.Industrial relevanceHigh hydrostatic pressure (HHP) processing, as opposed to heat treatment, has received much attention recently as a means of processing milk proteins. This review examines the differences in the denaturation pathways that give rise to different final products.     

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.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

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.     

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.     

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.