Efficient reduction ofβ-lactoglobulin allergenicity in milk using Clostridium tyrobutyricum Z816

Milk allergy is one of the most common food allergies,affecting 6%of young children,andβ-lactoglobulin(β-LG)is the main milk allergen.Clostridium tyrobutyricum Z816 was selected for the degradation ofβ-LG,which was successfully reduced by about 90%using permeabilized bacteria under the optimized conditions.The hydrolyzed peptides were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS)and analyzed by molecular modeling,which indicated that C.tyrobutyricum Z816 could effectively degrade the antigenic epitopes ofβ-LG.Finally,the concentration and digestibility ofβ-LG in actual samples was quantified using enzyme-linked immunosorbent assay(ELISA)and gastrointestinal digestion simulation experiments.The results showed more than 92%ofβ-LG in actual samples was hydrolyzed,and the gastric and total digestibility of whey protein isolate(WPI)was improved by 85.96%and 64.51%,respectively.Therefore,C.tyrobutyricum Z816 offers an effective method to degradeβ-LG and reduce the occurrence of milk allergies,which has great significance for the development of hypoallergenic dairy products.

从WPC中分离β-lactoglobulin的方法

β-Lactoglobulin是乳清中的主要蛋白,已经有很多方法应用于分离提取β-Lg,但是这些方法或技术大多数比较贵或有时候比较复杂,不能够达到足够的产量或纯度。介绍一种简便易行,重复性强,低成本的方法从浓缩乳清蛋白(WPC)中分离β-Lg,并且利用BCA法测定蛋白的质量浓度表明提取β-Lg的质量浓度很高,而SDS-PAGE表明提取的β-Lg有较高的纯度。

Study on the Simultaneously Quantitative Detection for β-Lactoglobulin and Lactoferrin of Cow Milk by Using Protein Chip Technique

Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.

In Vitro Refolding Process of Bovine Allergen β-lactoglobulin by Multispectroscopic Method

Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the

HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C_4 and C_(18) Column

Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.

Fabrication and evaluation of a portable and reproducible quartz crystal microbalance immunochip for label-free detection ofβ-lactoglobulin allergen in milk products

In this study,a label-free,portable and reproducible immunochip based on quartz crystal microbalance(QCM)was developed for the qualitative detection ofβ-lactoglobulin(β-LG),an allergen,in milk products.Experimental parameters in the fabrication and regeneration procedure such as pH of the coupling microenvironment,amount of anti-β-LG antibody and regeneration reagent were optimized in detail.Under optimal conditions,the proposed QCM immunochip exhibited good recognition of β-LG,with a calibration curve of ΔF=12.877 C_(β-LG)^(0.4809)(R^(2)=0.9982)and limit of detection of 0.04μg/mL.Additionally,this portable QCM immunochip had good stability,high specificity,and no obvious cross-reaction to three other milk proteins(α-casein,α-lactalbumin,and lactoferrin).It could compete a qualitative measurement within5 min,and could be reused at least ten times.In the β-LG analysis of actual milk samples,the developed QCM immunochip yielded reliable and accurate results,which correlated strongly with those from the standard HPLC method(R^(2)=0.9969).Thus,the portable,stable,and reproducible QCM immunochip developed in this study allowed the rapid,cost-effectively and sensitively measure theβ-LG in milk products.

β-Lactoglobulin stabilized lipid nanoparticles enhance oral absorption of insulin by slowing down lipolysis

Lipid-based nanocarriers have staged a remarkable comeback in the oral delivery of proteins and peptides, but delivery efficiency is compromised by lipolysis. β-Lactoglobulin(β-lg) stabilized lipid nanoparticles, including nanoemulsions(NE@β-lg) and nanocapsules(NC@β-lg), were developed to enhance the oral absorption of insulin by slowing down lipolysis due to the protection from β-lg. Cremophor EL stabilized nanoemulsions(NE@Cre-EL) were prepared and set as a control. The lipid nanoparticles produced mild and sustained hypoglycemic effects, amounting to oral bioavailability of 3.0% ± 0.3%, 7.0% ± 1.1%, and7.7% ± 0.8% for NE@Cre-EL, NE@β-lg, and NC@β-lg, respectively. Aggregation-caused quenching(ACQ)probes enabled the identification of intact nanoparticles, which were used to investigate the in vivo and intracellular fates of the lipid nanoparticles. In vitro digestion/lipolysis and ex vivo imaging confirmed delayed lipolysis from β-lg stabilized lipid nanoparticles. NC@β-lg was more resistant to intestinal lipolysis than NE@β-lg due to the Ca^(2+)-induced crosslinking. Live imaging revealed the transepithelial transport of intact nanoparticles and their accumulation in the liver. Cellular studies confirmed the uptake of intact nanoparticles. Slowing down lipolysis via food proteins represents a good strategy to enhance the oral absorption of lipid nanoparticles and thus co-formulated biomacromolecules.

牛乳β-乳球蛋白变异体检测方法研究进展

β-乳球蛋白(β-LG)是牛乳中重要的天然活性营养物质,也是牛乳中主要的过敏原之一。β-乳球蛋白约为乳清蛋白总量的50%,是牛乳中主要的乳清蛋白,具有较强的热敏感性和致敏性,在优质乳品工业中起关键作用。其不仅作为牛奶热处理强度的评估指标,还作为食品中牛乳过敏原的标志蛋白,有效识别和检测牛乳β-乳球蛋白非常重要。已报道的牛乳β-乳球蛋白有13种变异体,其中A和B是牛乳β-LG的常见变异体,且含量最高。根据不同的检测原理,文章简述近年来牛乳β-乳球蛋白变异体的三大类检测方法,总结电泳法、色谱法和免疫法的原理、优缺点及部分应用实例,重点介绍液相色谱法和毛细管电泳法两种定量方法,并展望牛乳β-乳球蛋白未来的发展方向。

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,分离过程中一价阳离子在膜两侧分布较均匀(脱除率大于80%),而二价阳离子则主要集中在截留液一侧(钙离子脱除率为38%)。透过液中的每克蛋白质所对应的金属离子比例远远大于其在截留液中的比例。

β-乳球蛋白-多酚纳米颗粒稳定的百香果籽油Pickering乳液及其性质

本研究以β-乳球蛋白负载3种多酚(阿魏酸、槲皮素、香草酸)制备的β-乳球蛋白三配体复合物纳米颗粒(β-lactoglobulin triple ligand complex nanoparticles,β-LGCNPs)作为乳化剂,采用超声波破碎法制备百香果籽油Pickering乳液,考察Pickering乳液在不同粒子质量分数和油相比例条件下的粒径、稳定性、微观结构、抗氧化性、消化性、流变特性及脂质氧化产物。结果表明,百香果籽油Pickering乳液是由β-LGCNPs吸附在油水界面稳定的水包油型(O/W)乳液,其粒径与颗粒含量呈正比,与油相比例呈反比,当颗粒质量分数为1.5%、油相比例为30%(体积分数)时,乳液平均粒径达到(5.78±0.10)μm。在不同离子强度和pH值条件下乳液稳定性好,展现出比百香果籽油更强的抗氧化性,且自由基的清除作用与颗粒含量呈剂量依赖关系。肠道消化2 h后,乳液中游离脂肪酸释放率达到(65.17±1.52)%,较百香果籽油提高了26.65%。流变学分析展现剪切变稀现象,表明该乳液属于非牛顿流体,随着剪切频率的提高,弹性模量和黏性模量都增大。在15 d储藏期内,Pickering乳液中脂质氧化产物(氢过氧化物和丙二醛)的生成量和脂质氧化速率降低。综上所述,由β-LGCNPs稳定的Pickering乳液改善了百香果籽油的稳定性、抗氧化活性、消化性及脂质氧化,有利于拓宽百香果籽油在食品领域的应用。

重组牛乳过敏原β-乳球蛋白的可溶性表达及其特性分析

目的实现β-乳球蛋白(β-lactoglobulin,BLG)的可溶性表达,并比较重组BLG和天然BLG在结构和免疫原性两个方面的差异性。方法将pET-30a-BLG重组质粒转入大肠杆菌BL21(DE3)感受态细胞中,用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)进行体外诱导表达,并对表达条件进行优化,采用十二烷基硫酸钠-聚丙烯酰胺电泳方法分析重组蛋白的可溶性。利用荧光光谱和圆二色谱比较重组BLG空间结构变化,同时采用竞争抑制酶联免疫吸附实验评价重组BLG致敏性能力。结果在Tris-HCl溶液体系中重组BLG的可溶性表达可达到50%以上,可溶性表达的最优条件为:在Tris-HCl溶液体系中,诱导温度为37℃,IPTG终浓度1.0 mmol/L,诱导4 h。重组BLG表面疏水性下降,可溶性重组BLG的α-螺旋结构减少,经除盐处理后致敏性下降,包涵体重组BLG无规则卷曲结构减少、致敏性上升。结论本研究成功从上清液中纯化得到可溶性重组BLG,且可溶性表达获得的BLG空间结构更接近天然蛋白。

β-乳球蛋白与红曲色素非共价相互作用及其对色素稳定性的影响

为探究在不同pH值(2.6、6.2、7.1、8.2)条件下,β-乳球蛋白(β-lactoglobulin,β-LG)与红曲色素(monascus pigments,Mps)提取物之间的结合特性,构建了4种复合体系,并利用荧光光谱、圆二色谱以及分子对接方法对β-LG与Mps之间的互作行为进行研究,同时评价了复合物(β-LG&Mps)的抗氧化活性、热稳定性以及光稳定性。结果表明,在实验pH值范围内,Mps与β-LG的结合导致β-LG内源荧光发生猝灭,二者主要借助范德华力、疏水相互作用以及氢键等非共价作用力结合,但β-LG的二级结构变化不显著;β-LG&Mps的DPPH自由基清除能力高于单独任一组分,但低于两组分之和,即β-LG与Mps在DPPH自由基清除方面表现出拮抗作用。与β-LG形成复合物后,在pH值为6.2、7.1、8.2,温度为50℃时,与未复合的Mps相比,Mps的保留率分别提高了58%、30%和24%;在pH值为6.2、7.1、8.2,温度为25℃,光照强度为600 Lux,光照时间为36 h时,与未复合的Mps相比,Mps的保留率分别提高了65%、43%和43%。在pH值为2.6时Mps热稳定性和光稳定性仍然较差,即β-LG提高了Mps在pH值为6.2、7.1、8.2时的热稳定性与光稳定性。通过食品组分相互作用对改善Mps稳定性的探讨,旨在为Mps相关产品的开发提供有益参考。

迷迭香酸共价偶联对β-乳球蛋白结构及性质的影响

以β-乳球蛋白(β-lactoglobulin,β-LG)为对象,探究膳食多酚迷迭香酸(rosmarinic acid,RA)共价偶联对蛋白结构和性质的影响。采用自由基法和碱法制得β-LG-RA共价偶联复合物;利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、傅里叶变换红外光谱、紫外吸收光谱、荧光光谱、圆二色光谱等技术,分析RA共价偶联后β-LG结构变化规律;通过对1,1-二苯基-2-三硝基苯肼自由基和2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基清除率检测及酶联免疫吸附测试,评价β-LG与RA共价偶联前后抗氧化性和与血清特异性免疫球蛋白G(immunoglobulin G,IgG)结合能力的变化。结果表明:RA共价偶联后β-LG的二级结构由α-螺旋向β-折叠和无规卷曲转变,蛋白的二、三级结构发生改变。此外,与β-LG相比,β-LG-RA共价偶联复合物的抗氧化性显著增强,与血清特异性Ig G的结合能力显著减弱。综上,RA共价偶联通过引入酚羟基增强了蛋白的抗氧化性,改变了β-LG的结构,降低了其与血清特异性IgG的结合能力。

DSC study of denaturation of β-lactoglobulin B

The denaturation of bovine β-lactoglobulin B (β-Lg B) has been studied in phosphate solutions with various concentrations of GuHCl with differential scanning calorimetry The experiments demonstrated that the presence of GuHCl made the β-Lg B undergo both cold denaturation and heat denaturation under the condition of a high concentration of the protein. The enthalpy changes of both kinds of denaturation exhibit opposite signs. Both the cold denaturation and the renaturation of the protein are reproducible, but its heat denaturation is irreversible. The cooperation among monomer molecules of the protein is involved in its heat denaturation The heat denaturation of the protein can be represented by the thermodynamic model Nc D→F. The activation energy of heat denaturation is 285 kJ/mol, which imples that the depression of temperature and enthalpy of heat denaturation of the P-Lg B does not result from decreasing considerably the activation energy by GuHCl As for the cold denaturation of the protein,

Biopolymer-stabilized emulsions on the basis of interactions betweenβ-lactoglobulin andι-carrageenan

ι-Carrageenan andβ-lactoglobulin(β-lg)stabilized oil-in-water(O/W)emulsions,which can be used for the oral administration of bioactive but environmentally sensitive ingredients,have been successfully prepared.The effects of protein/polysaccharide ratios,total biopolymer concentration,environmental stress(thermal processing and sonication),and pH on the complex formation betweenι-carrageenan andβ-lactoglobulin have been investigated.We found thatβ-lactoglobulin andι-carrageenan stabilized emulsions can be formed at pH values of 6.0,4.0,and 3.4.However,the microstructures of emulsions stabilized byβ-lactoglobulin andι-carrageenan was identified by optical microscopy,and it indicated that the emulsion prepared at pH 6.0 flocculated more extensively,while its hydrodynamic radius was much bigger than those prepared at pH 4.0 and 3.4.Regarding rheological properties,the emulsion of pH 6.0 showed a more solid-like behavior but with a lower viscosity than those of pH 4.0 and 3.4.The optimum concentration ranges forβ-lg andι-carrageenan to form stable emulsions at pH 4.0 and 3.4 were 0.3 wt-%-0.6 wt-%and 0.4 wt-%-0.7 wt-%,respectively.

作为姜黄素递送载体的黄原胶-β乳球蛋白纤维复合凝胶的制备

该研究旨在用黄原胶(xanthan gum,XG)和β-乳球蛋白纤维(β-lactoglobulin fiber,Fblg)制备多糖-蛋白纤维复合水凝胶作为生物活性物质姜黄素(curcumin,Cur)的递送载体。电位、浊度和原子力显微镜证实了XG与Fblg分子之间发生了络合作用,静电相互作用是XG和Fblg络合的主要驱动力。通过用葡萄糖酸内酯(glucono delta-lactone,GDL)调控复合体系pH至4.0,成功得到稳定的XG-Fblg凝胶。扫描电子显微镜显示,XG-Fblg凝胶形成三维网孔结构并随Fblg的增加而变得更致密。流变和质构测试显示,XG-Fblg凝胶的机械强度随Fblg的含量增加而增强。采用体外胃肠道释放实验研究Cur的释放行为,XG-Fblg凝胶比XG凝胶和Fblg更适合作为Cur肠道递送的载体,在肠道的Cur释放量最多,Cur的释放主要通过凝胶降解控制。该研究结果可为多糖-蛋白纤维复合凝胶的制备以及合理设计水凝胶作为营养物质递送载体提供新的思路。

β-乳球蛋白与二十二碳六烯酸互作效应对β-乳球蛋白致敏性机制的影响

β-乳球蛋白(β-lactoglobulin,β-LG)是牛乳中的主要过敏原,以β-LG为原料,采用三维荧光光谱法、圆二色谱法、分子模拟对接法和间接竞争酶联免疫吸附测定法探究二十二碳六烯酸(docosahexaenoic acid,DHA)与β-LG互作效应及其对β-LG致敏性的影响。结果表明:DHA与β-LG结合后显著改变了β-LG中芳香族残基的微环境,使其极性增加;DHA通过疏水作用力与β-LG结合形成稳定复合物,且结合后,β-LG的二级结构发生改变,蛋白质骨架发生折叠,随着DHA浓度的增加,β-折叠相对含量先增加后减少,α-螺旋相对含量的变化与β-折叠相反,β-转角、无规卷曲相对含量变化没有明显规律;此外,在两者结合后,β-LG的抗原性降低,DHA浓度为3 mmol/L时,免疫球蛋白E结合能力抑制率约为29%。

DSC study of cold and heat denaturation of β-lactoglobulin A with urea

IN the field of thermodynamics of protein solutions, there are two kinds of denaturation induced by temperature changes. One of them is the commonest heat denaturation induced by temperature increasing and accompanied by a heat absorption and an increase of enthalpy and entropy. The other is cold denaturation, predicted by Brandts over thirty years ago: the compact ordered structure of protein molecules in the native state is induced into a

High voltage electrical treatments can eco-efficiently promote the production of high added value peptides during chymotryptic hydrolysis of β-lactoglobulin

Chymotryptic hydrolysis of β-lactoglobulin (β-lg) leads to the release of a wide range of biofunctional peptides.However,the proteolysis of native β-lg is limited due to its compact globular structure,thus ending with low yields of peptides.Whereas our previous study showed that eco-efficient High Voltage Electric Treatments (HVET),namely Pulsed Electric Fields (PEF) and Electric Arc (ARC),of β-lg substantially improved its enzymatic hydrolysis,no information regarding the nature of released peptides is available up to date.Hence,the time-dependent release of peptides and the dynamics of this release were discussed in the current study.Chromatographic analysis of the final hydrolysates showed that chymotrypsinolysis of HVET samples released 19% more peptides with higher concentration than the native and preheated ones.Furthermore,1-min HVET were found to be more efficient than 10-min ones as they led to the release of bioactive peptides (even without chymotrypsin) with higher eco-efficiency scores.

A proteomic approach to investigate the qualitative and quantitative polymorphism of <i>β</i>-lactoglobulin in ovine milk: Inference on gene copy-number variations

The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.