Dietary polyphenols reduced the allergenicity ofβ-lactoglobulin via non-covalent interactions:a study on the structure-allergenicity relationship

Studies showed that complexation of polyphenols with milk allergens reduced their immunogenic potential.However,the relationship between structures of polyphenols and their hypoallergenic effects on milk allergens in association with physiological and conformational changes of the complexes remain unclear.In this study,polyphenols from eight botanical sources were extracted to prepare non-covalent complexes withβ-lactoglobulin(β-LG),a major allergen in milk.The dominant phenolic compounds bound toβ-LG with a diminished allergenicity were identified to investigate their respective role on the structural and allergenic properties ofβ-LG.Extracts from Vaccinium fruits and black soybeans were found to have great inhibitory effects on the IgE-and IgG-binding abilities ofβ-LG.Among the fourteen structure-related phenolic compounds,flavonoids and tannins with larger MWs and multi-hydroxyl substituents,notably rutin,EGCG,and ellagitannins were more potent to elicit changes on the conformational structures ofβ-LG to decrease the allergenicity of complexedβ-LG.Correlation analysis further demonstrated that a destabilized secondary structure and protein depolymerization caused by polyphenol-binding were closely related to the allergenicity property of formed complexes.This study provides insights into the understanding of structure-allergenicity relationship ofβ-LG-polyphenol interactions and would benefit the development of polyphenol-fortified matrices with hypoallergenic potential.

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.

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.

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.

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.

牛奶过敏原β-乳球蛋白的单克隆抗体制备及其双抗体夹心法的建立

目的制备与鉴定牛奶主要过敏原β-乳球蛋白(β-LG)的单克隆抗体,并建立双抗体夹心检测法。方法以β-LG为抗原免疫BALB/c小鼠,融合免疫鼠脾细胞和小鼠骨髓瘤NS-1。半固体培养基法结合有限稀释法筛选稳定分泌抗体的杂交瘤细胞株。杂交瘤细胞株诱生小鼠腹水,采用蛋白A亲和层析法获得纯化抗体。利用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型。间接ELISA方法和Western Blot鉴定抗体效价和特异性以及与其他过敏原的交叉反应性。建立双单克隆抗体夹心法,检测β-LG。结果共获得抗β-LG细胞株6株,分别命名为1H8,4A7,4C3,1F9,1G5,3D11,效价均高于20万。经抗体亚型鉴定,6株抗体均为Ig G1型。Western Blot的结果表明6株抗体能识别β-LG。在特异性检测实验中,6株抗体与其他种类食物过敏原无交叉反应,而1G5和3D11与牛奶酪蛋白过敏原有交叉反应性。通过建立双单克隆抗体夹心ELISA法,发现牛奶β-LG蛋白的检出低限为:15.625 ng/m L,标准曲线在15.625~250 ng/m L范围内线性良好。结论获得高效价抗体6株,建立了高效、高特异性的牛奶过敏原β-LG的检测方法,为食品中牛奶过敏原的检测提供了依据。

高效液相色谱法测定α-乳白蛋白和β-乳球蛋白

采用HPLC法测定牛乳中主要过敏蛋白α-乳白蛋白及β-乳球蛋白的含量,其测定条件为:Agilent-C8(4.6 mm×150 mm,5μm)色谱柱,流动相A为含0.5%三氟乙酸的乙腈,流动相B为含0.1%三氟乙酸的超纯水,采用梯度洗脱的方法,流速1 mL/min,柱温25℃,检测波长280 nm。结果表明,α-乳白蛋白和β-乳球蛋白的回收率均>92%,RSD<5%。该方法适合测定样品中的α-乳白蛋白和β-乳球蛋白。

动态高压微射流协同糖基化处理对β-乳球蛋白热稳定性和结构的影响

采用动态高压微射流(dynamic high pressure microfluidization,DHPM)协同葡聚糖糖基化处理对β-乳球蛋白(β-lactoglobulin,β-Lg)进行改性,研究其热稳定性和结构的变化。结果表明,β-Lg的峰顶温度为73. 48℃,经DHPM不同压力(40、80、120 MPa)处理后,其热稳定性先下降后上升,但经DHPM协同糖基化处理后,其热稳定性均呈上升趋势。理化分析结果显示,80 MPa DHPM协同糖基化处理的β-Lg具有最低的游离氨基酸含量(2. 20 mg/m L)和最高的褐变程度(A294=1. 092,A420=0. 062),说明DHPM预处理可以促进β-Lg-葡聚糖的糖基化反应,且80 MPa为最佳处理压力。结构分析表明,DHPM处理可明显提高β-Lg的表面疏水性和自由巯基含量,降低其内源荧光强度,使其发生二级结构变化。经DHPM协同糖基化处理后,β-Lg的表面疏水性有所降低,但仍高于天然β-Lg的表面疏水;自由巯基含量呈现先降低后升高趋势,在80 MPa时明显高于天然β-Lg,内源荧光强度随着压力的增加呈先降低后上升的趋势,但均明显低于天然β-Lg的内源荧光强度。因此,DHPM 80 MPa预处理样品具有最高的热稳定性和糖基化程度,且β-Lg的糖基化程度越高,其热稳定性越好。

β—lactoglobulin与磷脂复合膜形成过程中相互作用的动态研究

利用ADSA系统分别研究了蛋白质β-lactoglobulin在不同pH下与三种磷脂DPPC(中性头部基因)、DPPE(部分正电头部基团)、DPPA(部分带负电头部基因)的吸附动力学.结合AFM技术,讨论在弯曲的液/液界面上蛋白质与磷脂之间各种相互作用对复合膜生成的影响.认为这些相互作用的影响是动态变化着的;在吸附反应的不同阶段各类作用分别成为主导因素.

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.

Aqueous Two-Phase Systems Applied to Partition Proteins from Goat Milk Whey In-Nature

The proteins coming from the milk whey have numerous functional properties. Among the proteins with high bioactivity, α-lactoalbumin (α-La) and β-lactoglobulin (β-Lg) are present in large quantities in the milk whey. In the separation process of proteins, it is important to choose techniques which besides ensuring purity and high yield will not affect the molecule biological activity. The aqueous two-phase systems (ATS) have been utilized with success in the partition of these proteins, however, the studies were performed using protein in its pure form. Studies using milk whey in-nature and goat milk whey have not been found yet. In this context, the objective of this study was to evaluate the liquid liquid equilibrium of aqueous two-phase systems (ATS) in the partition of α-La and β-Lg from goat milk whey in-nature. Equilibrium data were performed considering ATS comprised of polyethylene glycol, potassium phosphate and water at 25°C and pH 7.0. The influence of the polymer molecular weight and amount of goat milk whey in-nature on the partition coefficient of these proteins were assessed. The partition coefficient, selectivity, process yield and purity of α-lactoalbumin and β-lactoglobulin proteins were determined. The results showed that the separation technique by aqueous biphasic systems is applicable indicating high efficiency in the whey proteins separation process.

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, especially in the solvent with 3.10 mol/L of GuHCl, it can be described by the two-state model N D.

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.

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.

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

Edible films based on milk proteins release effectively active immunoglobulins

Objectives:The goal of this study was to develop novel compositions of edible protein coatings based on immunoglobulin(Ig)fraction from bovine milk.Protein coatings can be used to protect foods against microbial,chemical,and physical damage.We developed novel compositions of edible protein coatings based on immunoglobulin(Ig)fraction from bovine milk.A lot of Ig could be obtained from under-utilized side streams of dairy industry.To the best of our knowledge,such use of the Ig fraction has not been published earlier.Materials and Methods:Bovine colostral Ig’s were incorporated in edible films based on various milk proteins and investigated the characteristics of the films including solubility of Ig’s and nisin and on technological properties of films.Ig’s specific to cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus were produced to colostrums by hyperimmunizing cows before parturition.Results:The milk Ig rich fraction suited well as a component of milk protein films.The Ig’s dissolved from the films very rapidly.Nisin,commoly used for food protection,was used as a model of antimicrobial peptide.Nisin was released biologically active from bothβ-lactoglobulin(β-lg)andβ-lg/Ig films.Nisin exerted its bactericidal effect at clearly lower concentrations in theβ-lg/Ig films when compared withβ-lg film.Nisin also retained its activity better in film containing Ig-enriched whey.Incorporating Ig-enriched whey into films enhanced adhesion and tensile strength of the films.The Ig-enriched whey also affected strongly on the appearance of films based on commercial whey protein concentrate in a dose-dependent way by making the films more smooth,transparent,and clear which are all favoured properties in most food and pharmaceutical applications.Conclusions:Biologically active Ig’s can be successfully incorporated in and released from milk protein based edible films.The content of Igs in films affected considerably technological properties of these films.Composition of other proteins in films had effect on preservability and release of Igs.