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.

Structure and allergenicity of a-lactalbumin:effects of ultrasonic prior to glycation and subsequent phosphorylation

Bovineα-lactalbumin(BLA)induced severe cow's milk allergy.In this study,a novel strategy combining ultrasonication,performed before glycation,and phosphorylation was proposed to reduce BLA allergenicity.Result showed that IgE-and IgG-binding capacities and the release rates of histamine and interleukin-6 from RBL-2 H3 were reduced.Moreover,intrinsic fluorescence intensity and surface hydrophobicity were decreased,whereas glycated sites(R10,N44,K79,K108,N102 and K114)and phosphorylated sites(Y36 and S112)of BLA were increased.Minimum allergenicity was detected during BLA treatment after ultrasonic prior to glycation and subsequent phosphorylation because of considerable increase in glycated and phosphorylated sites.Therefore,the decrease in allergenicity of BLA,the effect correlated well with the shielding effect of glycated sites combined with phosphorylated sites and the conformational changes.This study provides important theoretical foundations for improving and using the ultrasonic technology combined with protein modification in allergenic protein processing.

膜分离耦合离子交换层析制备高纯度α-乳白蛋白

以生牛乳为原料,经膜分离及喷雾干燥制得鲜乳乳清蛋白粉,结合离子交换层析制备高纯度α-乳白蛋白。结果表明:在浓缩3倍、微滤1次、洗滤2次的膜分离条件下,50 nm陶瓷膜渗透液中α-乳白蛋白占真蛋白的21.04%,高于100 nm陶瓷膜渗透液(15.84%);对50 nm陶瓷膜渗透液进行喷雾干燥,并进行离子交换层析,层析时,在10倍柱体积内,Na Cl溶液浓度由0 mol/L增至0.5 mol/L,α-乳白蛋白与β-乳球蛋白能较好地分离,得到的α-乳白蛋白和β-乳球蛋白纯度分别为98.18%和97.82%。本研究结果可为工业化制备高纯度α-乳白蛋白乳基料提供技术支持。

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。

An expression profile of human α-lactalbumin in the milk of transgenic mouse

Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human a-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female ex-pressed human a-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold in-crease in the total a-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the ha-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concen-trations of a-lactalbumin might boost more milk output.

基于分子对接法α-乳白蛋白缓解丙烯酰胺对小鼠血液毒性作用的研究

为了探讨α-乳白蛋白(α-lactalbumin,α-LA)降低丙烯酰胺血液学毒性作用机理,通过分子对接法探究α-LA与丙烯酰胺(Acrylamide,ACR)能否相互作用及其对接结合位点,并运用分子动力学仿真方法分析α-LA-ACR结合物稳定性。分子的准备和对接采用UCSF Chimera和其中的Auto Dock Vina,分子动力学仿真以ubuntu虚拟机中Gromacs为基础进行模拟计算。α-LA和ACR分子对接结果表明二者间有相互作用的可能。在分子动力学模拟中,分别对α-LA均方根误差(RMSD)、回转半径(Rg)和均方根浮动(RMSF)进行分析。将ICR小鼠随机分为4组,分别为对照组、ACR组、α-LA组、(α-LA+ACR)组,每天1次连续灌胃21 d,结束后,心脏取血,对全血进行分析,并测定红细胞的渗透脆性。结果表明,α-LA与ACR的相互作用对α-LA结构产生了较大影响。ACR能显著改变小鼠多项血液指标,并且能显著增大红细胞渗透脆性,而α-LA对ACR血液学毒性有抑制作用。文章为探究α-LA与ACR相互作用时如何降低ACR对人体负面影响提供了理论依据。

Bioaccessibility and Cell Metabolic Activity Studies of Antioxidant Low Molecular Weight Peptides Obtained by Ultrafiltration of <i>α</i>-Lactalbumin Enzymatic Hydrolysates

α-lactalbumin (α-LA) might increase its antioxidant potential after hydrolysis. In particular, low molecular weight (LMW) peptides showed greater antioxidant capacity. Different hydrolysis conditions with Alcalase enzyme were optimized with a composite central design and surface methodology. Sample obtained after 0.1% (w/w enzyme:substrate), 60 min hydrolysis, ultrafiltrated with membranes of 3 kDa (named 4 LMW), showed the greatest antioxidant values: 1.574 ± 0.060 and 1.636 ± 0.076 μmolTE/mg of protein for ABTS and ORAC-FL, respectively. Sample 4 LMW produced mild ACE inhibition capacity, 22% related to Captopril. 4 LMW was submitted to in vitro gastrointestinal conditions using α-amylase, pepsin, pancreatin and bile-extract;its antioxidant capacity was enhanced by the shorter peptides released, confirmed by SE-HPLC. Antioxidant capacity of digested 4 LMW sample (D 4 LMW) was 1.743 ± 0.086 and 2.542 ± 0.245 μmolTE/mg of protein for ABTS and ORAC-FL, respectively, showing improvement on bioaccessibility. Intestinal cells viability was higher for D 4 LMW.

Au@Ag-labeled SERS lateral flow assay for highly sensitive detection of allergens in milk

Casein andα-lactalbumin(α-LA)are the main allergens in cow's milk,which can affect the skin,respiratory system,and gastrointestinal tract,even cause anaphylactic shock.Developing a rapid and sensitive detection method of casein andα-LA is still pursued.Herein,a surface-enhanced Raman scattering based lateral flow assay(SERS-LFA)method for rapid and highly sensitive detection of milk allergens in food was established for effectively preventing allergic symptoms.Gold@silver nanoparticles(Au@Ag NPs)were synthesized as SERS active substrate to prepare the antibody-modified SERS Probe and SERS-LFA strips toward casein andα-LA were assembled according to the sandwich mode.The detection results were calculated according to colorimetric and Raman signal.The introduction of SRES signal significantly increased the sensitivity of detection with the limit of detection(LOD)of 0.19 ng/mL and 1.74 pg/mL,and exhibited an excellent linear relationship within the range of 0.55-791.50 ng/mL and 0.1 pg/mL-100.0 ng/mL for casein andα-LA,respectively.Furthermore,SERS-LFA strips was highly specific with the recovery rates for corresponding80.36%-105.12%and 85.73%-118.22%for casein andα-LA,respectively.Therefore,the SERS-LFA could be in great potential to develop a unique allergen detection method.

Unleashing the healing potential:Exploring next-generation regenerative protein nanoscaffolds for burn wound recovery

Burn injury is a serious public health problem and scientists are continuously aiming to develop promising biomimetic dressings for effective burn wound management.In this study,a greater efficacy in burn wound healing and the associated mechanisms ofα-lactalbumin(ALA)based electrospun nanofibrous scaffolds(ENs)as compared to other regenerative protein scaffolds were established.Bovine serum albumin(BSA),collagen type I(COL),lysozyme(LZM)and ALA were separately blended with poly(ε-caprolactone)(PCL)to fabricate four different composite ENs(LZM/PCL,BSA/PCL,COL/PCL and ALA/PCL ENs).The hydrophilic composite scaffolds exhibited an enhancedwettability and variablemechanical properties.The ALA/PCL ENs demonstrated higher levels of fibroblast proliferation and adhesion than the other composite ENs.As compared to PCL ENs and other composite scaffolds,the ALA/PCL ENs also promoted a better maturity of the regenerative skin tissues and showed a comparable wound healing effect to Collagen sponge^(■)on third-degree burn model.The enhanced wound healing activity of ALA/PCL ENs compared to other ENs could be attributed to their ability to promote serotonin production at wound sites.Collectively,this investigation demonstrated that ALA is a unique protein with a greater potential for burn wound healing as compared to other regenerative proteins when loaded in the nanofibrous scaffolds.

α-乳白蛋白对婴幼儿生长发育的影响研究进展

乳白蛋白是哺乳动物和人类乳汁中的一种钙结合蛋白,占母乳总蛋白质的20%~25%,其结构与溶菌酶相似,含有丰富的赖氨酸、亮氨酸、异亮氨酸、半胱氨酸和色氨酸等必需氨基酸,具有多种营养保健功能,在食品生产中具有广阔的应用发展前景,受到大众广泛的关注。目前,我国6月龄以下纯母乳喂养率低,婴幼儿配方乳粉与母乳对婴儿健康生长至关重要。大量研究表明α-乳白蛋白能够促进神经发育、调节睡眠节律并改善抑郁情绪;还能够提高铁和锌的吸收利用率、促进消化道功能完善和胃肠道发育,对于婴幼儿早期的营养至关重要。本文从α-乳白蛋白的氨基酸组成和结构,对婴幼儿体格、大脑神经以及胃肠道发育的影响等方面综述了国内外相关研究进展,以期为婴幼儿配方乳粉的升级和“母乳化”的推广应用提供理论参考。

不同热加工工艺对巴氏杀菌乳中乳清蛋白的影响

以牛乳乳清蛋白为研究对象,探究不同热加工工艺(72℃/15 s、75℃/15 s、80℃/15 s、85℃/15 s)对巴氏杀菌乳乳清蛋白中3种活性蛋白(α-乳白蛋白、β-乳球蛋白和乳铁蛋白)的影响,以及测定并分析杀菌温度对各样品菌落总数和嗜冷菌的影响。结果表明:随着热加工强度的提升,牛乳中的菌落总数随之减少,当杀菌温度在80℃以上时牛乳中的菌落总数小于10 CFU/mL;当杀菌温度在72℃以上时样品中的嗜冷菌数均小于10 CFU/mL;72℃/15 s和75℃/15 s对α-乳白蛋白、β-乳球蛋白和乳铁蛋白影响较小,当杀菌温度达到80℃以上时,巴氏杀菌乳中的α-乳白蛋白、β-乳球蛋白和乳铁蛋白含量显著下降(P<0.05)。综上,热加工的时间和温度与乳清蛋白的关系密切,72~75℃/15 s的热加工工艺能更好地保留乳清蛋白中的3种活性蛋白。

鲟鱼头汤对产后缺乳大鼠泌乳功能的促进作用

目的:研究经蒸煮后高压液化熬制鲟鱼头汤的催乳功效。方法:将Wistar母鼠随机分为空白组(生理盐水)、缺乳模型组(溴隐亭)、阳性对照组(乳清蛋白鲈鱼粉)和实验组(鲟鱼头汤),每组6只,对哺乳期Wistar母鼠进行连续14 d灌胃,考察其体质量和泌乳量变化。通过苏木精伊红染色、免疫组化分析、酶联免疫吸附测定与实时荧光定量聚合酶链式反应等方法测定母鼠乳腺组织形态、相关激素与蛋白表达水平。结果:实验组母鼠乳腺的腺管与腺泡中可以观察到棕黄色阳性显色蛋白分泌物数量增多,乳腺小叶间脂肪结缔组织数量减少,相较于模型组有明显恢复。与模型组相比,实验组母鼠每小时泌乳量、乳腺相对质量以及仔鼠窝重净增率均显著提高(P<0.05),母鼠血清与乳腺组织中催乳素(prolactin,PRL)、催乳素受体(prolactin receptor,PRLR)、β-酪蛋白(β-casein,β-CN)、α-乳清蛋白(α-lactalbumin,α-LA)水平及乳糖合成酶(lactose synthase,LS)和脂肪酸合成酶(fatty acid synthase,FAS)活力也显著提升(P<0.05)。乳腺中β-CN、α-LA和FAS的mRNA表达分析结果表明,控制β-CN和FAS合成的基因表达水平与模型组相比明显上调。结论:鲟鱼头汤对产后缺乳母鼠具有促进泌乳的作用,其机制可能与增加PRL和PRLR水平以及促进β-CN、α-LA和FAS的表达有关。

不同长度糖链对α-乳白蛋白过敏表位^(13)KDLKGYGGVSLPEW^(26)结构和致敏性的影响

α-乳白蛋白的过敏表位是影响其致敏性强弱的分子基础,本文研究不同长度的糖链对其过敏表位结构和致敏性的影响。先合成α-乳白蛋白的IgE线性过敏表位(^(13)KDLKGYGGVSLPEW26,13K-W^(26)),选用葡萄糖、麦芽糖、麦芽三糖、麦芽五糖对其进行糖基化修饰,采用光谱、色谱分析其结构的变化;运用ELISA和细胞实验等方法来评价其IgE结合能力和KU812细胞释放组胺、白介素-4和白介素-6的能力。结果表明:不同长度的糖链与13K-W26发生糖基化反应后,降低其游离氨基含量,改变其荧光强度和紫外吸收强度,同时降低其IgE结合能力和KU812细胞中组胺、白介素-4和白介素-6的释放能力。因此,不同长度的糖链能改变13K-W26的结构,降低其致敏性,顺序为麦芽五糖>麦芽三糖>麦芽糖>葡萄糖。糖链可遮蔽α-乳白蛋白的线性表位13K-W26,糖链越长,遮蔽作用越强,致敏性消减效果越好。

α-乳白蛋白与β-酪蛋白预防炎症的协同作用研究

牛乳中酪蛋白比例高,而母乳中乳清蛋白含量高,α-乳白蛋白、β-酪蛋白作为母乳中主要的乳清蛋白和酪蛋白,以α-乳白蛋白和β-酪蛋白为研究对象,利用脂多糖(LPS)诱导RAW264.7细胞炎症反应,探究不同比例的组合蛋白对细胞炎症的预防作用,探索乳清蛋白和酪蛋白的最佳配比,使婴配粉更贴合母乳。结果表明,添加α-乳白蛋白和β-酪蛋白单体能改变炎症因子mRNA和蛋白水平的表达,然而当混合比例为1:0.17时效果最佳,能显著降低白介素6(IL-6)、白介素10(IL-10)、髓样分化基因88(MyD88)、Toll样受体4(TLR4)在mRNA水平的表达,在蛋白水平降低IL-61.99倍、白介素1β(IL-1β)3.91倍、肿瘤坏死因子α(TNF-α)2.72倍。通过抑制非TLR4介导炎症信号通路关键蛋白NF-κB p65的磷酸化抑制炎症的发生。本研究明确了α-乳白蛋白和β-酪蛋白预防炎症发生的最佳比例,初步阐明了两者协同抗炎的作用机理,为其在婴幼儿配方奶粉中的精准添加提供理论支持和技术指导。

乳蛋白高效液相色谱检测方法的优化及甘南牦牛乳指纹图谱的建立

建立多种乳蛋白同时检测的高效液相色谱法,为乳及乳制品乳蛋白的分离检测提供方法,并建立甘南牦牛乳的指纹图谱。样品采用缓冲液使蛋白溶解变性,采用高效液相色谱检测,ZORBAX 300SB-C8色谱柱,检测波长为214 nm,柱温40℃,流速0.6 mL/min,梯度洗脱。所建方法能够分离乳中的κ-酪蛋白、α-酪蛋白、β-酪蛋白、α-乳白蛋白、β-乳清蛋白,峰保留时间RSD<1.3%,峰面积RSD<3.3%。10批甘南牦牛乳样品得到的指纹图谱相似度大于0.99,指认了其中9个乳蛋白色谱峰。该方法操作简便、具有良好的重现性和专属性,适用于乳及乳制品中多种乳蛋白的分离检测。所建立的甘南牦牛乳指纹图谱为牦牛乳的质量控制提供参考依据。

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。

热处理对液态乳中乳清蛋白的影响研究进展

牛乳中的乳清蛋白主要包括β-乳球蛋白、α-乳白蛋白、牛血清白蛋白和免疫球蛋白。在牛乳加工过程中,热处理会使乳中乳清蛋白发生变性,影响了乳清蛋白的结构和活性,进而降低了牛乳的营养价值。本文对乳业发达国家液态乳的主要加工方式以及热处理过程对4种乳清蛋白的影响进行了综述。

牦牛α-乳清蛋白基因的克隆与序列分析

根据奶牛α 乳清蛋白基因序列设计引物 ,用PCR方法扩增并克隆了牦牛 (Poephagensgrunnieus)α 乳清蛋白基因的全序列。结果表明 ,牦牛α 乳清蛋白基因长 2 999bp ,其中 1～ 670bp为 5′侧翼序列 ,2 690～2 999bp为 3′侧翼序列 ,在 671～ 2 689bp之间 ,共有 4个外显子和 3个内含子 ,牦牛α 乳清蛋白基因共编码 14 2个氨基酸 ,其中第 1～ 19氨基酸之间的短肽为信号肽序列。牦牛的α 乳清蛋白基因有较高水平的表达可能与基因内非编码序列碱基突变引起的回文结构消失有关。该基因 5′侧翼序列在结构上牦牛和牛基本相同 ,只有MGF因子识别位点稍有差别。且牦牛的该序列更符合Groenen等 1994年总结的该因子识别位点的模式序列 ,因此牦牛的该基因

α-乳白蛋白提高β-胡萝卜素乳液稳定性

为了提高β-胡萝卜素乳液稳定性,该研究利用α-乳白蛋白(α-LA)为乳化剂,考察了不同α-LA添加量(0.25%~3.00%)对10%的水包油(O/W)β-胡萝卜素乳液粒径、电位、快速稳定性、包埋率、界面α-LA含量和化学稳定性的影响。结果表明:随着α-LA添加量的增加,β-胡萝卜素乳液粒径减小,电位增加,包埋率提高;当α-LA添加量大于1.50%时,乳液粒径、电位和包埋率不再随着α-LA添加量的增加而变化(P>0.05)。乳液的Turbiscan扫描指数(TSI)在α-LA添加量大于1.50%之后没有显著性变化,β-胡萝卜素乳液趋于稳定。随着α-LA添加量的增加,水油界面上α-LA含量显著增加(P<0.05)。当α-LA添加量大于1.00%时,β-胡萝卜素在乳液中的保留率不再随着α-LA添加量的增加而增加。研究结果表明α-LA是一种可以应用在β-胡萝卜素乳液中的乳化剂,在α-LA添加量为1.50%时,可以得到物理和化学稳定性较好的β-胡萝卜素乳液,为食品工业中应用β-胡萝卜素提供了参考。