单酶水解豆芽蛋白及其产物的潜在致敏性

目的优化豆芽蛋白酶水解的条件,并探讨其致敏性的变化。方法利用Alcalase 2.4L碱性蛋白酶水解豆芽蛋白,以水解度为评价指标,根据单因素实验优化豆芽蛋白的酶水解条件,并通过IgG、IgE的结合实验评估酶解产物潜在致敏性的变化。结果酶水解豆芽蛋白的优化工艺条件:底物浓度为8%、酶与底物比(E/S)为1:20(m:m)、酶解时间为4 h。豆芽蛋白酶水解产物的抗原性低于大豆蛋白酶解产物的抗原性,但豆芽蛋白水解产物的IgE结合能力高于大豆蛋白酶解产物的IgE结合能力。结论大豆经过发芽处理后再用Alcalase2.4L轻度水解能有效降低大豆蛋白的潜在致敏性。     

臭氧处理对牛乳乳清蛋白的结构及致敏性的影响

以牛乳乳清蛋白为研究对象,探究臭氧处理对乳清蛋白的结构及致敏性的影响。分别测定臭氧处理0、5、10、15、20 s和25 s后乳清蛋白氨基酸组分、巯基和二硫键含量的变化,并采用圆二色光谱仪、紫外分光光度计和荧光分光光度计等光谱学技术分析其结构变化,最后利用间接竞争酶联免疫吸附剂测定（enzyme linked immunosorbentassay,ELISA）分析体外特异性抗体的结合能力变化,用于评估潜在的致敏性。结果表明,臭氧处理会使乳清蛋白中部分氨基酸的含量降低,游离巯基含量由5.78μmol/g减少至2.13μmol/g,总巯基含量由14.98μmol/g减少至12.97μmol/g,二硫键含量则由4.60μmol/g升高至5.42μmol/g。光谱学分析表明,臭氧处理后乳清蛋白的二级结构改变、三级结构变得松散,但随着处理时间的延长,部分蛋白分子之间相互聚集,二硫键的增加也使乳清蛋白的空间结构重归有序。间接竞争ELISA的结果表明臭氧处理后乳清蛋白的致敏性明显下降。综上所述,臭氧处理在降低乳清蛋白致敏性、提高乳蛋白食品安全性上具有良好的研究潜力和开发前景。     

双蛋白酶解法制备低抗原性乳清蛋白肽及其抗氧化性能研究

通过双蛋白酶分步水解法制备了一种低抗原性乳清蛋白肽,并对其抗氧化活性进行了实验研究.通过响应面法实验对乳清蛋白水解的工艺技术条件如双酶复配比例、底物浓度、酶解温度、时间、酶解pH值等进行了优化.结果表明:碱性蛋白酶与风味蛋白酶之比为1∶1,酶解温度54℃,pH值7.1条件下水解,此时乳清蛋白肽的抗原抑制率仅为10.02...     

羊乳清蛋白部分和深度水解工艺、水解物致敏性及肽谱分析

利用羊乳清蛋白制备低致敏性配料是目前乳品工业的研究热点。乳清蛋白是乳中主要的蛋白质之一,也是引起婴幼儿过敏反应的主要成分,将蛋白质水解为小分子肽是降低其致敏性的有效方法。以山羊乳清蛋白为原料,研究了部分水解乳清蛋白和深度水解乳清蛋白的水解工艺和水解物特性(水解度、分子质量分布和β-乳球蛋白抗原性),并利用液相色谱串联质谱法(LC-MS/MS) 比较了部分水解和深度水解工艺中过敏表位酶切位点的差异。研究结果表明,中性蛋白酶和碱性蛋白酶对羊乳清蛋白水解效果较好,其中碱性蛋白酶的水解度最高,达21.26%。经电泳分析,单酶水解后的产物中仍存在大分子多肽链,深度水解工艺需要复合酶水解。在酶底比为4000U/g时,使用碱性蛋白酶在pH值为10.0、温度为55℃条件下水解羊乳清蛋白1.0h,部分水解产物的水解度为12.31%,分子质量在5kDa以下的多肽占95.18%,β-乳球蛋白抗原性下降率为9.40%。中性蛋白酶和碱性蛋白酶的质量比为1∶1,酶底比为6000U/g,在pH值为8.5、温度为50℃条件下,水解羊乳清蛋白3.0h,深度水解产物的水解度为35.58%,分子质量低于3kDa的多肽为97.26%,β-乳球蛋白抗原性下降率为40.97%。部分水解和深度水解均能破坏β-乳球蛋白的大部分过敏表位,但相较于部分水解,深度水解能更大程度地降低乳清蛋白的致敏性。研究旨在为低致敏性羊水解乳清蛋白的生产提供一定的理论参考。     

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％.     

适度水解乳清蛋白婴儿配方奶粉的致敏性评价

牛奶是婴儿饮食中最早引入的食物之一,也是引起食物过敏最常见的原因之一。因此,低致敏奶粉的研制具有重要意义。该文采用4周龄雌性BALB/c小鼠,以灌胃方式致敏。将乳清蛋白浓缩物作为阳性对照,通过观察过敏症状,计算脾脏系数,测定血清中总免疫球蛋白E(IgE)和组胺含量,对适度水解乳清蛋白婴儿配方奶粉进行致敏性评价。结果表明:乳清蛋白浓缩物组出现了明显过敏症状,血清中总免疫球蛋白E(IgE)、组胺含量以及脾脏系数显著高于空白及佐剂对照组,说明乳蛋白致敏模型成功建立;适度水解乳清蛋白婴儿配方奶粉组无过敏症状,与空白组及致敏佐剂组相比,血清中总免疫球蛋白E(IgE)、组胺含量以及脾脏系数均无显著差异,说明适度水解乳清蛋白婴儿配方奶粉未引起小鼠过敏;同样,由于深度水解破坏了乳清蛋白抗原表位,深度水解乳清蛋白粉也未引起小鼠致敏;而普通脱脂奶粉致敏小鼠血清中总免疫球蛋白E(IgE)、组胺含量以及脾脏系数均显著高于空白组及致敏佐剂组,说明普通脱脂奶粉导致小鼠致敏。因此,适度水解乳清蛋白婴儿配方奶粉可有效降低或缓解牛奶过敏症状,但仍有待于更多临床试验的验证。     

水产品致敏蛋白的研究进展

水产品因营养丰富、味道鲜美而深受消费者喜爱,但水产品也是容易引起食物过敏反应的一类食品。目前, 全球水产品的加工难以满足过敏人群对食用安全性的需求,且针对水产品过敏的治疗,也尚无特效药物。因此,食用水产品能危害潜在过敏人群的健康,降低其生活水平。近年来,随着全球过敏发病率的上升,水产品致敏蛋白的研究已成为全球关注的公共卫生问题之一。本文介绍了水产品中的钙结合蛋白、原肌球蛋白、精氨酸激酶、肌球蛋白轻链、血蓝蛋白等主要致敏蛋白的生化特性及其抗原表位,阐述了基于致敏蛋白及其DNA为基础的酶联免疫吸附法、质谱法、生物传感器法、聚合酶链式反应检测水产品致敏蛋白的方法,同时介绍了利用物理、化学和生物方法消减致敏蛋白致敏性的研究现状,在此基础上提出了水产品致敏蛋白研究和应用中存在的问题及其发展趋势,旨为进一步认识水产品致敏蛋白,开发低致敏或无致敏性水产食品提供参考。     

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%。     

低致敏食品制备技术及其工业化应用研究进展

食物过敏是联合国粮农组织和世界卫生组织认定的全球性食品安全问题之一。在食品加工多元化的背景下,食物过敏患者要完全避免过敏原十分困难,研发低致敏食品对食物过敏患者的安全膳食至关重要。总结了低致敏食品制备技术的加工技术原理;以蒸煮、微波和烘烤为主的热加工技术通过加热诱导蛋白质变性的方式破坏致敏性构象性表位;高压、脉冲电场、脉冲光、低温等离子体、辐照和超声等非热加工技术可以通过过敏原蛋白结构修饰、多肽链断裂、新化学键的产生等方式直接破坏致敏性表位;酶水解、酶交联、糖基化、微生物发酵等其他加工方法则通过改变蛋白质构象或将蛋白质与糖类物质结合,破坏或隐藏过敏原致敏性表位。另外,对工业化低致敏蛋白配料的加工方法和生产现状进行了阐述分析。基于酶法水解的部分水解乳蛋白和深度水解乳蛋白已经可以工业化生产,其他消减食物致敏性的方法以及其他低致敏蛋白配料值得进一步研究。希望可以为工业化生产低致敏食品提供参考。     

大豆过敏原蛋白及致敏性消减技术的研究进展

大豆是我国重要的粮食作物之一,其蛋白质含量高达35%~40%（m/m）。与此同时,大豆蛋白是人们日常生活中最常见的一类食物过敏原,大豆过敏已经成为了急需解决的公共安全问题。β-伴大豆球蛋白（β-Conglycinin,7S）、大豆球蛋白（Glycinin,11S）、Gly m Bd 28K和Gly m Bd 30K（P34）被认为是大豆过敏原中引发机体发生过敏反应的主要成分。迄今为止,国内外对于大豆过敏尚无根治办法,唯一的预防策略是严格避免摄入来防止过敏反应的发生。但研究指出,通过特殊的加工方法或技术手段可以降低大豆过敏原的致敏性,其中以热加工法、超高压法、酶处理法和基因工程法等方法为代表的消减技术得到广泛关注。因此,该文综述了大豆过敏原的类型,常用的过敏蛋白致敏性消减技术及各项技术的优缺点,以期为低敏性大豆食品的开发提供参考。     

不同酶切方式对乳清蛋白疏水性和乳化性的影响

采用不同的蛋白酶对乳清蛋白进行水解,考察了肽键断裂方式对乳清蛋白肽疏水性和乳化性的影响,以及乳清蛋白不同酶解产物的疏水性和乳化性的关系。结果表明:不同蛋白酶作用于乳清蛋白得到的水解产物疏水性不同,6种蛋白酶解液的疏水性均随水解度的增大而降低,其中以胰凝乳蛋白酶酶解液的疏水性下降的最慢。研究还发现,乳化性随着水解度增加而先升高后下降,以双酶复合酶解液最差。不同蛋白酶水解液的乳化性指数随疏水性指数的降低而升高,乳化性指数与疏水性氨基酸质量分数成正相关。     

Production of low antigenic cheese whey protein hydrolysates using mixed proteolytic enzymes

This study examined the effect of different proteolytic enzymes on the production of cheese whey protein (CWP) hydrolysates with low antigenicity. Four enzyme combinations (1:1) trypsin + papain W‐40 (TP), trypsin + neutrase 1.5 (TN), papain W‐40 + protease S (PP) and papain W‐40 + neutrase 1.5 (PN) were added at the rate of 1% of the CWP and it was incubated for 15, 30, 60, 90, 120 and 180 min at 50 °C. CWP hydrolysis and its non‐protein nitrogen concentrations were higher with TP and TN compared with PP and PN at all incubation times. The SDS‐PAGE revealed complete removal of α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) from hydrolysates produced by trypsin‐containing enzyme mixtures. Reverse‐phase HPLC analysis ascertained the CWP hydrolysis and SDS‐PAGE results. The lowest antigenicity in CWP hydrolysates was observed with the use of trypsin‐containing enzyme mixtures compared with other enzyme combinations. Present results suggested that TP and TN combinations were the most effective for CWP hydrolysis for the removal of β‐LG from CWP. Further research is warranted to identify the peptides in CWP hydrolysates produced with these enzyme combinations that may help enhance the utilisation of whey protein in human food. Copyright © 2007 Society of Chemical Industry     

Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity

This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100°C) WPC (2% protein solution) were incubated at 50°C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The α-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and α-lactalbumin. The β-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The β-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of β-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that incubation of heated WPC with 1% pepsin and then with 1% trypsin was the most effective for producing low-antigenic hydrolysates by WPC hydrolysis and obtaining low molecular weight small peptides. Further research is warranted to identify the low molecular weight small peptides in the WPC hydrolysates produced by pepsin and trypsin, which may enhance the use of whey.     

限制性酶解乳清蛋白功能性质研究

探究乳清蛋白在碱性蛋白酶限制性水解下功能性质变化。以乳清蛋白的溶解性,乳化性、乳化稳定性,起泡性、起泡稳定性为考察指标,确定乳清蛋白的等电点及分析不同水解度下乳清蛋白功能性质在p H调控下的变化。结果表明:乳清蛋白的等电点为4.8。乳清蛋白进行限制性酶解后功能性质有了很大提高,其中溶解性在DH14、p H10下达到最大值,较原蛋白提高了14.55%;起泡性在DH14、p H4下达到最大值,较原蛋白提高了107.5%;起泡稳定性在DH4、p H4下达到最大值,比原蛋白提高了8.66%;乳化性在DH14、p H12下达到最大值,比原蛋白提高了56.1%;乳化稳定性DH4、p H12下达到最大值,比原蛋白提高了50.42%。      

响应面设计法优化花生分离蛋白酶解条件的研究

以花生粕为原料,利用响应面设计对其花生分离蛋白的酶解条件进行了优化。通过对花生分离蛋白水解的最佳用酶进行了筛选,确定以碱性蛋白酶Alcalase水解效果最好。在单因素实验基础上,以水解度为指标,设计了响应面分析方案,通过数学推导及实验分析,得出Alcalase可控酶解花生分离蛋白的动力学模型及相关参数:Alcalase水解花生分离蛋白的最优工艺参数为反应温度53.11℃、酶浓度为135.94μL/g、pH为8.05、预测蛋白水解度为24.15%,实际结果与拟合方程预测结果(24.57%)基本吻合。      

Incorporated glucosamine adversely affects the emulsifying properties of whey protein isolate polymerized by transglutaminase

Glucosamine (GlcN) and microbial transglutaminase (Tgase) are used separately or together to improve the emulsifying properties of whey protein isolate (WPI). However, little is known about how the emulsifying properties change when GlcN residues are incorporated into WPI cross-linked by Tgase. We used Tgase as a biocatalyst to cross-link WPI in the presence of GlcN in a liquid system for 12 h at a moderate temperature (25°C). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses indicated that protein polymerization and GlcN conjugation occurred simultaneously, phenomena also supported by the loss of free amines (9.4–20.5%). Addition of 5 U Tgase/g protein improved the emulsifying properties of moderately cross-linked WPI polymers. The Tgase-treated WPI polymers had a larger particle size (～2.6-fold) than native WPI, which may have reduced coalescence and flocculation in an oil-in-water emulsion system. However, the incorporation of GlcN residues into WPI, predominantly via enzymatic glycation, partly inhibited the cross-links between the WPI molecules catalyzed by Tgase, reducing the size of the WPI polymers 0.81- to 0.86-fold). Consequently, WPI+GlcN conjugates provided less stability to the emulsion. Moreover, high NaCl concentration (0.2 M) decreased the emulsifying properties of the WPI+GlcN conjugates by neutralizing negative electric charges in the glycoconjugates. However, the improved emulsifying properties of WPI cross-linked by Tgase may be useful in food processing at higher NaCl concentrations due to the formation of the thicker steric barrier at the oil-water interface.     

Evaluation of bitterness in enzymatic hydrolysates of soy protein isolate by taste dilution analysis

ABSTRACT:  Although enzymatic hydrolysates of soy protein isolate (SPI) have physiological functionality, partially hydrolyzed SPI exhibits bitter taste depending on proteases and degree of hydrolysis (DH). To determine proteolysis conditions for SPI, it is important to evaluate bitterness during enzymatic hydrolysis. Taste dilution analysis (TDA) has been developed for the screening technique of taste-active compounds in foods. The objectives of the present study were to evaluate bitterness of enzyme-hydrolyzed SPI by TDA and to compare bitterness of SPI hydrolysates with respect to kinds of proteases and DH. SPI was hydrolyzed at 50 °C and pH 6.8 to 7.1 to obtain various DH with commercial proteases (flavourzyme, alcalase, neutrase, protamex, papain, and bromelain) at E/S ratios of 0.5%, 1%, and 2%. The DH of enzymatic hydrolysates was measured by trinitrobenzenesulfonic acid method. The bitterness of enzymatic hydrolysates was evaluated by TDA, which is based on threshold detection in serially diluted samples. Taste dilution (TD) factor was defined as the dilution at which a taste difference between the diluted sample and 2 blanks could be detected. As DH increased, the bitterness increased for all proteases evaluated. Alcalase showed the highest TD factor at the same DH, followed by neutrase. Flavourzyme showed the lowest TD factor at the entire DH ranges. At the DH of 10%, TD factor of hydrolysate by flavourzyme was 0 whereas those by protamex and alcalase were 4 and 16, respectively. These results suggest that TDA could be applied for the alternative of bitterness evaluation to the hedonic scale sensory evaluation.     

Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes

The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), β-Lg A and β-Lg B were observed as hydrolysis proceeded to a 5% degree of hydrolysis (DH) in both unheated and heat-treated (80 °C, 10 min) WPI dispersions (100 g L⁻¹). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC–MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate – the majority of which were derived from β-Lg. The mapping of the detected regions in α-La, β-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.