乳与乳制品中主要乳清蛋白组分的定量分析检测方法研究
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摘要
乳清蛋白作为蛋白质源和他特有的生物学功能,被广泛应用于乳制品和配方食品中,在这些食品加工过程中一般会使用不同程度的热处理。而这些热处理过程不可避免地会导致乳清蛋白主要功能性组分α-乳白蛋白、β-乳球蛋白和乳铁蛋白等发生变性,其变性程度在很大程度上依赖于热处理的时间和温度。热处理变性导致乳清蛋白中的生物活性性组分的活性损失甚至丧失,因而会影响人体对乳制品中α-乳白蛋白等主要功能性乳清蛋白组分的生物利用率。现有的检测方法由于操作繁琐检测周期长,且分离度较差不能准确定量,无法对产品中的乳清蛋白组分进行有效准确的评价。因此有必要建立分别可以检测主要功能性乳清蛋白组分的方法,为准确评价乳制品中有效营养价值提供参考依据和准确的检测方法。
本课题应用超高效液相色谱-质谱联用仪,建立了分别检测乳与乳制品中非变性α-乳白蛋白和非变性β-乳球蛋白的方法,两个方法分别以人乳a-乳白蛋白作为内标和空白基质加标进行定量以尽可能降低基质干扰的影响,分别为以0.1%三氟乙酸水溶液/乙腈溶液作为流动相,经梯度洗脱后,进入质谱采用选择区间扫描方式进行定量检测。其各自的选择扫描区间分别为α-牛乳白蛋白：2357-2368m/z；人乳α-乳白蛋白内标：2340-2350m/z；p-牛乳球蛋白A：1525-1535m/z；p-牛乳球蛋白B：1518-1528m/z。在2-50μg/mL浓度范围内,两个方法都具有良好的线性关系,相关系数均>0.999,两个方法在高、中、低三个浓度水平,其平均回收率分别为95.33-98.67%(RSD%<6.41)、95.14-98.15%(RSD%<7.64)和94.28-98.70%(RSD%<4.53)。将其应用于实际样品检测以进一步检验所建立方法的适用于与有效性结果表明,所建立的方法可适用于婴幼儿食品和乳制品中非变性牛乳α-乳白蛋白和牛乳β-乳球蛋白A和牛乳β-乳球蛋白B的定量测定。
本研究建立了在肽水平上分别用于检测总a-乳白蛋白、总牛乳铁蛋白和总牛乳p-乳球蛋白的方法。将三种蛋白分别用牛胰蛋白酶酶解后,选择优化其各自的特异标签肽,设计合成适当的内标物,使其具有与蛋白本身相似的酶解效率和色谱质谱行为。通过蛋白与标签肽之间的等摩尔定量关系,实现在肽水平上对蛋白进行准确定量,并对所建立的方法分别进行了方法学验证,三个方法的平均回收率分别在95.8-100.6%(RSD％<5.1％).92.04-100.67(RSD％<8.6％)和92.04-109.91%(RSD%<11.2%)之间。将其应用于实际样品检测以进一步检验其适用于与有效性。本法具有灵敏度和准确度高,重现性好,处理简便的优点,本方法可用于婴幼儿食品和乳制品中总的(热变性和非变性的)牛乳α-乳白蛋白、牛乳铁蛋白和牛乳β-乳球蛋白的定量测定。
Whey protein is widely used in dairy products and formula food industry because of its its nutritional and biological functions. The heat treatment during processing these products will inevitably denature the major components of whey protein, such as bovine α-lactalbumin, β-lactoglobulin, lactoferrin, and so on. The degree of denaturation depends on the temperature and heat time. The active components of whey protein will lose their biological activities when they are denatured. The denatured whey protein will impact on the absorption and utilization. The previous methods can not accurately quantify the major whey protein in dairy products due to their low resolution. Thus, it is necessary to establish the methods to detect the main functional components in whey protein in order to evaluate the effective nutritional value of dairy products.
In the present study, the native bovine α-lactalbumin and β-lactoglobulin were quantified at protein level using ultra-high performance liquid chromatography coupled to tandem mass spectrometer, respectively. The accurate quantitation of native bovine a-lactalbumin was achieved by employing human α-lactalbumin as the internal standard. Compared to the previous approaches, the present two independent methods with mass spectrometer under selected area monitoring mode offered shorter analysis time and lower detection limit. The two genetic variants of bovine β-lactoglobulin were successfully separated by employing an Acquity UPLC BEH300C18column under a linear gradient elution. The ranges of mass-to-charge ratio for selected area monitoring were2357-2368m/z,2340-2350m/z and1525-1535m/z,1518-1528m/z for bovine α-lactalbumin, human α-lactalbumin and bovine β-lactoglobulin A, bovine β-lactoglobulin B, respectively. The developed methods for analysis of bovine α-lactalbumin and β-lactoglobulin were extensively validated by determining the linearity (R2>0.99), recovery and repeatability. The recovery rates were95.33-98.67%,95.14-98.15%and94.28-98.70%for bovine α-lactalbumin and β-lactoglobulin, respectively. The repeatability was RSD%<6.41%, RSD%<7.64and RSD%<4.53%for bovine α-lactalbumin and β-lactoglobulin, respectively. The current validated method was successfully applied to the major whey protein quantification of dairy products and infant formulas.
Three independent reliable ultra-high-performance liquid chromatography-mass spectrometry methods were developed for determination of total bovine α-lactalbumin, bovine lactoferrin or total bovine β-lactoglobulin, respectively. After tryptic digestion, the independent signature peptides of bovine α-lactalbumin, lactoferrin and β-lactoglobulin were selected based on the theoretical tryptic peptide fragments, respectively. Three independent internal standards were successfully designed and synthesized responding to bovine α-lactalbumin, bovine lactoferrin or bovine β-lactoglobulin, respectively. They showed the similar digestion efficiency and chromatographic behavior to the responding whey protein components during the digestion and analysis procedures. The three components of whey protein were quantified at peptide level by employing mass spectrometry operated under multiple reaction monitoring mode. They were determined based on the molar equivalent relationship among the responding protein, internal standard and their signature peptides. The recovery rates were95.8-100.6%,92.04-100.67%, and92.04-109.91%for bovine α-lactalbumin, lactoferrin and β-lactoglobulin, respectively. Their relative standard deviation was RSD%<5.1%, RSD%<8.6%, and RSD%<11.2%, respectively. Comparing with the previous methods for analysis of only native protein, the current independent methods proved to be highly suitable for determination of bovine α-lactalbumin, lactoferrin or β-lactoglobulin in dairy products and infant formulas, avoiding forgoing the thermally induced denatured protein during the different processing.
本课题应用超高效液相色谱-质谱联用仪,建立了分别检测乳与乳制品中非变性α-乳白蛋白和非变性β-乳球蛋白的方法,两个方法分别以人乳a-乳白蛋白作为内标和空白基质加标进行定量以尽可能降低基质干扰的影响,分别为以0.1%三氟乙酸水溶液/乙腈溶液作为流动相,经梯度洗脱后,进入质谱采用选择区间扫描方式进行定量检测。其各自的选择扫描区间分别为α-牛乳白蛋白：2357-2368m/z；人乳α-乳白蛋白内标：2340-2350m/z；p-牛乳球蛋白A：1525-1535m/z；p-牛乳球蛋白B：1518-1528m/z。在2-50μg/mL浓度范围内,两个方法都具有良好的线性关系,相关系数均>0.999,两个方法在高、中、低三个浓度水平,其平均回收率分别为95.33-98.67%(RSD%<6.41)、95.14-98.15%(RSD%<7.64)和94.28-98.70%(RSD%<4.53)。将其应用于实际样品检测以进一步检验所建立方法的适用于与有效性结果表明,所建立的方法可适用于婴幼儿食品和乳制品中非变性牛乳α-乳白蛋白和牛乳β-乳球蛋白A和牛乳β-乳球蛋白B的定量测定。
本研究建立了在肽水平上分别用于检测总a-乳白蛋白、总牛乳铁蛋白和总牛乳p-乳球蛋白的方法。将三种蛋白分别用牛胰蛋白酶酶解后,选择优化其各自的特异标签肽,设计合成适当的内标物,使其具有与蛋白本身相似的酶解效率和色谱质谱行为。通过蛋白与标签肽之间的等摩尔定量关系,实现在肽水平上对蛋白进行准确定量,并对所建立的方法分别进行了方法学验证,三个方法的平均回收率分别在95.8-100.6%(RSD％<5.1％).92.04-100.67(RSD％<8.6％)和92.04-109.91%(RSD%<11.2%)之间。将其应用于实际样品检测以进一步检验其适用于与有效性。本法具有灵敏度和准确度高,重现性好,处理简便的优点,本方法可用于婴幼儿食品和乳制品中总的(热变性和非变性的)牛乳α-乳白蛋白、牛乳铁蛋白和牛乳β-乳球蛋白的定量测定。
Whey protein is widely used in dairy products and formula food industry because of its its nutritional and biological functions. The heat treatment during processing these products will inevitably denature the major components of whey protein, such as bovine α-lactalbumin, β-lactoglobulin, lactoferrin, and so on. The degree of denaturation depends on the temperature and heat time. The active components of whey protein will lose their biological activities when they are denatured. The denatured whey protein will impact on the absorption and utilization. The previous methods can not accurately quantify the major whey protein in dairy products due to their low resolution. Thus, it is necessary to establish the methods to detect the main functional components in whey protein in order to evaluate the effective nutritional value of dairy products.
In the present study, the native bovine α-lactalbumin and β-lactoglobulin were quantified at protein level using ultra-high performance liquid chromatography coupled to tandem mass spectrometer, respectively. The accurate quantitation of native bovine a-lactalbumin was achieved by employing human α-lactalbumin as the internal standard. Compared to the previous approaches, the present two independent methods with mass spectrometer under selected area monitoring mode offered shorter analysis time and lower detection limit. The two genetic variants of bovine β-lactoglobulin were successfully separated by employing an Acquity UPLC BEH300C18column under a linear gradient elution. The ranges of mass-to-charge ratio for selected area monitoring were2357-2368m/z,2340-2350m/z and1525-1535m/z,1518-1528m/z for bovine α-lactalbumin, human α-lactalbumin and bovine β-lactoglobulin A, bovine β-lactoglobulin B, respectively. The developed methods for analysis of bovine α-lactalbumin and β-lactoglobulin were extensively validated by determining the linearity (R2>0.99), recovery and repeatability. The recovery rates were95.33-98.67%,95.14-98.15%and94.28-98.70%for bovine α-lactalbumin and β-lactoglobulin, respectively. The repeatability was RSD%<6.41%, RSD%<7.64and RSD%<4.53%for bovine α-lactalbumin and β-lactoglobulin, respectively. The current validated method was successfully applied to the major whey protein quantification of dairy products and infant formulas.
Three independent reliable ultra-high-performance liquid chromatography-mass spectrometry methods were developed for determination of total bovine α-lactalbumin, bovine lactoferrin or total bovine β-lactoglobulin, respectively. After tryptic digestion, the independent signature peptides of bovine α-lactalbumin, lactoferrin and β-lactoglobulin were selected based on the theoretical tryptic peptide fragments, respectively. Three independent internal standards were successfully designed and synthesized responding to bovine α-lactalbumin, bovine lactoferrin or bovine β-lactoglobulin, respectively. They showed the similar digestion efficiency and chromatographic behavior to the responding whey protein components during the digestion and analysis procedures. The three components of whey protein were quantified at peptide level by employing mass spectrometry operated under multiple reaction monitoring mode. They were determined based on the molar equivalent relationship among the responding protein, internal standard and their signature peptides. The recovery rates were95.8-100.6%,92.04-100.67%, and92.04-109.91%for bovine α-lactalbumin, lactoferrin and β-lactoglobulin, respectively. Their relative standard deviation was RSD%<5.1%, RSD%<8.6%, and RSD%<11.2%, respectively. Comparing with the previous methods for analysis of only native protein, the current independent methods proved to be highly suitable for determination of bovine α-lactalbumin, lactoferrin or β-lactoglobulin in dairy products and infant formulas, avoiding forgoing the thermally induced denatured protein during the different processing.
引文
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Wal J.M. Bovine milk allergenicity. Ann. Allergy Asthma Immunol.2004,93 (5 Suppl 3):S2-11.
Xiao R, Carter JA, Linz AL, Ferguson M, Badger TM, Simmen FA. Dietary whey protein lowers serum C-peptide concentration and duodenal SREBP-lc mRNA abundance, and reduces occurrence of duodenal tumors and colon aberrant crypt foci in azoxymethane-treated male rats. J Nutr Biochem. 2006,17 (9):626-634.
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