Quantitative Determination Of The Major Whey Protein Components In Milk And Dairy Products

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.