6. AOACSPIFANMethods-2018Awards

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F eng et al .: J ournal of AOAC I nternational V ol . 100, N o . 2, 2017  513

Figure 2016.15B. Separation of the protein MW size standard.

and BSA peak; perform a manual integration from the valley between the end of the κ-casein peak and the peak of Ig H and BSA to the end of the last peak in the e-grams (at least 9 min after the peak of the 10 kDa IS). (j)  To determine the casein region, set the start time for casein integration just before the β-casein peak in the e-gram of the SMP (about 3.1 min after the peak of the 10 kDa IS). Referencing the SMP, identify the β-casein peak in the infant formula samples, then set the start time of the casein region in the infant formula to just before the β-casein peak. Set the end time at the valley between the end of the κ-casein peak and the Ig H and BSA peak (about 7.0 min after the 10 kDa IS). (a)  To calculate whey protein content, separately sum the peaks in the following three regions: two at each end of the e-gram (smaller and larger whey proteins) and one in the middle. The middle region corresponds to casein proteins (A cn ), and the two others are summed together to obtain the whey proteins (A w ). (b)  Whey protein content is calculated using the following equations: F. Calculations

Results and Discussion

Specificity

(a)  Reagent blank .—Each sample sequence was started with purified water as a blank. The blank e-gram is shown in Figure 1 (gray line). No peaks were detected after the 10 kDa peptide internal standard (IS). There was no significant interference from other components in the protein region. (b)  Placebo test .—To test for the presence of interference from nonprotein components in infant formulas, a placebo infant formula trial sample that contained all of the ingredients that are typical first-age formulas, except protein (vitamins, minerals, fat, and carbohydrates), was manufactured. SDS-CGE did not detect significant peaks at any of the protein regions in the e-gram (Figure 1, black line). (c)  Specific protein migration time and migration pattern of whey proteins and caseins .—The SDS-CGE method can separate individual whey and casein protein standards very well, as demonstrated with standard solutions containing five major whey proteins (Figures 2 and 3) or four casein proteins (Figure 4), as well as with fresh raw milk (Figure 5). Protein phosphorylation and glycosylation delay casein migration times relative to their molecular sizes (Figure 6). Protein glycation—the nonenzymatic sugar modification of amines and the early stage of a Maillard reaction—occurs during the mixing and heating of milk proteins with lactose (3), which results in the splitting of several individual milk proteins into several peaks representing the modified protein glycoforms. This was seen for α-Lac and β-Lg, where splitting was observed in a commercial sweet whey protein ingredient (Figure 7) and by comparing the casein peaks in fresh milk and in an SMP sample (Figures 5 and 8). Although glycation prevents the complete separation of all proteins individually, whey proteins

A A A w,c w,c +

=

Percentage of whey protein

(1)

cn

A A 1.4 w,c w = ×

(2)

where A w = total integrated areas of whey components; A w,c = corrected integrated area of whey components; A cn = integrated area of casein components; and 1.4 = CF to account for the difference between the mass-to-area ratio of whey and  casein proteins.