Short communication: Separation and quantification of caseins and casein macropeptide using ion-exchange chromatography

A) Elution chromatogram (data are average of 3 runs) for raw milk and B) SDS-PAGE of the corresponding peaks (P). NaCas=sodium caseinate; WP=whey protein isolate. Arrow indicates direction of migration.

Calibration curves for purified casein fractions resuspended in buffer for A) β-casein; B) κ-casein; C) α_S1-casein; and D) α_S2-casein. Lines indicate 95% confidence interval and points are the average of 3 different replicate samples. Regression coefficients b(0) (intercept), b(1) are also shown in the graph. Peak area in mAU·mL.

Elution chromatograms of raw skim milk with added amounts of purified A) β-casein; B) κ-casein; C) α_S1-casein; and D) α_S2-casein. Abs=absorbance.

Calibration curves for purified casein fractions added to raw skim milk: A) β-casein; B) κ-casein; C) α_S1-casein; and D) α_S2-casein. Lines indicate 95% confidence interval and points are the average of 3 different replicate samples. Regression coefficients b(0) (intercept) and b(1) are also shown in the graph. Peak area in mAU·mL.

Elution chromatograms for milk treated with chymosin and incubated for different times.

Decrease in κ-casein area compared with the initial area (○) and increase in para-κ-casein released (■), as a function of time of hydrolysis after addition of chymosin.

Amount of casein macropeptide (CMP) released during chymosin hydrolysis. ●=para-κ-casein area percentage measured using reverse phase-HPLC; ○=CMP area percentage measured using ion-exchange chromatography; ■=amount of κ-casein hydrolyzed (1 – κ-casein initial area).