Separation and determination of beta-lactoglobulin variants A and B in cow's milk by capillary free zone electrophoresis.

beta-Lactoglobulin is a whey protein that can be present in at least two genetic variant forms that determine milk composition and product functionality. A free zone capillary electrophoresis (CZE) method was optimized to separate, identify, and quantify beta-lactoglobulin (beta-Lg) A and B variants in milk. Whey proteins were prepared by casein precipitation at pH 4.6. The experimental conditions such as sample preparation, injection size, pH, voltage, and capillary length and temperature were determined after a univariate optimization process. alpha-Lactoalbumin (alpha-La), beta-Lg A, and beta-Lg B were separated in an uncoated capillary using 0.05 M borate buffer containing 0.1% Tween 20 (Sigma Chemical Co., St. Louis, MO, U.S.A.) at pH 8.0 by applying 25 kV. Repeatability was excellent, since variation coefficients for migration times and peak areas were <0.98 and <1.33%, respectively. Identification of the individual proteins was confirmed by spiking with commercially purified standards. Linearity of the method was demonstrated by constructing calibration curves that followed linear relationships with highly significant (p < 0.01) correlation coefficients. Optimized conditions were used for phenotyping and quantifying beta-Lg in milk collected from Holstein cows. The concentration ranges of the individual beta-Lg A and B variants determined in the AA and BB phenotypes were 5.9-6.02 and 3.43-5.48 mg/mL, respectively. In the AB phenotypes, the range was 1.05-5.46 mg/mL for beta-Lg B and 0.37-4.05 mg/mL for beta-Lg A. The frequency of beta-Lg AA, BB, and AB phenotypes were 0.03, 0.10, and 0.86, respectively. The quantitative determination of beta-Lg variants may be useful in establishing statistical correlations between genetic polymorphism of this protein and milk composition.