QUANTIFICATION OF GENETIC VARIANTS OF CASEINS IN MILK OF GIRGENTANA GOAT BREED

Abstract

In the milk of ruminants, more than 95% of proteins are synthesized by six structural genes, four caseins (alphas1, beta, alphas2 and κ-caseins) and two whey proteins (alpha-lactalbumin and beta-lactoglobulin). Goat casein genes have been found to show high polymorphism, which influences not only the quantity of casein in milk but also the structural and nutritional characteristics and technological properties of milk. The aim of this work was to separate and quantify the most common allelic variants of caseins in milk of Girgentana goat breed, a Sicilian autochthonous breed, and to evaluate the effect of casein polymorphisms on casein content. The alleles at different casein genes were detected using PCR, PCR-RFLP, AS-PCR and sequencing analysis. Milk samples were prepared following the method proposed by Bobe et al. (1998) and analyzed by RP-HPLC method. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5µm, 300Å, 150×4.6 I.D.) was used and the detection was made at wavelength of 214 nm. The procedure was developed using individual raw milk samples of Girgentana goats. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes, considering that commercial standards for goat allelic variants were not available. In particular, were used animals with AA, BB, FF and NN genotypes at alphas1-casein; CC and C'C' genotypes at beta-casein; AA and FF genotypes at alphas2-casein; and AA and BB genotypes at κ-casein. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. For Girgentana goat breed the following levels of caseins for allele were obtained: alphas1-casein A 3.2±0.4 g/L, B 5.4±0.5 g/L and F 0.7±0.1 g/L; beta-casein C 3.0±0.8 g/L and C' 2.0±0.7 g/L; alphas2-casein A 2.9±0.8 g/L and F 1.8±0.4 g/L; and κ-casein A 1.6±0.3 g/L and B 1.1±0.2 g/L.