Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins

Abstract

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of β-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 °C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for β-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of β-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between β-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5–95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.