Separation and determination of denatured alpha(s1)-, alpha(s2)-, beta- and kappa-caseins by hydrophobic interaction chromatography in cows', ewes' and goats' milk, milk mixtures and cheeses.
Caseins alpha(s1)-, alpha(s2)-, beta- and kappa- from raw cows', ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alpha-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analyzed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (alpha(s1)/kappa, alpha(s2)/beta, beta/kappa and alpha(s2)/alpha(s1)) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.
Bramanti
Emilia
E
Italian National Research Council, CNR, Istituto per i Processi Chimico-Fisici, Laboratory of Instrumental Analytical Chemistry, Via G. Moruzzi 1, 56124 Pisa, Italy. emilia@icas.pi.cnr.it
Sortino
Chandra
C
Onor
Massimo
M
Beni
Francesca
F
Raspi
Giorgio
G
D000818:Animals; D002364:Caseins / Q000032:analysis / Q000302:isolation & purification* / Q000592:standards; D002417:Cattle; D002611:Cheese / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006041:Goats; D008892:Milk / Q000737:chemistry*; D011489:Protein Denaturation; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012756:Sheep
D002364:Caseins