Measurement of casein in milk by Kjeldahl and sodium dodecyl sulfate–polyacrylamide gel electrophoresis

摘要

Our objectives were to determine if milk casein asa percentage of true protein (CN%TP) estimated bysodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE) is equivalent to CN%TP estimatedby Kjeldahl, and to determine the proportion of casein(CN), casein proteolysis products (CNPP), and serumprotein (SP) from milk true protein (TP) that goesinto the Kjeldahl noncasein nitrogen (NCN) filtrateand the proportion that stays in the NCN precipitateusing SDS-PAGE. Raw milk samples were collectedfrom 16 mid-lactation Holstein cows twice a week for2 wk. These milks were analyzed for Kjeldahl totalnitrogen, nonprotein nitrogen, and NCN content in duplicate,and by SDS-PAGE. The CN%TP determinedby Kjeldahl was compared with the CN%TP estimatedby SDS-PAGE calculated in 2 ways: as a percentage ofonly intact caseins divided by TP and as a percentageof both intact caseins and CNPP divided by TP. Threemilks varying in fat, lactose, TP, CN, and SP contentwere formulated. These milks were analyzed in duplicatefor Kjeldahl total nitrogen, nonprotein nitrogen,and NCN content, and each of the NCN filtrate andNCN precipitate were analyzed in duplicate by SDSPAGEfor relative quantity (%) of CN, CNPP, and SP.We found that the estimate of CN%TP by Kjeldahl washigher than the estimate of CN%TP by SDS-PAGEthat was calculated as only intact CN divided by thetotal of all protein bands. However, no difference wasdetected in the estimate of CN%TP by Kjeldahl comparedwith CN%TP by SDS-PAGE when CNPP wereincluded as CN in the calculation of SDS-PAGE results.Based on SDS-PAGE results, we found that a majority(89%) of the CNPP from the milk (approximately10.13 out of 11.41% TP) were retained in the KjeldahlNCN precipitate. Thus, CN%TP measured by Kjeldahlunderestimates the amount of proteolytic damage thathas been done to CN in milk. It is important for thedairy industry to correctly and rapidly measure the extentof proteolytic damage to milk protein to correctlyvalue milk from a product quality and yield point ofview. A rapid and quantitative measure of proteolyticdamage to milk protein is needed.