Antioxidant capacity of bovine milk as assayed by spectrophotometric and amperometric methods

The total antioxidant capacity (TAC) of bovine milk, whey, and low-molecular-weight (LMW) fractions of whey was investigated using spectrophotometric methods including an ABTS-based method (reduction of the cation radical of 2,2′-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid)) and a FRAP method (reduction of Fe³⁺). Significant antioxidant capacity in milk and whey was demonstrated by the ABTS method (TAC(ABTS)), and it apparently increased with increasing pH. TAC (ABTS) was several-fold higher in milk than in whey, which had a slightly higher TAC than a LMW fraction prepared from it. Also the FRAP method could be used to demonstrate TAC in whey, although the low pH necessary for this method led to some protein precipitation. Most of the ferric-reducing activity of whey was found in the LMW fraction. The TAC values obtained using these methods were also compared to those obtained using a newly developed flow-injection amperometric (FIAmp) procedure for LMW fractions. High correlations were found for the TAC values of LMW samples obtained by the ABTS, FRAP and FIAmp methods (R²>0.8, P<0.001). Furthermore, to identify the major antioxidants in the LMW fraction, it was treated by uricase. Since most of the TAC (using ABTS, FRAP and FIAmp methods) was removed by uricase treatment, it could be concluded that urate is the major antioxidant in the LMW fraction. Also the effect of heat treatment on TAC in milk and whey was monitored. After heating of whey at 63°C for 1 h, TAC(ABTS) tended to increase by at least 20%, while TAC(FRAP) was not significantly changed. In milk, TAC(ABTS) remained constant during the same heat treatment. It is concluded that the methods tested gave the most reliable results for the LMW fraction of whey and that the use of several methods is necessary to characterise the antioxidant capacity of milk.