Effect of fat on protein estimation in milk and its correlation with lactose in different milk types: A small-scale study

Estimation of macronutrients like protein and lactose is important to assess the quality of milk. To estimate these two macronutrients, ten raw milk samples obtained from each group of different animals (cow, goat, buffalo), ten pasteurized cow milk and ten human milk samples were analysed.

Bicinchoninic acid (BCA) method was used to estimate protein from different milk samples. Four different sample preparation protocols were compared to check the effect of fat on BCA based protein estimation: dilution (D), fat removal-protein precipitation (FR&PP), fat removal-dilution (FR&D) and dilution-fat removal (D&FR). For lactose quantification, ultrahigh-performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was developed and validated using ¹³C₆ lactose as internal standard (ISTD).

Among these four different protocols, D&FR method showed consistent data for total protein content in animal milk (cow- 3.16%, goat-3.21%, buffalo-3.81%, pasteurized-2.98%) and FR&PP showed consistent data in human milk samples (1.2%). Though BCA method is simple to use, proper sample preparation protocol has to be applied prior to protein estimation to avoid the interference due to fat or lactose. In case of lactose, inter-day validation showed the accuracy ranging from 97.13 to 100.54%, coefficient of variation varying between 0.1 to 1.53%, correlation R²=0.999. Lactose is in the range of 4.1 to 4.8% in animal milk and 6.6% in human milk samples. The internal ratio of lactose/protein (1.28 to 1.55 in animal milk and 5.33 in human milk) will be useful to differentiate human milk from animal milk type and to assess the milk quality.