Lipoprotein lipase (LPL) activity and free fatty acid levels were studied in freshly milked, uncooled milk from individual Danish Holstein or Jersey cows, or after storage for up to 24h at either a cooling temperature (4°C) or at the milking temperature (31°C). Upon cooling for up to 24h, LPL activity increased in the cream phase, whereas the activity in the skim milk was steady, as observed for Jersey cows, or increased, as seen for the Holsteins. Storage at 31°C decreased the LPL activity in both the cream phase and the skim milk phase. The increase in free fatty acid levels was found to depend on LPL activity, incubation temperature, substrate availability, and incubation time. Furthermore, the migration of milk proteins between the skim milk phase and the cream phase upon cooling of milk from Jersey cows or from Danish Holstein cows was studied using proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry. Proteins associated with the milk fat globules were isolated from all milk fractions and analyzed. Major changes in the distributions of proteins between the skim milk phase and the cream phase were observed after cooling at 4°C for 4h, where a total of 29 proteins between the 2 breeds was found to change their association with the milk fat globule membrane (MFGM) significantly. Among these, the MFGM proteins adipophilin, fatty acid-binding protein, and lactadherin, as well as the non-MFGM proteins β-casein, lactoferrin, and heat shock protein-71, were identified. Adipophilin, lactadherin, and lactoferrin were quantitatively more associated with the MFGM upon cold storage at 4°C, whereas β-casein, fatty acid-binding protein, and heat shock protein-71 were found to be less associated with the MFGM upon cold storage.

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This study demonstrated that homogenization did not increase the activity of lipoprotein lipase (LPL) in spite of a fast accumulation of free fatty acids (FFA). Two homogenization pressures (100 and 170 bar) and two temperatures (40℃and 50℃) were examined. The activity of LPL was analyzed and the formation of FFA was measured with two different methods, the B.D.I.-method and a nonesterified fatty acids (NEFA) method. A homogenization temperature of 50℃ resulted in a decreased LPL activity compared to 40℃. No effect of homogenization pressure was found. Analyzing FFA concentration with the B.D.I.-method resulted in significant effect of homogenization temperature and no effect of pressure. The largest formation of FFA was found in milk homogenized at 40℃. Using the NEFA method, another result was obtained, indicating no effect of homogenization temperature and a larger FFA accumulation at 100 bar than at 170 bar. Both analytic methods demonstrated significant production of FFA during 60 min incubation at homogenization temperature after treatment. The level of FFA in the milk samples immediately after homogenization was very high, demonstrating that LPL cleaves the triglycerides very rapidly when the native membrane was damaged. The regression between the B.D.I.-method and the NEFA was fair in the interval between 4 and 14 mmol/100 g fat, whereas at higher concentrations, the correlation was poor