Properties of proteases from milk somatic cells and blood leukocytes.

Proteolytic activity of proteases associated with somatic cells isolated from high SCC milk and proteases associated with leukocytes isolated from bovine blood was assayed at pH 6.6 and 5.2 in a model system consisting of a beta-casein (.96%) substrate in Jenness/Koops buffer. Intact beta-casein and casein proteolysis products were measured by densitometric analysis of SDS-PAGE gels. Relative proteolytic activity was expressed as percentage degradation of beta-casein after 24 h at 37 degrees C per 1 x 10(6) cells/ml. Proteolytic activity associated with somatic cells isolated from bovine milk was 27.5 and 13.6% at pH 6.6 and 5.2, respectively. Proteolytic activity associated with leukocytes isolated from bovine blood was 16.0 and 8.4% at pH 6.6 and 5.2, respectively. Proteolytic activity at pH 6.6 was significantly higher for the somatic cells isolated from milk than for leukocytes isolated from blood. The reason for the difference in proteolytic activity of leukocytes isolated from blood of a healthy cow versus somatic cells isolated from milk produced by a cow with mastitis is not known. Further work is needed to determine whether the difference may be caused by a higher proportion of activated macrophages in somatic cells isolated from milk produced by cows with mastitis than in the leukocytes isolated from blood of healthy cows.

Effect of mastitis on proteolytic activity in bovine milk.

Proteolytic activity of milk was studied before, during, and after experimental-induced mastitis. An inoculum of Streptococcus agalactiae was infused into one quarter of each udder of six cows to elicit an infection. Bacteriological cultures and SCC of milk were used to monitor infection status. Sodium dodecyl sulfate-PAGE was used to measure proteolytic activity of milk. Inhibitor 6-amino-n-hexanoic acid was used to determine the relative proportion of plasmin and nonplasmin proteolytic activity of milk. Somatic cell count, total milk proteolytic activity, and nonplasmin proteolytic activity were higher in infected quarters than in quarters preinfection. After elimination of infections, SCC and nonplasmin proteolytic activity decreased to preinfection amounts. Total proteolytic activity of milk decreased after infections were cured but remained significantly higher than preinfection activity. This postinfection proteolytic activity in milk may be due to an increase in milk plasmin activity. Our data suggest that detrimental effects of mastitis on milk quality can continue after infection has been eliminated and milk SCC have returned to low values.

Preliminary investigation of the properties of somatic cell proteases.

Effect of the plasmin inhibitor 6-amino-n-hexanoic acid on somatic cell proteases (equivalent to 2.3 X 10(6) cells/ml) was determined using a model system of casein micelles dispersed in Jenness/Koops buffer (1.5% wt/vol). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to quantitate casein. There was no effect of 120 mM 6-amino-n-hexanoic acid on casein proteolysis by somatic cell proteases. Molecular weights of casein proteolysis products produced by somatic cell proteases were different from those produced by plasmin. Quantitative and qualitative analyses of pasteurized mastitic milk by SDS-PAGE indicated that a portion of the somatic cell proteolytic activity survived pasteurization. Because 6-amino-n-hexanoic acid does not inhibit somatic cell proteases, it can be used to establish the relative contribution of somatic cell proteases and plasmin to total proteolytic activity in mastitic milk.

Variability in true protein, casein, nonprotein nitrogen, and proteolysis in high and low somatic cell milks.

Monthly variation in milk protein (total nitrogen X 6.38) and milk fat was determined for 115 farms over 2 yr. Yearly average milk protein and fat tests in each year were 3.16 and 3.62%, respectively. The mean regression coefficient for milk protein with respect to milk fat was .47 for the entire period. Twenty-four farms were selected and grouped high or low based on their previous 2-yr somatic cell history. Monthly milk samples for each farm were tested for direct microscopic somatic cell count, total nitrogen, noncasein nitrogen, and nonprotein nitrogen. No differences in monthly nonprotein nitrogen, true protein, and casein were found between groups. Casein as a percent of total nitrogen was significantly higher for the low somatic group for seven of the 12 mo studied but was significantly higher for 9 mo when expressed as a percent of true protein. The average increase in tyrosine value for incubated preserved milk was significantly higher for the high somatic cell milk, indicating higher proteolytic activity in high somatic cell milk. Electrophoretic analysis of high and low somatic cell milk indicated that there was substantial proteolytic breakdown of alpha S-casein and beta-casein by proteases associated with elevated somatic cell counts.