Fate of lysostaphin in milk from individual cows through pasteurization and cheesemaking¹

Flowchart of the processing of the milk from the 4 shipments: a) 1L for heat treatment and b) 50L for UHT heat treatment and cheesemilk.

Spot-on-lawn assay showed similar activity of lysostaphin in 1) raw skim milk, and skim milk pasteurized at 2) 63°C for 30min, and 3) 72°C for 15s; 2-fold serial dilution of samples (top to bottom).

Spot-on-lawn assay showed the changes in recombinant lysostaphin acitivity in different fractions collected durning cheesemaking; 2-fold serial dilution (top to bottom). Fractions: 1) raw milk, 2) raw skim milk, 3) raw cream, 4) raw milk standardized to 3% fat, 5) pasteurized milk standardized to 3% fat, 6) ripened cheese milk, 30min after the addition of the starter culture, 7) milk gel, 30min after the addition of chymosin, 8) whey drained from the cheese vat, 9) fresh curd after draining whey, 10) whey from overnight pressing, 11) cheese curd after overnight pressing, and 12) cheese after 30 d of storage at 4°C; solid samples were homogenized 1:10 before serial dilution.

Minimum inhibitory concentration assay showed lysostaphin activity in milk and cheese made with 3% milk from transgenic cows; 2-fold serial dilution (left to right). Milk from the transgenic cow secreting 8.3μg of lysostaphin/mL of milk [row 5) raw whole milk, and 6) pasteurized 3% fat] was made into cheese and aged at 4°C for 1) 0-d, 2) 30-d, 3) 60-d, and 4) 90-d. Milk from the transgenic cow secreting 1.7μg of lysostaphin/mL milk [row 7) raw whole milk, and 8) raw 3% fat milk] is shown for comparison.

Spot-on-lawn assay showed the decrease in activity of bacterial lysostaphin after incubation with chymosin in 10mM phosphate buffer, pH 6.0, 90min at 37°C. Lysostaphin concentration decreased from left to right [1) 50, 2) 25, and 3) 12.5μg of lysostaphin/ mL], and chymosin concentration increased from top to bottom [a) 0, b) 7.8, c) 15.5, d) 31μg of chymosin/mL].

Protein profiles of different fractions from a) 3% fat milk from nontransgenic cow 310_T; b) 3% fat milk from transgenic cow 312_C; and c) cheeses made from 3% milk without (lanes 8 to 11) and with lysostaphin (lanes 12 to 15). Lanes: 1) raw milk, 2) HTST pasteurized milk, 3) cheesemilk incubated with starter cultures, 4) gelled milk gel after the addition of rennet, 5) whey, 6) fresh curd, and 7, 8, and 12) cheese after 1 d of manufacture, 9 and 13) cheese after 30d of aging, 10 and 14) cheese after 60 d of aging, and 11 and 15) cheese after 90 d of aging. Protein bands are identified as α_s2-CN, α_s1-CN, β-CN, κ-CN, β-LG, and α-LA.