Bovine Lf was purified from cheese whey or concentrated cheese whey by the expanded bed absorption chromatography method . Iron content of native Lf was approximately 8%–15% . Lf was stored frozen at -20°C and sterile-filtered (32 mm Acrodisc PT Syringe Filters 0.8/0.2 μm, Gelman Laboratory REF: 4658) before use. The final concentration of Lf in the product was 35.5 mg/ml . Purity of Lf was tested in SDS-page (sodium dodecyl sulphate polyacrylamide gel electrophoresis) . Apo-Lf was prepared from Lf by citrate dialyzing , and its iron content was approximately 4%.
Two growth media were used for bacterial cultures: the commercial Iso Sensitest-Broth (ISB CM473, Oxoid Ltd., Basingstoke, Hampshire, England), and whey. Whey was prepared from 3 liters of fresh raw milk obtained from the university dairy herd by high-speed centrifugation of defatted milk (32600 g for 60 min at 4°C). Aliquots of 40-ml whey were sterile-filtered and frozen immediately after preparation for later use.
Bacterial isolates and the preparation of inoculums
Five isolates of E. coli, S. aureus, and coagulase negative-staphylococci (CNS), 2 isolates of P. aeruginosa and 2 isolates of K. pneumoniae originally isolated from subclinical or clinical cases of bovine mastitis were used. These isolates were received from the mastitis laboratory of the Faculty of Veterinary Medicine and the National Veterinary and Food Research Institute, Helsinki. One of the S. aureus isolates was the reference isolate M60 kindly provided by Dr. A. J. Guidry (Immunology and Disease Resistance Laboratory USDA, Beltsville, USA). During the experiment, bacteria were maintained on blood agar plates at 8°C. To adapt the bacterial isolates to grow in whey, they were grown overnight at 37°C in a growth medium consisting of 2/3 Iso Sensitest-Broth and 1/3 sterile whey. The cultures were tested using Gram-staining for purity. Bacteria were harvested by centrifugation (5000 g for 10 min) and washed twice between centrifugations using sterile saline (0.9% NaCl, 20°C). A suspension containing approximately 109colony-forming units (CFU) in 0.9% NaCl was prepared according to the McFarland standard (bioMérieux sa, 69280 Marcy I'Etoile, France) by spectrophotometry (550 nm, Hitatchi U-2000, Hitachi, Ltd., Tokyo, Japan). Bacterial suspension was diluted to the final concentration of 1.5 × 103CFU/ml used in each well.
Analysis of bacterial growth by turbidometry
Bacterial growth was measured using turbidometry (Bioscreen instrument, Labsystems, Helsinki, Finland). The instrument is a fully automated analyzing system for measuring bacterial growth using the vertical light bath with wide band absorption principle; 200 individual samples can be run simultaneously. Each well contained 100 μl of ISB broth or 150 μl of whey as the growth medium, 50 μl of bacterial suspension, and 50 μl of Lf concentrate. Physiological saline was added to bring the final volume to 300 μl: 50 μl and 100 μl in ISB and whey wells, respectively. Tested amounts of Lf were 200 μg (the final Lf concentration in the well was 0.67 mg/ml), 500 μg (1.67 mg/ml), and 800 μg (2.67 mg/ml). Control wells contained all components except Lf, which was replaced by 50 μl of 0.9% saline. Five parallel wells were used. Wells between whey and ISB wells were filled with 0.9% NaCl to prevent cross-contamination. The wells on 2 100-well plates were covered and preincubated in the Bioscreen instrument for 30 min at 37°C. The change in turbidity was monitored automatically every hour for 20 h at 37°C. The plates were shaken 10 min before each measurement. Two E. coli and 2 S. aureus isolates were also tested with Apo-Lf. The lag time (time from beginning of incubation until the time-point when absorbance began to increase), slope (slope of the growth curve in logarithmic growth phase), and maximum absorbance (highest absorbance value measured during the 20-h period) were used as variables describing the bacterial growth. After the 20-h incubation period, bacterial survival and bactericidal effect of Lf in the wells were confirmed by culturing aliquots of 10 ml on blood agar plates and incubating the plates overnight at 37°C.
The effect of different Lf concentrations on lag time, slope, and maximum absorbance was tested by repeated measures analysis of variance with concentration as a within factor. The significance of concentration was evaluated by Greenhouse-Geisser adjusted p-values. Concentrations of 0.67, 1.67, and 2.67 mg/ml of Lf were further compared with a negative control.