Milk Quality and Production by Using Antimicrobial Milk-Derived Peptides and Proteins to Enhance the Immune System

Non-Technical Summary: The antimicrobial activity of the collectively molecules comprising milk reflects an evolutionary successful paradigm to preventing and limiting infection. In milk, the most abundant antimicrobial components are lactoferrin, lactoperoxidase, and lysozyme. Lactoferrin is an iron-binding antimicrobial polypeptide found in mucosal secretions (milk, tears, saliva) and within the granules of neutrophils. Plasma lactoferrin has been found to be elevated significantly in a variety of infections such as pneumonia, meningitis, and sepsis (Phadke et al., 2005). Antimicrobial peptides derived from milk proteins constitute a new field in the combined use of antimicrobial agents in foods, cosmetics and pharmaceuticals. The best examples of milk-derived peptides are those constituted by bovine lactoferricin and bovine ?s2-casein. To our knowledge, no synergism has been described among milk-derived peptides and lactoferrin in targeting bacterial infections i.e., sub-clinical mastitis in commercially important dairy farm animals. <P> Approach: In vitro Antimicrobial: Activity Antimicrobial activity will be determined using CryoTubes Vials (Fisher Scientific, USA). Single colonies of bacteria grown on tryptic soy agar (TSA) plates (E. coli, Staph. epidermidis) will be inoculated with 10 ml of tryptic soy broth (TSB) and grown overnight at 37 oC. A total of 300 ?l of bacterial suspension will be diluted 1/50 with TSB. Bacteria will be grown at 37 oC, and logarithmic phase organism will be harvested at a density of 1 to 4 x 108 cfu/ml. The culture will be then centrifuged at 2000 x g for 10 min. Bacteria will be washed twice with 10 mM Na-phosphate buffer, pH 7.4, and adjusted to 105 cfu/ml approximately. A total of 50 ?l of the bacterial suspension will be mixed with 50 ?l of the antimicrobial sample to be investigated together with 100 ?l of 2% TSB in 10 mM phosphate buffer, pH 7.4 and with 800 ?l of 10 mM phosphate buffer, pH 7.4. The mixture will be incubated at 37 oC with agitation and then plated on TSA plates. The plates will be incubated at 37 oC for 24 h before the colonies are counted. The assays will be conducted in triplicate. The antimicrobial activity will be expressed as the concentration of antimicrobial agent that gives a log (No/Nf) value between 0.25 and 0.5. In vivo Antimicrobial Activity: Goats Ten (n = 10) French-Alpine dairy goats that are in their first lactation, clinically healthy, and free of mastitis will be selected. Before the study begins, the mammary glands of the dairy goats will be examined clinically, and milk samples will be examined bacteriologically. The somatic cell content (SCC) will be determined. Only those goats with a SCC lower than 2 x 105/ml that are negative for major mastitis pathogens, such as gram-positive and gram-negative bacteria will be selected for this study. During the experiment, the goats will be housed in groups of five animals. Goats will be fed a daily ration of approximately 4 kg of concentrate and have ad libitum access to water and hay. Goats will be fed twice daily at 9:00 AM and 4:00 PM. Goats will be milked twice daily at 8:00 AM and 5:00 PM with a device that measures individual mammary gland milk production. Mean milk production before the first treatment with the antimicrobial sample or the excipient (d -7) will be recorded (baseline). It should be noted here that the experimental work in goats will be repeated as needed. Experimentally induced Staph. dermiditis and/or E. coli mastitis in goats during transition from a lactation period to a dry period will be also studied.