Concentrations and anti-Haemophilus influenzae activities of beta-casein phosphoforms in human milk

Background: The distribution and concentrations of six phosphorylated forms of human beta-casein, a major source of nutrition among breast-fed infants , have not been examined in milt samples without prior fractionation. In th is study, the levels of beta-casein phosphoforms in untreated human milk sa mples were analyzed and their antiadhesion activities determined against Ha emophilus influenzae, a pathogen implicated in middle ear infection in infa nts. Methods: Human milk samples were analyzed using urea-polyacrylamide gel ele ctrophoresis of whole-milk samples and scanning densitometry to determine t he concentrations of beta-casein and its phosphoforms. A nontypable H. infl uenzae strain was radiolabeled to monitor its attachment to human pharyngea l cells in microtiter plates. Purified phosphoforms of beta-casein were pre incubated for 15 minutes with radiolabeled bacteria to determine their anti adhesion activities. Results: The average beta-casein concentration in 151 human milk samples wa s 5.37 +/- 2.26 mg/ml. On average, the phosphoforms in untreated milk are p resent in the following order ranked by concentration: tetra- > di- > non- > mono- > tri- > pentaphosphorylated beta-casein. The tri-, tetra-, and pen taphosphorylated forms of human beta-casein exhibited more than 60% inhibit ion of H. influenzae in the antiadhesion assay when used at a concentration of 0.6 to 0.9 mg/ml. Conclusion: The beta-casein level in untreated human milk is significantly higher than previously reported. The phosphoform distribution of beta-casei n in individual donors varies widely. Anti-H. influenzae activity was detec ted in vitro among human beta-casein molecules with three or more phosphate groups.