Concentrations and Anti-Haemophilus influenzae Activities of β-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 milk samples without prior fractionation. In this study, the levels of beta-casein phosphoforms in untreated human milk samples were analyzed and their antiadhesion activities determined against Haemophilus influenzae, a pathogen implicated in middle ear infection in infants. METHODS: Human milk samples were analyzed using urea-polyacrylamide gel electrophoresis of whole-milk samples and scanning densitometry to determine the concentrations of beta-casein and its phosphoforms. A nontypable H. influenzae strain was radiolabeled to monitor its attachment to human pharyngeal cells in microtiter plates. Purified phosphoforms of beta-casein were preincubated for 15 minutes with radiolabeled bacteria to determine their antiadhesion activities. RESULTS: The average beta-casein concentration in 151 human milk samples was 5.37+/-2.26 mg/ml. On average, the phosphoforms in untreated milk are present in the following order ranked by concentration: tetra- > di- > non- > mono- > tri- > pentaphosphorylated beta-casein. The tri-, tetra-, and pentaphosphorylated forms of human beta-casein exhibited more than 60% inhibition 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-casein in individual donors varies widely. Anti-H. influenzae activity was detected in vitro among human beta-casein molecules with three or more phosphate groups.