Purification and Characterization of the Agglutinating Factor for Lactic Streptococci from Bovine Milk: IgM-Immunoglobulin

Abstract

An agglutinating factor for lactic streptococci of very high potency was isolated from bovine milk. Crude euglobulin was fractionated further by gel filtration on Bio Gel A-50m, followed by three filiations on A-15m. The agglutinating activity against Streptococcus cremoris strain HP increased from 1/7.3 per 100mg protein for skim milk to 1/520 per 100mg for the crude euglobulin to 1/2830 per 100mg after Bio Gel A-50m and 1/4100 per 100mg after Bio Gel A-15m. Agglutinating activity of the purified agglutinin differed among strains of Streptococcus cremoris. Activity was maximum in the pH range of 6.5 to 8.5; it increased linearly with concentration of purified agglutinin. Heating for 10min at 70C or reduction with mercaptoethanol caused a loss of most of the agglutinating activity. Purified agglutinin was homogeneous on immunoelectrophoresis against bovine antisera and antisera against bovine milk euglobulin. The rate of acid production in heated skim milk by lactic streptococci was retarded by added purified agglutinin. Physico-chemical analyses showed purified agglutinin was homogeneous by analytical centrifugation with: molecular weight = 960,000 daltons; 11.02% of total carbohydrate, 6.09% hexoses, 3.09% hexosamines, 1.51% sialic acid, and 0.36% fucose. These properties were close to those of IgM-immunoglobulin from bovine serum and colostrum and from other species. The IgM-immunoglobulin is the component in normal milk causing agglutination of lactic streptococci.