Isolation of Hyaluronic Acid from Broth Cultures of Streptococci.

A readily available source of pure hyaluronic acid is a necessity in studies involving the ground substance of connective tissue and its relation to diverse spreading factors thought to be important in the pathogenesis of many bacterial diseases. Any objection that contamination with other mucopolysaccharides or protein of hyaluronic acid prepared from mammalian sources is generally likely, unless additional purification and separation procedures are resorted to, can be overcome by using certain bacterial species as a source of pure hyaluronic acid. Isolation of hyaluronic acid from group A hemolytic streptococci was first reported by Kendall, Heidelberger, and Dawson (l), who used acetate and ethanolic precipitation on their culture supernatants; between 60 and 140 mg of hyaluronic acid per liter of culture were obtained. Similar methods and results were reported by Seastone (2) and Topper and Lipton (3) with group A streptococcal strains in broth media. Roseman, Moses, Ludowieg, and Dorfman (4) obtained yields of purified hyaluronic acid from fluid cultures of group A streptococci, strain Alll, of from 200 to 300 mg from 4 liters of medium with an acetone-phosphomolybdic acid-acetone precipitation technique. Working with the same strain of microorganism, Cifonelli and Mayeda (5) described a simplified method of obtaining purified hyaluronic acid from streptococci and other biological sources by passing ethanol-precipitated crude mucopolysaccharide repeatedly through Darco G-60 cellulose pads. They obtained 300 to 400 mg of hyaluronic acid per liter of culture medium. Warren and Gray (6) developed a semisynthetic medium for the cultivation of group A streptococci in large batches. Their methods for rapid, reproducible isolation of partially purified hyaluronic acid from the fluid media filtrates of group A streptococci consisted of acetone, acetate, and ethanolic precipitation with yields of 250 mg per liter of culture medium. Our experiments concern the isolation and purification of hyaluronic acid from strains of group A hemolytic streptococci with 65% (NH,)2S04 concentration of the broth cultures for precipitation of mucopolysaccharide as suggested by Stock and Lynn (7). The method is relatively easy, reproducible, and gives a satisfactory amount of a highly purified hyaluronic acid.