The pathway of formation of acetate from pyruvate in the homofermentative organism L. bulgaricus was studied. Three pathways for the formation of acetate were investigated. These were the formation of acetyl CoA by the pyruvate dehydrogenase (lipoate) system, the formation of acetaldehyde by pyruvate decarboxylase, and the formation of acetyl phosphate by pyruvate oxidase. The first two pathways were eliminated when it was found that the formation of acetate was not inhibited by arsenite and that acetaldehyde was not converted to acetate by L. bulgaricus. The formation of acetyl phosphate and acetate by dialysed cell-free extracts indicated the presence of pyruvate oxidase in L. bulgaricus. The pyruvate oxidase system, unlike the pyruvate dehydrogenase (lipoate) system, was not inhibited by unsaturated fatty acids. The organism was shown to possess both acetate kinase and phosphate acetyltransferase which suggested that acetyl phosphate could be converted to acetate or acetyl CoA.

Frozen concentrated cheese starters were prepared from 7 Streptococcus cremoris and 2 Str. lactis strains by continuous culture. pH control with NH 4 0H and Ca(OH) 2 increased the yield of Str. cremoris strains as compared with NaOH, but the resultant cells were smaller and less active. Maximum yields for both species were obtained at a dilution rate of 0-6 and pH 6-3, but with Str. cremoris cell activity was reduced above pH 60.Degeneration in continuous culture was observed in the 2 Str. lactis strains, due apparently in one case to the action of a bacteriophage and in the other possibly to selection or to loss of adaptive enzymes.Concentrated starters retained full viability and activity when stored for 10-12 weeks at -110 °C, and 94% activity after storage for 4 weeks at -40 °C.Cheese made with the concentrated starters ripened normally and was comparable in quality with control cheese made with normal bulk starter.Cheese-making in Australia is mostly confined to the Cheddar variety and the starter cultures used are single strains of the mesophilic lactic streptococci.Two strains of Streptococcus lactis and 10 strains of Str. cremoris are currently used in the manufacture of Cheddar cheese in Australia. These strains have been selected because there is no evidence that they are phage-related. A daily rotation of such strains is advised to help control phage levels in the factory. The commercial development of frozen concentrated starters in Australia would therefore need to be based on the supply of a number of strains to allow for at least a 4-day rotation (Czulak, 1966) and some degree of preference by individual factories.Concentrated starters prepared from Str. lactis grown in continuous culture in a trypsin-digested whey medium gave promising results, but with Str. cremoris poor yields were obtained (Lloyd & Pont, 1970). As several Str. cremoris strains would be required for a 4-day starter rotation, we have attempted to improve their yields in continuous culture.Using a trypsin-digested milk medium, some of the more important parameters affecting the growth of Str. cremoris and Str. lactis in continuous culture were studied, particularly the yield and biological activity and storage stability of frozen concentrates. The results are presented in this paper .

Equipment and methods are described for the production, on the laboratory scale, of frozen concentrated cheese starters. A single-stage Porton-type fermenter with a working volume of 3-5 1 was used for the continuous culture of the Streptococcus lactis and Str. cremoris starter strains. The cells grown in a trypsindigested cheese-whey or trypsin-digested skim-milk, both containing autolysed yeast, were harvested with a Sharpies laboratory super-centrifuge, resuspended in skim-milk and layer-frozen in liquid N 2 . The frozen culture was crushed to a granular freeflowing form facilitating direct addition to and ready dispersion in cheese milk. The cultures were stored at -196 °C.