Isolation of Tyrosine Decarboxylaseless Mutants of a Bacteriocin-Producing Enterococcus faecalis Strain and Their Application in Cheese

Abstract

Enterococcus faecalis INIA 4, a bacteriocin-producing strain, was exposed to the mutagenic agent ethyl methanesulfonate (EMS). A modified differential plating medium permitted the isolation of mutants INIA 4-01 to 4-09 with reduced tyrosine decarboxylase activity. All mutant strains displayed rapid growth in milk, while bacteriocin production ranged from 15 to 125% as compared to the parental strain.Enterococcus faecalis INIA 4, INIA 4-07 (less than 0.1% residual tyrosine decarboxylase activity) and INIA 4-03 (10% residual activity) were used in cheese-making experiments. In cheese made from pasteurized milk, using INIA 4-07 as adjunct starter, tyramine was not produced (< 5 mg/kg of cheese after 4 months of ripening). Suppression of Listeria innocua in raw-milk cheeses made using INIA 4-03 and INIA 4-07 was equal to that observed in the cheese made using the parental strain INIA 4. After 2 months of ripening, no free tyrosine and 195 mg/kg of tyramine were detected in the cheese with INIA 4. The tyrosine content of the cheeses made with INIA 4-03 and INIA 4-07 and of the control cheese was approximately 95 mg/kg. The cheeses with INIA 4-03 and 4-07 contained 116 and 120 mg/kg of tyramine respectively, values comparable to the level of 101 mg/kg detected in a control raw-milk cheese to which no enterococci were added. The relatively high tyramine formation in the cheeses made with INIA 4-03 and INIA 4-07 and in the control cheese was probably caused by the decarboxylating activity of the indigenous flora of the raw milk.