Development of a milk-based medium for the selection of urease-defective mutants of Streptococcus thermophilus

Streptococcus thermophilus strains are used in fermented dairy products for their capacity to metabolize lactose into lactic acid. The rate of lactic acid production in milk is of major economic importance, as rapid acidification prevents growth of undesirable microorganisms. It is also of paramount significance for aroma, texture and flavor of the end product. Besides achieving customer satisfaction, improvement of production rate and operational costs incite industrials into selecting fast acidifying strains. Another important trait of S. thermophilus influencing acidification is the urease, which catabolizes urea into ammonia and has a detrimental effect on acidification. Unfortunately, most of the S. thermophilus strains possess the urease, and the urease-negative ones are necessary for industrial applications. Urease activity is a widely distributed activity in S. thermophilus species, and urease-negative strains are rare. The later are however interesting from an industrial point of view, as they may give faster acidification in dairy applications, because lactic acid is not buffered by urea-derived ammonia. Nowadays, the efforts to improve the characteristics of strains for industrial applications are based on natural strategies such as random mutagenesis. This implies the need of a screening method that is efficient in terms of time and success. In this context, the aim of this study was the development of a new medium that allows selection of urease-defective mutants based on S. thermophilus colony morphology. Discrimination capacity of the new medium was verified using previously characterized urease-negative recombinant strains. The new milk-based medium, applied to industrial S. thermophilus strains subjected to UV mutagenesis, allowed the selection of 3 mutants, partially or completely defective in urease activity. Genetic characterization of urease-defective mutants highlighted the presence of nonsense or missense mutations in the ureA, ureC and ureG genes, thus supporting their phenotype. Evaluation of milk acidification revealed increased performance for one out of three urease-defective mutants compared to wild-type strains.