One of the characteristics of Roquefort cheese is the presence of irregularly
shaped openings. Although many factors affect the development of opening in blue-veined
cheeses (Martley & Crow, 1996), the limiting step in the case of Roquefort
cheese is the production of CO2 by lactic acid bacteria (J.-P. Reverbel, pers. comm.).
Most of the opening occurs after moulding; the process is difficult to control and
many manufacturing runs result in cheeses with an insufficient opening. Concentrated
suspensions of Leuconostoc strains are used to increase the production of CO2
(Devoyod & Muller, 1969), but it would clearly be useful to have microorganisms that
produce larger quantities of the gas. McKay & Baldwin (1974) isolated a spontaneous
mutant of Lactococcus lactis that produced more acetoin and CO2 than the parent
strain. This mutant was lactate dehydrogenase (LDH)-deficient, which favoured the
conversion of pyruvate into end products other than lactate. Following nitrosoguanidine
mutagenesis, we isolated three Lc. lactis mutants whose LDH activities
were reduced to varying extents and which produced varying amounts of CO2
(Boumerdassi et al. 1997). Subsequent work showed that these mutants were
unstable on successive subculture in milk or synthetic broth (El Attar et al. 2000).
The aim of our current work was to select a large number of Lc. lactis mutants
producing excess CO2. This would increase the probability of selecting stable
mutants and also provide a collection of strains with differing gas production
activities. Currently available screening methods are, however, unsuitable for
processing large numbers of mutants, which is why we have developed an improved
screening method.