Competitive Growth of Genetically Marked Malolactic-Deficient Lactobacillus plantarum in Cucumber Fermentations

Procedures were developed for the differential enumeration of an added strain of Lactobacillus plantarum and indigenous lactic acid bacteria (LAB) during the fermentation of brined cucumbers. The added strain was an N,N-nitrosoguanidine-generated mutant that lacked the ability to produce CO₂ from malic acid (MDC^-). The MDC^- phenotype is desirable because CO₂ production from malic acid decarboxylation has been shown to contribute to bloater formation in fermented cucumbers. A basal medium containing malic acid and adjusted to pH 4.0 permitted growth of indigenous LAB (predominantly MDC⁺), but not growth of the added MDC^- culture. Transformation of the MDC^- culture by electroporation with cloning vector pGK12 conferred chloramphenicol resistance, which permitted selective enumeration of this culture. The reversion frequency of the MDC^- mutation was determined by a fluctuation test to be less than 10^-10. The level of retention of plasmid pGK12 was greater than 90% after 10 generations in cucumber juice medium at 32°C. With the procedures developed, we were able to establish the ratio of MDC^- to MDC⁺ LAB that results in malic acid retention in fermentations of filter-sterilized cucumber juice and unsterilized whole cucumbers under specified conditions.