Use of the gfp gene in monitoring bacteriocin-producing Lactobacillus plantarum N014, a potential starter culture in nham fermentation

Lactobacillus plantarum N014 is a bacteriocin-producing lactic acid bacteria originally isolated from nham, a traditional Thai fermented sausage, and in the process of development to be used as a starter culture for nham fermentation. During the fermentation process, there is a need to identify the starter culture among several naturally occurring bacteria. In this study, a new plasmid carrying the gfp (green fluorescent protein) gene was constructed based on pGKV210, an Escherichia coli/Lactococcus shuttle vector containing an erythromycin resistance marker. The gfp gene derived from pGFPuv was placed under the control of an L-lactate dehydrogenase promoter and then inserted at the EcoRI site of pGKV210, leading to pN014-GFP. The novel plasmid was used to transform L. plantarum N014, which is a bacteriocin-producing lactic acid bacteria isolated from nham. The resulting transformant, L. plantarum N014-GFP(+), was brightly fluorescent and harbored the expected plasmid. A plasmid stability test revealed that pNO14-GFP was stable after 100 generations of growth under nonselective pressure. L. plantarum N014-GFP(+) and its parent strain were shown to be very similar in growth rate, bacteriocin production, and lactate production. L. plantarum NO14-GFP(+) was able to survive in a nham model. The survival clones were still fluorescent and harbored pNO14-GFP.