Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Abstract

Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of L. plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L. plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.

Arginine metabolism in wineLactobacillus plantarum: in vitro activities of the enzymes arginine deiminase (ADI) and ornithine transcarbamilase (OTCase)

“…Lactobacillus plantarum strains Lp90, Lp65, Lp60, Lp61, Lp77 and Lp21 previously identified by Spano et al (2004Spano et al ( , 2006 isolated from red wine undergoing malolactic fermentation were used for enzymes analysis.…”

“…The ecological flexibility of L. plantarum is reflected by the observation that this species has one of the largest genomes known among lactic acid bacteria (LAB) (Kleerebezem et al, 2003;Molenaar et al, 2005). Although in wine L. plantarum is capable of malolactic fermentation, it usually contributes to production of undesirable substances such as biogenic amine and precursors of ethyl carbamate during and after winemaking and is therefore of general concern because of its spoilage nature (Lonvaud-Funel, 1999;Liu, 2002;Spano et al, 2004Spano et al, , 2006. Ethyl carbamate (or urethane), a well known animal carcinogen (Zimmerli and Schlatter, 1991) found in many fermented foods, including wine (Canas et al, 1994, Kodama et al, 1994, may be produced from precursors such as urea which is produced by yeasts, while citrulline and carbamyl phosphate are produced by LAB through the arginine deiminase (ADI) pathway (Liu and Pilone, 1998;Mira de Orduña et al, 2000Liu, 2002;Spano et al, 2002).…”

“…We previously reported the presence of genes (arcABC) coding for enzymes involved in the ADI pathway in wine L. plantarum (Spano et al, , 2006. The high identities among arginine deiminase (ADI), ornithine transcarbamylase (OTCase) and carbamate kinase (CK) protein sequences between Oenococcus oeni and L. plantarum and the induction of arcABC genes by arginine suggested that the putative genes cloned controlled arginine catabolism in L. plantarum.…”

Validation of an internal control gene to apply reverse transcription quantitative PCR to study heat, cold and ethanol stresses in Lactobacillus plantarum

“…Although these results confirm (especially for lag phase and the exponential phase) the RT-PCR analysis previously reported (Spano et al 2005) a difference was noted at stationary phase. Using the RT-PCR analysis a decrease, rather then an increase, in the hsp 18.55 transcript levels was noted at the beginning of the stationary phase (Spano et al 2006a). The explanation may be related to the sensitivity of the RT-qPCR analysis compared to the RT PCR analysis.…”

“…The most frequent strategy implies the determination of an internal control gene to normalize mRNA fractions for each study. In this study we presented the validation of a housekeeping gene suitable for the analysis of gene expression during abiotic stresses in Lactobacillus plantarum, a versatile bacterium that is found in a variety of ecological niches, ranging from vegetable and plant fermentations to the human gastrointestinal tract (Ercolini et al 2003;Aymerich et al 2003;Spano et al 2004a;Spano et al 2006a). Moreover, L. plantarum is extensively used as model microorganism among Lactobacillus spp.…”