A real-time PCR assay for detection and quantification of 2-branched (1,3)-β-D–glucan producing lactic acid bacteria in cider

Abstract

Ropiness in natural cider is a relatively frequent alteration, mainly found after bottling, leading to consumer rejection. It is derived from the production of exopolysaccharides (EPS) by some lactic acid bacteria most of which synthesize a 2-branched (1,3)-β-D-glucan and belong to the genera Pediococcus, Lactobacillus and Oenococcus. This polysaccharide synthesis is controlled by a single transmembrane glycosyltransferase (GTF). In this work, a method based on quantitative PCR (qPCR) and targeting the gtf gene was developed for detection and quantification of these bacteria in cider. The newly designed primers GTF3/GTF4 delimit a 151 bp fragment within the 417 bp amplicon previously designed for conventional PCR. The inclusivity and exclusivity of the qPCR assay were assessed with 33 cider isolates belonging to genus Lactobacillus, Oenoccocus and Pedioccocus, together with reference strains of 16 species and five genera including β-glucan,α-glucan and heteropolysaccharide (HePS) producing strains and non-EPS producers. The qPCR assay,followed by the melting curve analysis, confirmed the generation of a single PCR product from the β-glucan producers with a Tm of 74.28± 0.08 and CT values (10 ng DNA) ranging between 8.46 and 16.88 (average 12.67± 3.5). Some EPS− LAB strains rendered CT values ranging from 28.04 to 37.75 but they were significantly higher (P(CTb28.54)= 0.05) than those of the β-glucan producers. The assay showed a wide quantification range of 5 log units using calibrated cell suspensions of Pediococcus parvulus 2.6 and Oenococcus oeni I4. The linearity was extended over 7 log orders when calibration curves were obtained from DNA. The detection limit for β-glucan producing LAB in artificially contaminated cider was about 3× 102 CFU per ml. The newly developed qPCR assay was successfully applied to monitor the cidermaking process, in 13 tanks from two cider factories, revealing a decrease in CT values derived from an increase in β-glucan producing LAB populations. In addition, 8 naturally spoiled bottled cider were tested for the quantification of these organisms using the five standard curves constructed: P. parvulus 2.6 genomic DNA and gtf amplicon (417 bp), calibrated cell suspensions of Pediococcus parvulus 2.6, Lactobacillus diolivorans G77 and Oenococcus oeni I4 and results were compared to LAB total counts on MRS. Levels obtained from the different approaches were within a log range and showed no significant differences. Therefore, the amplicon-derived standard curve is proposed for the routine estimation of gtf+populations in cider.