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

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)-beta-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 151bp fragment within the 417bp 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 beta-glucan, alpha-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 beta-glucan producers with a T(m) of 74.28+/-0.08 and C(T) values (10ng DNA) ranging between 8.46 and 16.88 (average 12.67+/-3.5). Some EPS(-) LAB strains rendered C(T) values ranging from 28.04 to 37.75 but they were significantly higher (P(C(T)<28.54)=0.05) than those of the beta-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 beta-glucan producing LAB in artificially contaminated cider was about 3x10(2)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 C(T) values derived from an increase in beta-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 (417bp), 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.

Exopolysaccharide production by Pediococcus damnosus 2.6 in a semidefined medium under different growth conditions.

Ropy Pediococcus damnosus (strain 2.6) was used for production of exopolysaccharide (EPS) in a semidefined medium. From a kinetic point of view, an experiment conducted in SMD medium containing 30 g l(-1) glucose and 5 g l(-1) Bacto casamino acids (Difco Laboratories, Detroit, MI), without pH control, showed that EPS production took place mainly during the growth phase. The viscosity of the cultures developed in parallel to the EPS synthesis until 94 h of incubation; after 200 h of fermentation, viscosity decreased. The effect of glucose, Bacto casamino acid concentrations and temperature on growth and EPS production was determined by using a full factorial design. Within the domain of experimental conditions considered, the concentration of glucose and Bacto casamino acids has a significant effect on the production of exopolysaccharide. The incubation at 12 degrees C produced a prolonged lag phase and due to the lower growth yield, higher specific EPS production was found at this temperature. At 25 degrees C the EPS production was mainly enhanced by the increase in glucose concentration. The increase in nitrogen concentration from 5 to 15 g l(-1) did not yield greater EPS production. However, at 12 degrees C optimal EPS production was obtained when both higher glucose and nitrogen concentrations were used.