Integrated approach baased on 16S rDNA PCR-DGGE and sp60 qPCR assay for monitoring dynamics of a L. rhamnosus probiotic strain in table olive fermentation.

Recently, vegetarian fermented foods have reached an increasing attention as working base alternative to milk-based foods for the development of probiotic-type functional foods. Table olives have been demonstrated an excellent food carrier suitable for delivering probiotics to humans (Lavermicocca et al., 2005; Abriouel et al., 2011). Management of fermentation for developing probiotic-carrier table olives is critical since the use of a different starter strain, in addition to probiotic one, could hamper probiotic's multiplication and survival by depletion of nutrients in brine. The use of probiotic strains as starter to pilot the fermentation can overcome this drawback [3]. Alternatively, probiotic strains could be added during fermentation process. Here, we developed an integrated approach based on 16S rDNA PCR-DGGE profiling and a L. rhamnosus-specific qPCR assay to detect the dominant species in Sicilian table olives and to monitor the implantation of the L. rhamnosus probiotic H25 strain, inoculated both in presence and in absence of a indigenous L. plantarum starter. The amount of H25 strain was compared to that of LGG strain. Based on PCR-DGGE profiling, few microbial shifts throughout fermentation time were revealed in brine samples, while many different dominant bands among samples were achieved, indicating that both starter and probiotic inoculums could influence brine microbiota. In particular, DGGE results of brine samples revealed that samples without L. plantarum starter exhibited a high biodiversity with more pronounced dominant bands, including the dominance of the L. rhamnosus probiotic strain H25 until the end of fermentation. Brine samples inoculated with probiotic LGG strain showed the presence of few dominant bands and a weak presence of the probiotic strain up to the end of the process. DGGE results of olive samples revealed a marked microbial shifts during fermentation time and a higher biodiversity in samples without L. plantarum inoculum. Nevertheless, the presence of H25 and LGG probiotic strains was not detected. In order to species specifically quantify L. rhamnosus, a SYBR Green qPCR assay was designed with a primer set targeting chaperonin hsp60 gene sequence. qPCR 254 assay was effective in detecting the L. rhamnosus target DNA. L. rhamnosus genome copy number shifted from 6.13 (±0.04) to 6.40 (±0.01) log10/ml in brine sample with starter, after 60 days of inoculum with L. rhamnosus probiotic H25 and from 6.72 (±0.03) to 7.86 (±0.02) log10/ml in brine samples without L. plantarum starter. Similarly, LGG genome copy number was from 6.54 (±0.03) to 7.53 (±0.02) log10/ml after 60 days of inoculum in brine samples without starter. Evaluating olive samples, H25 L. rhamnosus genome copy number was registered at 2.91±0.04 and at 3.99±0.05 log10/g after 60 days of inoculum in both samples with and without L. plantarum starter, respectively. These values were slightly higher than those found for LGG (3.07±0.03 log10/g). In conclusion, the integrated approach based 16S rRNA gene PCR-DGGE and hsp60 qPCR, was effective in monitoring implantation of a L. rhamnosus probiotic strain on microbial population inhabiting olives and brines. Preliminary results also suggest that olives could support the survival of L. rhamnosus probiotic H25 strain.