title: Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR.
abstract: Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Martha Lissete Morales
authors.initials: ML
authors.lastname: Villarreal
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Marina
authors.initials: M
authors.lastname: Padilha
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Antonio Diogo Silva
authors.initials: AD
authors.lastname: Vieira
authors.affiliation: Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Bernadette Dora Gombossy de Melo
authors.initials: BD
authors.lastname: Franco
authors.affiliation: Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Rafael Chacon Ruiz
authors.initials: RC
authors.lastname: Martinez
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Susana Marta Isay
authors.initials: SM
authors.lastname: Saad
mesh_terms: D001386:Azides; D058491:Bacterial Load; D002611:Cheese / Q000382:microbiology*; D005516:Food Microbiology; D041981:Gastrointestinal Tract / Q000382:microbiology*; D050296:Microbial Viability; D019936:Probiotics / Q000302:isolation & purification*; D011419:Propidium / Q000031:analogs & derivatives; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium