Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Author(s)

Morales Villarreal, Martha Lissete ^[1] ; Padilha, Marina ^[1] ; Silva Vieira, Antonio Diogo ^[1] ; Gombossy de Melo Franco, Bernadette Dora ^[2] ; Ruiz Martinez, Rafael Chacon ^[2] ; Isay Saad, Susana Marta ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Pharmaceut Sci, Dept Biochem & Pharmaceut Technol, Sao Paulo - Brazil

^[2] Univ Sao Paulo, Fac Pharmaceut Sci, Dept Food & Expt Nutr, Sao Paulo - Brazil

• Document type: Journal article
• Source: PLoS One; v. 8, n. 12 DEC 17 2013.
• Web of Science Citations: 20

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. (AU)

• FAPESP's process: 12/13535-7 - Impact of the addition of inulin and fructooligosaccharides on Bifidobacterium animalis and Lactobacillus acidophilus survival in a dairy product before and after in vitro simulated gastrointestinal stress
• Grantee: Susana Marta Isay Saad
• Support type: Regular Research Grants
• FAPESP's process: 11/05203-1 - Probiotic and symbiotic petit-suisse: technological features and use of culture-independent methods for the evaluation of probiotic survival in the product and in vitro essays
• Grantee: Marina Padilha
• Support type: Scholarships in Brazil - Master