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Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR.
PLoS One. 2013; 8(12):e82102.Plos

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.

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Authors+Show Affiliations

Villarreal ML
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Padilha M
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Vieira AD
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Franco BD
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Martinez RC
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Saad SM
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil.

MeSH

AzidesBacterial LoadCheeseFood MicrobiologyGastrointestinal TractMicrobial ViabilityProbioticsPropidiumReal-Time Polymerase Chain Reaction

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24358142

Citation

Villarreal, Martha Lissete Morales, et al. "Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-suisse Cheeses Under in Vitro Gastrointestinal Conditions By Propidium Monoazide--qPCR." PloS One, vol. 8, no. 12, 2013, pp. e82102.
Villarreal ML, Padilha M, Vieira AD, et al. Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. PLoS One. 2013;8(12):e82102.
Villarreal, M. L., Padilha, M., Vieira, A. D., Franco, B. D., Martinez, R. C., & Saad, S. M. (2013). Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. PloS One, 8(12), e82102. https://doi.org/10.1371/journal.pone.0082102
Villarreal ML, et al. Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-suisse Cheeses Under in Vitro Gastrointestinal Conditions By Propidium Monoazide--qPCR. PLoS One. 2013;8(12):e82102. PubMed PMID: 24358142.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. AU - Villarreal,Martha Lissete Morales, AU - Padilha,Marina, AU - Vieira,Antonio Diogo Silva, AU - Franco,Bernadette Dora Gombossy de Melo, AU - Martinez,Rafael Chacon Ruiz, AU - Saad,Susana Marta Isay, Y1 - 2013/12/17/ PY - 2013/09/06/received PY - 2013/10/30/accepted PY - 2013/12/21/entrez PY - 2013/12/21/pubmed PY - 2014/10/1/medline SP - e82102 EP - e82102 JF - PloS one JO - PLoS One VL - 8 IS - 12 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/24358142/Advantageous_direct_quantification_of_viable_closely_related_probiotics_in_petit_suisse_cheeses_under_in_vitro_gastrointestinal_conditions_by_Propidium_Monoazide__qPCR_ DB - PRIME DP - Unbound Medicine ER -