Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR.
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 bacte...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3866109?pdf=render |
| _version_ | 1818487130359332864 |
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| author | Martha Lissete Morales Villarreal Marina Padilha Antonio Diogo Silva Vieira Bernadette Dora Gombossy de Melo Franco Rafael Chacon Ruiz Martinez Susana Marta Isay Saad |
| author_facet | Martha Lissete Morales Villarreal Marina Padilha Antonio Diogo Silva Vieira Bernadette Dora Gombossy de Melo Franco Rafael Chacon Ruiz Martinez Susana Marta Isay Saad |
| author_sort | Martha Lissete Morales Villarreal |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-10T16:33:14Z |
| format | Article |
| id | doaj.art-c5689e8c1e75463296f1013ae6b197d9 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T16:33:14Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-c5689e8c1e75463296f1013ae6b197d92022-12-22T01:41:30ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01812e8210210.1371/journal.pone.0082102Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR.Martha Lissete Morales VillarrealMarina PadilhaAntonio Diogo Silva VieiraBernadette Dora Gombossy de Melo FrancoRafael Chacon Ruiz MartinezSusana Marta Isay SaadSpecies-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.http://europepmc.org/articles/PMC3866109?pdf=render |
| spellingShingle | Martha Lissete Morales Villarreal Marina Padilha Antonio Diogo Silva Vieira Bernadette Dora Gombossy de Melo Franco Rafael Chacon Ruiz Martinez Susana Marta Isay Saad Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. PLoS ONE |
| title | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. |
| title_full | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. |
| title_fullStr | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. |
| title_full_unstemmed | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. |
| title_short | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR. |
| title_sort | advantageous direct quantification of viable closely related probiotics in petit suisse cheeses under in vitro gastrointestinal conditions by propidium monoazide qpcr |
| url | http://europepmc.org/articles/PMC3866109?pdf=render |
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