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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| 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 |
| doi_str_mv | 10.1371/journal.pone.0082102 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0082102</identifier><identifier>PMID: 24358142</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Azides ; Bacteria ; Bacterial Load ; Bacteriocins ; Bifidobacterium animalis ; Cheese - microbiology ; Colonies ; Dairy products ; Electron microscopy ; Enumeration ; Flow cytometry ; Food ; Food Microbiology ; Gastrointestinal system ; Gastrointestinal tract ; Gastrointestinal Tract - microbiology ; In vitro methods and tests ; Inulin ; Lactobacillus ; Lactobacillus acidophilus ; Listeria ; Listeria monocytogenes ; Methods ; Microbial Viability ; Microbiota ; Microorganisms ; Nutrition ; Pharmaceutical sciences ; Population levels ; Populations ; Prebiotics ; Probiotics ; Probiotics - isolation & purification ; Propidium - analogs & derivatives ; Real-Time Polymerase Chain Reaction ; Scanning electron microscopy ; Shelf life ; Species ; Strains (organisms) ; Survival ; Viability ; Yogurt</subject><ispartof>PloS one, 2013-12, Vol.8 (12), p.e82102</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Villarreal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Villarreal et al 2013 Villarreal et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-97c33898852953ad7e8f5e44a18742f7ac42acc611a17cdce9635b69eb3603083</citedby><cites>FETCH-LOGICAL-c692t-97c33898852953ad7e8f5e44a18742f7ac42acc611a17cdce9635b69eb3603083</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866109/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866109/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793,79600,79601</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24358142$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Zhou, Dongsheng</contributor><creatorcontrib>Villarreal, Martha Lissete Morales</creatorcontrib><creatorcontrib>Padilha, Marina</creatorcontrib><creatorcontrib>Vieira, Antonio Diogo Silva</creatorcontrib><creatorcontrib>Franco, Bernadette Dora Gombossy de Melo</creatorcontrib><creatorcontrib>Martinez, Rafael Chacon Ruiz</creatorcontrib><creatorcontrib>Saad, Susana Marta Isay</creatorcontrib><title>Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR</title><title>PloS one</title><addtitle>PLoS One</addtitle><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.</description><subject>Azides</subject><subject>Bacteria</subject><subject>Bacterial Load</subject><subject>Bacteriocins</subject><subject>Bifidobacterium animalis</subject><subject>Cheese - microbiology</subject><subject>Colonies</subject><subject>Dairy products</subject><subject>Electron microscopy</subject><subject>Enumeration</subject><subject>Flow cytometry</subject><subject>Food</subject><subject>Food Microbiology</subject><subject>Gastrointestinal system</subject><subject>Gastrointestinal tract</subject><subject>Gastrointestinal Tract - microbiology</subject><subject>In vitro methods and tests</subject><subject>Inulin</subject><subject>Lactobacillus</subject><subject>Lactobacillus acidophilus</subject><subject>Listeria</subject><subject>Listeria monocytogenes</subject><subject>Methods</subject><subject>Microbial Viability</subject><subject>Microbiota</subject><subject>Microorganisms</subject><subject>Nutrition</subject><subject>Pharmaceutical sciences</subject><subject>Population levels</subject><subject>Populations</subject><subject>Prebiotics</subject><subject>Probiotics</subject><subject>Probiotics - isolation & purification</subject><subject>Propidium - analogs & derivatives</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Scanning electron microscopy</subject><subject>Shelf life</subject><subject>Species</subject><subject>Strains (organisms)</subject><subject>Survival</subject><subject>Viability</subject><subject>Yogurt</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk11r2zAUhs3YWLtu_2BsgsFgF84sy5blm0II-wh0tHQft0KWjxMFR3IkOSz7Q_ubkxu3xLDB8IXM0XNevX7lE0UvcTLDpMDvN6a3WrSzzmiYJQlLcZI-is5xSdKYpgl5fPJ-Fj1zbpMkOWGUPo3O0ozkDGfpefR7Xu-F9mIFpneoVhakR7s-lFSjpPDKaGQatFeiagHJ1jhoD8hCKzzUqLOmUsYr6ZDSqAOvfOx65VxA1wAOHOp1DXbY3StvDVoJFxalPTivgn0kja7VcIxD1QHdWNOpWvVb9MVoI36pGuJ4d7O4fR49aUTr4MW4XkTfP374tvgcX11_Wi7mV7GkZerjspCEsJKxPC1zIuoCWJNDlgnMiixtCiGzVEhJMRa4kLWEkpK8oiVUhCYkYeQien3U7cKn8jFjx3FGGWOUUByI5ZGojdjwzqqtsAduhOJ3BWNXXNgQSQtc5pSVwCSGIs8oSUva4AQTAbTCZVGXQetyPK2vthDsaG9FOxGd7mi15iuz58M94mQQeDMKWLPrQ6b_sDxSKxFcKd2YICa3ykk-zwqWZsEZCdTsL1R4atiqcE3QqFCfNLybNATGw0-_Er1zfPn19v_Z6x9T9u0JuwbR-rUzbX_3l0zB7AhKa5yz0DwkhxM-DMl9GnwYEj4OSWh7dZr6Q9P9VJA_UNYQUQ</recordid><startdate>20131217</startdate><enddate>20131217</enddate><creator>Villarreal, Martha Lissete Morales</creator><creator>Padilha, Marina</creator><creator>Vieira, Antonio Diogo Silva</creator><creator>Franco, Bernadette Dora Gombossy de Melo</creator><creator>Martinez, Rafael Chacon Ruiz</creator><creator>Saad, Susana Marta Isay</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20131217</creationdate><title>Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR</title><author>Villarreal, Martha Lissete Morales ; Padilha, Marina ; Vieira, Antonio Diogo Silva ; Franco, Bernadette Dora Gombossy de Melo ; Martinez, Rafael Chacon Ruiz ; Saad, Susana Marta Isay</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-97c33898852953ad7e8f5e44a18742f7ac42acc611a17cdce9635b69eb3603083</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Azides</topic><topic>Bacteria</topic><topic>Bacterial Load</topic><topic>Bacteriocins</topic><topic>Bifidobacterium animalis</topic><topic>Cheese - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24358142</pmid><doi>10.1371/journal.pone.0082102</doi><tpages>e82102</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2013-12, Vol.8 (12), p.e82102 |
| issn | 1932-6203 1932-6203 |
| language | eng |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Azides Bacteria Bacterial Load Bacteriocins Bifidobacterium animalis Cheese - microbiology Colonies Dairy products Electron microscopy Enumeration Flow cytometry Food Food Microbiology Gastrointestinal system Gastrointestinal tract Gastrointestinal Tract - microbiology In vitro methods and tests Inulin Lactobacillus Lactobacillus acidophilus Listeria Listeria monocytogenes Methods Microbial Viability Microbiota Microorganisms Nutrition Pharmaceutical sciences Population levels Populations Prebiotics Probiotics Probiotics - isolation & purification Propidium - analogs & derivatives Real-Time Polymerase Chain Reaction Scanning electron microscopy Shelf life Species Strains (organisms) Survival Viability Yogurt |
| title | Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-04T23%3A30%3A54IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Advantageous%20direct%20quantification%20of%20viable%20closely%20related%20probiotics%20in%20petit-suisse%20cheeses%20under%20in%20vitro%20gastrointestinal%20conditions%20by%20Propidium%20Monoazide--qPCR&rft.jtitle=PloS%20one&rft.au=Villarreal,%20Martha%20Lissete%20Morales&rft.date=2013-12-17&rft.volume=8&rft.issue=12&rft.spage=e82102&rft.pages=e82102-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0082102&rft_dat=%3Cgale_plos_%3EA478242963%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1468886361&rft_id=info:pmid/24358142&rft_galeid=A478242963&rft_doaj_id=oai_doaj_org_article_c5689e8c1e75463296f1013ae6b197d9&rfr_iscdi=true |