Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UFPB |
Texto Completo: | https://repositorio.ufpb.br/jspui/handle/123456789/20155 |
Resumo: | Listeria monocytogenes is the etiological agent of a gastrointestinal disease known as listeriosis, and has been linked to several outbreaks involving ready-to-eat products, mainly animal origin products. Its importance in food is related to its ability to withstand adverse conditions found in food processing environments and in food matrices. Therefore, the aim of this study was to evaluate whether pre-exposure (24, 48 and 72 h) to sublethal concentrations (1/2 MIC, 1/4 MIC and 1/8 MIC) of organic acids (acetic acid and lactic acid) and salts (sodium chloride and potassium chloride) could induce the development of cross-tolerance to Origanum vulgare L. (OVEO) and Rosmarinus officinalis L. (ROEO) essential oils (EO) in three strains of L. monocytogenes. It was further investigated whether exposure to OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL) in PBS and meat broth could develop in L. monocytogenes a “viable but non culturable state (VBNC). The minimum inhibitory concentration (MIC) of organic acids, salts and EO was determined by the broth microdilution method. The induction of tolerance was assessed by MIC modulation and survival curves assay. Culturability loss/recovery was evaluated by the viable cell counts method. The effect of stressing agents and EO tested on different physiological parameters (membrane integrity and potential and respiratory, enzymatic and efflux activities) were monitored by flow cytometry (FC). The results obtained in the induction of tolerance assays showed that preexposure to organic acids and salts did not induce the development of cross-tolerance to OVEO, however, induced increased tolerance to OERO. In the MIC modulation assay, MIC of OVEO decreased up to 10 times the initial MIC value, while the MIC of OERO increased up to 4.8 times. In the survival curves assay, OVEO decreased the counts of cells pre-exposed and non pre-exposed to stressing agents, while ROEO decreased only non pre-exposed cell counts. The culturability loss/recovery assay showed that L. monocytogenes cells treated in PBS lost their culturability after 60 min of exposure to OVEO (2.5 and 5 µL/mL) and ROEO (10 µL/mL) and after 180 min to ROEO (5 µL / mL); when treated in meat broth, the loss of culturability occurred after 180 min for both OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL). Cells that were exposed to OVEO were unable to restore their culturability after recovery treatment in PBS added 0.2% glucose. However, cells exposed to ROEO in PBS (5 µL/mL) and meat broth (5 and 10 µL/mL) restored their culturability after 24 hours of recovery treatment. Results from FC analysis showed that all functions monitored in L. monocytogenes cells pre-exposed to AA or NaCl and treated with OVEO or ROEO were affected, although in different intensities. Cells that lost culturability after exposure to OVEO or ROEO showed that all monitored functions were damaged. However, after recovery treatment, for cells that did not recover culturability, an improvement was observed in most of the evaluated physiological parameters, indicating cell viability. These data indicate that exposure to stressing conditions imposed by organic acids or salts may result in the development of cross-tolerance to ROEO but not to OVEO, however, stressing conditions caused by both EO may induce a VBNC state in L. monocytogenes which, in turn, can restore its ability to multiply under favorable conditions, making it a hazard to consumers’ health. |
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Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenesListeriaConservantes de alimentosResposta adaptativaCulturabilidadeDano celularÓleos essenciaisOréganoAlecrimFood preservativesAdaptive responseCulturabilityEssential oilsOreganoRosemaryCNPQ::CIENCIAS DA SAUDE::NUTRICAOListeria monocytogenes is the etiological agent of a gastrointestinal disease known as listeriosis, and has been linked to several outbreaks involving ready-to-eat products, mainly animal origin products. Its importance in food is related to its ability to withstand adverse conditions found in food processing environments and in food matrices. Therefore, the aim of this study was to evaluate whether pre-exposure (24, 48 and 72 h) to sublethal concentrations (1/2 MIC, 1/4 MIC and 1/8 MIC) of organic acids (acetic acid and lactic acid) and salts (sodium chloride and potassium chloride) could induce the development of cross-tolerance to Origanum vulgare L. (OVEO) and Rosmarinus officinalis L. (ROEO) essential oils (EO) in three strains of L. monocytogenes. It was further investigated whether exposure to OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL) in PBS and meat broth could develop in L. monocytogenes a “viable but non culturable state (VBNC). The minimum inhibitory concentration (MIC) of organic acids, salts and EO was determined by the broth microdilution method. The induction of tolerance was assessed by MIC modulation and survival curves assay. Culturability loss/recovery was evaluated by the viable cell counts method. The effect of stressing agents and EO tested on different physiological parameters (membrane integrity and potential and respiratory, enzymatic and efflux activities) were monitored by flow cytometry (FC). The results obtained in the induction of tolerance assays showed that preexposure to organic acids and salts did not induce the development of cross-tolerance to OVEO, however, induced increased tolerance to OERO. In the MIC modulation assay, MIC of OVEO decreased up to 10 times the initial MIC value, while the MIC of OERO increased up to 4.8 times. In the survival curves assay, OVEO decreased the counts of cells pre-exposed and non pre-exposed to stressing agents, while ROEO decreased only non pre-exposed cell counts. The culturability loss/recovery assay showed that L. monocytogenes cells treated in PBS lost their culturability after 60 min of exposure to OVEO (2.5 and 5 µL/mL) and ROEO (10 µL/mL) and after 180 min to ROEO (5 µL / mL); when treated in meat broth, the loss of culturability occurred after 180 min for both OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL). Cells that were exposed to OVEO were unable to restore their culturability after recovery treatment in PBS added 0.2% glucose. However, cells exposed to ROEO in PBS (5 µL/mL) and meat broth (5 and 10 µL/mL) restored their culturability after 24 hours of recovery treatment. Results from FC analysis showed that all functions monitored in L. monocytogenes cells pre-exposed to AA or NaCl and treated with OVEO or ROEO were affected, although in different intensities. Cells that lost culturability after exposure to OVEO or ROEO showed that all monitored functions were damaged. However, after recovery treatment, for cells that did not recover culturability, an improvement was observed in most of the evaluated physiological parameters, indicating cell viability. These data indicate that exposure to stressing conditions imposed by organic acids or salts may result in the development of cross-tolerance to ROEO but not to OVEO, however, stressing conditions caused by both EO may induce a VBNC state in L. monocytogenes which, in turn, can restore its ability to multiply under favorable conditions, making it a hazard to consumers’ health.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESListeria monocytogenes é o agente etiológico de uma doença gastrointestinal conhecida como listeriose, e tem sido relacionado a diversos surtos envolvendo produtos prontos para o consumo, principalmente produtos de origem animal. Sua importância em alimentos está relacionada à sua capacidade de resistir a condições adversas encontradas em ambientes de processamento de alimentos e em matrizes alimentares. O objetivo deste estudo foi avaliar se a pré-exposição (24, 48 e 72 h) a concentrações subletais (1/2 CIM, 1/4 CIM e 1/8 CIM) de ácidos orgânicos (ácido acético e ácido lático) e sais (cloreto de sódio e cloreto de potássio) poderia induzir o desenvolvimento de tolerância cruzada aos óleos essenciais (OE) de Origanum vulgare L. (OEOV) e Rosmarinus officinalis L. (OERO) em três cepas de L. monocytogenes. Investigou-se, ainda, se a exposição ao OEOV (5 e 2,5 µL/mL) e OERO (0 e 5µL/mL), em PBS e caldo carne, poderia desenvolver em L. monocytogenes um estado “viável mas não cultivável” (viable but non culturable – VBNC). A concentração inibitória mínima (CIM) dos ácidos orgânicos, sais e OE foi determinada pelo método de microdiluição em caldo. A indução de tolerância foi avaliada por meio da modulação da CIM e dos ensaios de curva de sobrevivência. A avaliação da perda/recuperação da culturabilidade foi realizada pelo método de contagens de células viáveis. O efeito dos agentes estressores e OE testados sobre diferentes parâmetros fisiológicos (integridade e potencial de membrana e atividades respiratória, enzimática e de efluxo) foram monitorados por meio de citometria de fluxo (CF). Os resultados obtidos nos ensaios de indução de tolerância mostraram que a pré-exposição aos ácidos orgânicos e sais não induziu o desenvolvimento de tolerância cruzada ao OEOV, contudo, induziu aumento de tolerância ao OERO. No ensaio de modulação da CIM, a CIM do OEOV reduziu em até 10 vezes o valor da CIM inicial, enquanto que a CIM do OERO aumentou em até 4,8 vezes. No ensaio de curva de sobrevivência, o OEOV diminuiu a contagem tanto das células pré-expostas quanto das células não pré-expostas aos agentes estressores, enquanto o OERO diminuiu apenas as contagens de células não pré-expostas. O ensaio de perda/recuperação da culturabilidade mostrou que as células de L. monocytogenes tratadas em PBS perderam sua culturabilidade após 60 min de exposição ao OEOV (5 e 2,5 µL/mL) e ao OERO (10 µL/mL) e após 180 min ao OERO (5 µL/mL); quando tratadas em caldo carne, a perda da culturabilidade acorreu após 180 min tanto para o OEOV (5 e 2,5 µL/mL) quanto para o OERO (10 e 5 µL/mL). As células que foram expostas ao OEOV não foram capazes de restaurar sua culturabilidade após o tratamento de recuperação em PBS adicionado de 0,2% de glicose. No entanto, as células expostas ao OERO em PBS (5 µL/mL) e em caldo carne (10 e 5 µL/mL) restauraram sua cultura após 24 horas de tratamento de recuperação. Os resultados da análise de CF mostraram que todas as funções monitoradas em células de L. monocytogenes pré-expostas ao AA ou NaCl e tratadas com OEOV ou OERO foram afetadas, embora com intensidades diferentes. As células que perderam a culturabilidade após exposição ao OEOV ou OERO apresentaram dano em todas as funções monitoradas. Entretanto, após o tratamento de recuperação, para as células que não recuperaram a culturabilidade foi observada uma melhora na maioria dos parâmetros fisiológicos avaliados, sendo indicativo de viabilidade celular. Esses dados indicam que a exposição às condições de estresse impostas por ácidos orgânicos ou sais pode resultar no desenvolvimento de tolerância cruzada ao OERO, mas não ao OEOV, porém, as condições de estresse causadas por ambos os OE podem induzir um estado VBNC nas células de L. monocytogenes que, por sua vez, pode restaurar sua capacidade de se multiplicar em condições favoráveis, tornando-se um risco à saúde do consumidor.Universidade Federal da ParaíbaBrasilNutriçãoPrograma de Pós-Graduação em Ciências da NutriçãoUFPBSouza, Evandro Leite dehttp://lattes.cnpq.br/9103355732367822Barbosa, Isabella de Medeiros2021-06-01T19:53:02Z2020-12-042021-06-01T19:53:02Z2019-11-05info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesishttps://repositorio.ufpb.br/jspui/handle/123456789/20155porhttp://creativecommons.org/licenses/by-nd/3.0/br/info:eu-repo/semantics/embargoedAccessreponame:Biblioteca Digital de Teses e Dissertações da UFPBinstname:Universidade Federal da Paraíba (UFPB)instacron:UFPB2021-06-09T13:48:55Zoai:repositorio.ufpb.br:123456789/20155Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufpb.br/PUBhttp://tede.biblioteca.ufpb.br:8080/oai/requestdiretoria@ufpb.br|| diretoria@ufpb.bropendoar:2021-06-09T13:48:55Biblioteca Digital de Teses e Dissertações da UFPB - Universidade Federal da Paraíba (UFPB)false |
dc.title.none.fl_str_mv |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
title |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
spellingShingle |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes Barbosa, Isabella de Medeiros Listeria Conservantes de alimentos Resposta adaptativa Culturabilidade Dano celular Óleos essenciais Orégano Alecrim Food preservatives Adaptive response Culturability Essential oils Oregano Rosemary CNPQ::CIENCIAS DA SAUDE::NUTRICAO |
title_short |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
title_full |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
title_fullStr |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
title_full_unstemmed |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
title_sort |
Investigação do desenvolvimento de tolerância cruzada aos óleos essenciais de Origanum vulgare L. e Rosmarinus officinalis L. e perda da culturabilidade em células de Listeria monocytogenes |
author |
Barbosa, Isabella de Medeiros |
author_facet |
Barbosa, Isabella de Medeiros |
author_role |
author |
dc.contributor.none.fl_str_mv |
Souza, Evandro Leite de http://lattes.cnpq.br/9103355732367822 |
dc.contributor.author.fl_str_mv |
Barbosa, Isabella de Medeiros |
dc.subject.por.fl_str_mv |
Listeria Conservantes de alimentos Resposta adaptativa Culturabilidade Dano celular Óleos essenciais Orégano Alecrim Food preservatives Adaptive response Culturability Essential oils Oregano Rosemary CNPQ::CIENCIAS DA SAUDE::NUTRICAO |
topic |
Listeria Conservantes de alimentos Resposta adaptativa Culturabilidade Dano celular Óleos essenciais Orégano Alecrim Food preservatives Adaptive response Culturability Essential oils Oregano Rosemary CNPQ::CIENCIAS DA SAUDE::NUTRICAO |
description |
Listeria monocytogenes is the etiological agent of a gastrointestinal disease known as listeriosis, and has been linked to several outbreaks involving ready-to-eat products, mainly animal origin products. Its importance in food is related to its ability to withstand adverse conditions found in food processing environments and in food matrices. Therefore, the aim of this study was to evaluate whether pre-exposure (24, 48 and 72 h) to sublethal concentrations (1/2 MIC, 1/4 MIC and 1/8 MIC) of organic acids (acetic acid and lactic acid) and salts (sodium chloride and potassium chloride) could induce the development of cross-tolerance to Origanum vulgare L. (OVEO) and Rosmarinus officinalis L. (ROEO) essential oils (EO) in three strains of L. monocytogenes. It was further investigated whether exposure to OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL) in PBS and meat broth could develop in L. monocytogenes a “viable but non culturable state (VBNC). The minimum inhibitory concentration (MIC) of organic acids, salts and EO was determined by the broth microdilution method. The induction of tolerance was assessed by MIC modulation and survival curves assay. Culturability loss/recovery was evaluated by the viable cell counts method. The effect of stressing agents and EO tested on different physiological parameters (membrane integrity and potential and respiratory, enzymatic and efflux activities) were monitored by flow cytometry (FC). The results obtained in the induction of tolerance assays showed that preexposure to organic acids and salts did not induce the development of cross-tolerance to OVEO, however, induced increased tolerance to OERO. In the MIC modulation assay, MIC of OVEO decreased up to 10 times the initial MIC value, while the MIC of OERO increased up to 4.8 times. In the survival curves assay, OVEO decreased the counts of cells pre-exposed and non pre-exposed to stressing agents, while ROEO decreased only non pre-exposed cell counts. The culturability loss/recovery assay showed that L. monocytogenes cells treated in PBS lost their culturability after 60 min of exposure to OVEO (2.5 and 5 µL/mL) and ROEO (10 µL/mL) and after 180 min to ROEO (5 µL / mL); when treated in meat broth, the loss of culturability occurred after 180 min for both OVEO (2.5 and 5 µL/mL) and ROEO (5 and 10 µL/mL). Cells that were exposed to OVEO were unable to restore their culturability after recovery treatment in PBS added 0.2% glucose. However, cells exposed to ROEO in PBS (5 µL/mL) and meat broth (5 and 10 µL/mL) restored their culturability after 24 hours of recovery treatment. Results from FC analysis showed that all functions monitored in L. monocytogenes cells pre-exposed to AA or NaCl and treated with OVEO or ROEO were affected, although in different intensities. Cells that lost culturability after exposure to OVEO or ROEO showed that all monitored functions were damaged. However, after recovery treatment, for cells that did not recover culturability, an improvement was observed in most of the evaluated physiological parameters, indicating cell viability. These data indicate that exposure to stressing conditions imposed by organic acids or salts may result in the development of cross-tolerance to ROEO but not to OVEO, however, stressing conditions caused by both EO may induce a VBNC state in L. monocytogenes which, in turn, can restore its ability to multiply under favorable conditions, making it a hazard to consumers’ health. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-11-05 2020-12-04 2021-06-01T19:53:02Z 2021-06-01T19:53:02Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufpb.br/jspui/handle/123456789/20155 |
url |
https://repositorio.ufpb.br/jspui/handle/123456789/20155 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by-nd/3.0/br/ info:eu-repo/semantics/embargoedAccess |
rights_invalid_str_mv |
http://creativecommons.org/licenses/by-nd/3.0/br/ |
eu_rights_str_mv |
embargoedAccess |
dc.publisher.none.fl_str_mv |
Universidade Federal da Paraíba Brasil Nutrição Programa de Pós-Graduação em Ciências da Nutrição UFPB |
publisher.none.fl_str_mv |
Universidade Federal da Paraíba Brasil Nutrição Programa de Pós-Graduação em Ciências da Nutrição UFPB |
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reponame:Biblioteca Digital de Teses e Dissertações da UFPB instname:Universidade Federal da Paraíba (UFPB) instacron:UFPB |
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Universidade Federal da Paraíba (UFPB) |
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UFPB |
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UFPB |
reponame_str |
Biblioteca Digital de Teses e Dissertações da UFPB |
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Biblioteca Digital de Teses e Dissertações da UFPB |
repository.name.fl_str_mv |
Biblioteca Digital de Teses e Dissertações da UFPB - Universidade Federal da Paraíba (UFPB) |
repository.mail.fl_str_mv |
diretoria@ufpb.br|| diretoria@ufpb.br |
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1801842974962745344 |