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Desenvolvimento de filmes antimicrobianos para aplicação na indústria alimentar

Publication . Silva, Ângela Maria Leite da; Domingues, Fernanda da Conceição; Sousa, Sónia Cristina Lopes de

Campylobacter coli e Campylobacter jejuni são os principais agentes infeciosos causadores de doença gastrointestinal no mundo. Nos humanos, a principal via de contaminação é o manuseamento, a preparação e o consumo de carne de aves contaminada, sobretudo a carne de frango. As embalagens antimicrobianas são uma forma de embalagens ativas, que permitem a redução e inibição do crescimento de microrganismos que possam estar presentes nos alimentos ou no próprio material das embalagens. Os derivados de celulose são uma fonte importante de biomateriais para a formação de filmes antimicrobianos (AM) e a incorporação de compostos fenólicos nestes filmes, tem vindo a demonstrar grandes potencialidades. O resveratrol (RV) é um composto fenólico de origem natural que apresenta diversas propriedades benéficas para a saúde humana, incluindo propriedades antimicrobianas. No entanto a sua baixa solubilidade em meio aquoso e a sua baixa disponibilidade limitam a sua aplicação. Para ultrapassar estas limitações é feita a sua complexação com ciclodextrinas. Neste trabalho foram desenvolvidos filmes AM em monocamada baseados em carboximetilcelulose (CMC), e em bicamada baseados em acetato de celulose e hidroxietilcelulose. Como agente AM foi incorporado o RV e o seu complexo de inclusão com a hidroxipropil-?-ciclodextrina (HP-?-CD). Foram avaliadas as propriedades físicas destes filmes e a atividade antimicrobiana contra C. coli e C. jejuni pelo método de difusão em disco. Também foi quantificada a percentagem de libertação dos agentes AM em estudo nos filmes em bicamada para etanol a 10%. Como a regulação e a inibição de patogéneos alimentares pode ser regulada por fenómenos de quorum sensing (QS), foi estudada a interferência dos filmes com incorporação de RV e da sua forma complexada nesse processo de comunicação. Foi ainda avaliada a atividade anti-Campylobacter dos filmes em bicamada aplicados num modelo alimentar, usando para este efeito a carne de frango. Os resultados demonstraram que ambos os agentes AM tornaram os filmes de CMC e os filmes em bicamada mais elásticos e menos resistentes. Os filmes em bicamada com incorporação do complexo de inclusão de RV foram os mais hidrofílicos, comparativamente com os de RV na forma livre e o controlo. Os ensaios de libertação demonstraram que os agentes AM foram totalmente libertados dos filmes em bicamada, ao fim de 10 dias à temperatura de 4 e 20ºC. Também foi demonstrado que os filmes em bicamada inibiram o crescimento das estirpes testadas na área de contacto dos filmes com a superfície de agar e formaram um halo de inibição, enquanto os filmes de CMC apenas inibiram o crescimento na zona de contacto. Mostrou-se que os filmes em bicamada possuíram atividade anti-QS através da inibição da produção de violaceína superior a 70%. Por fim, foi observada uma redução das Unidades Formadoras de Colónias de C. coli e C. jejuni, dependente da temperatura de incubação, por ação dos filmes em bicamada testados no peito de frango. Em suma, este estudo demonstrou o elevado potencial dos filmes AM biodegradáveis com incorporação de RV e da sua forma complexada com HP-?-CD no controlo de Campylobacter em carne de frango.

2015-11-13Master thesis

Open access

Resveratrol against Arcobacter butzleri and Arcobacter cryaerophilus: Activity and effect on cellular functions

Publication . Ferreira, Susana; Silva, Filomena; Queiroz, João; Oleastro, Mónica; Domingues, F.C.

The frequent isolation of Arcobacter butzleri and Arcobacter cryaerophilus from food samples makes it imperative to search for potential compounds able to inhibit the development of these bacteria. Taking this into consideration, this study focuses on the antimicrobial activity of resveratrol and its mechanism of action against A. butzleri and A. cryaerophilus. The activity of resveratrol was assessed by a microdilution method and time-kill curves. Resveratrol effect on cellular functions was assessed by flow cytometry evaluating intracellular DNA content and metabolic activity. Ethidium bromide (EtBr) accumulation in the presence of resveratrol was also evaluated, as well as the susceptibility to resveratrol in the presence of phenylalanine-arginine β-naphthylamide (PAβN). Scanning electron microscopy (SEM) was used to further evaluate cell damage caused by resveratrol. Resveratrol presented MIC values of 100 and 50μg/mL to A. butzleri and A. cryaerophilus, respectively. Based on the time-kill curves, resveratrol exhibited bactericidal activity, leading to a ≥3log10CFU/mL reduction of initial inoculums, for A. butzleri exponential phase cells incubated for 6h with 1× MIC or with 2× MIC after 24h for stationary phase cells. For A. cryaerophilus cells in exponential growth phase, 99.9% killing was achieved after 24h incubation with 2× MIC, whereas, for stationary phase cells, bactericidal activity was only detected after incubation with 4× MIC. Incubation with resveratrol led to a decrease in both intracellular DNA content and metabolic activity. An increase in the accumulation of EtBr was observed in the presence of resveratrol, and the efflux pump inhibitor PAβN reduced the MIC of resveratrol. SEM analysis revealed disintegration of A. butzleri cells treated with resveratrol, whereas no morphological alteration was observed for A. cryaerophilus cells. Resveratrol has a good anti-Arcobacter activity, and the results obtained suggest that this compound could act through several different mechanisms in the inhibition of this microorganism. The results encourage the use of this compound for the development of potential strategies to control Arcobacter in food products.

2014Journal article

Restricted access

Clinical isolates of Acinetobacter baumannii from a Portuguese hospital: PFGE characterization, antibiotic susceptibility and biofilm-forming ability

Publication . Duarte, Andreia; Ferreira, Susana; Almeida, Sofia; Domingues, F.C.

Acinetobacter baumannii is an emerging pathogen associated with nosocomial infections that in addition has shown an increasing resistance to antibiotics. In this work the genetic diversity of A. baumannii isolates from a Portuguese hospital, their antibiotic resistance profiles and ability to form biofilms was studied. Seventy-nine clinical A. baumannii isolates were characterized by pulsed-field gel electrophoresis (PFGE) with 9 different PFGE profiles being obtained. Concerning the antimicrobial susceptibility, all A. baumannii isolates were resistant to 12 of the 17 tested antibiotics and classified as multidrug-resistant (MDR). In addition, 74.7% of the isolates showed biofilm formation ability, however no statistical significance with antibiotic resistance was observed. In contrast, urine samples isolates were more likely to form biofilms than strains isolated from other sources. Our findings highlight the high number of MDR A. baumannii isolates and the importance of the formation of biofilms as a potential virulence factor.

2016Journal article

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Resveratrol production in bioreactor: Assessment of cell physiological states and plasmid segregational stability

Publication . Afonso, Margarida S.; Ferreira, Susana; Domingues, F.C.; Silva, Filomena

Resveratrol is a plant secondary metabolite commonly found in peanuts and grapevines with significant health benefits. Recombinant organisms can produce large amounts of resveratrol and, in this work, Escherichia coli BW27784 was used to produce resveratrol in bioreactors while monitoring cell physiology and plasmid stability through flow cytometry and real-time qPCR, respectively. Initially, the influence of culture conditions and precursor addition was evaluated in screening assays and the data gathered was used to perform the bioreactor assays, allowing the production of 160 μg/mL of resveratrol. Cellular physiology and plasmid instability affected the final resveratrol production, with lower viability and plasmid copy numbers associated with lower yields. In sum, this study describes new tools to monitor the bioprocess, evaluating the effect of culture conditions, and its correlation with cell physiology and plasmid segregational stability, in order to define a viable and scalable bioprocess to fulfill the need for larger quantities of resveratrol.

2015Journal article

Open access

Genetic diversity, antibiotic resistance and biofilm-forming ability of Arcobacter butzleri isolated from poultry and environment from a Portuguese slaughterhouse

Publication . Ferreira, Susana; Fraqueza, Maria J.; Queiroz, João; Domingues, F.C.; Oleastro, Mónica

The genus Arcobacter is an emerging pathogen associated with several clinical symptoms. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, where poultry is considered the major source. In this study, forty three Arcobacter butzleri strains isolated from poultry and environment of a Portuguese slaughterhouse, were characterized by pulsed field gel electrophoresis (PFGE) and assessed for antimicrobial susceptibility and ability to form biofilms. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed high genetic diversity, with 32 distinct PFGE patterns. Most of A. butzleri isolates presented multiple antimicrobial resistance, exhibiting four different resistance profiles. All 43 isolates were susceptible to gentamicin and 2.3% were resistant to chloramphenicol, in contrast to twenty four (55.8%) that were resistant to ciprofloxacin. Among 36 selected isolates, 26 strains presented biofilm-forming ability, which was dependent on the atmosphere and initial inoculum density. Overall, the results showed that A. butzleri displays a high genetic diversity, and presents resistance to several antibiotics, which together with its biofilm formation ability may represent a potential hazard for foodborne infections and a considerable risk for human health.

2013Journal article

Restricted access