Utilização da espectroscopia de infravermelho para análise de fiambre

Mestrado em MicrobiologiaFace às crescentes exigências relativamente à qualidade alimentar, nomeadamente em termos microbiológicos, é importante que se desenvolvam métodos rápidos e eficazes para a detecção de contaminações. Neste trabalho foram testadas as potencialidades da Espectroscopia de Infravermelho com Transformadas de Fourier na detecção de alterações em fiambre ao longo do tempo provocadas por microrganismos, bem como na distinção de diferentes colónias de microrganismos Foi ainda testada a capacidade deste método de análise permitir a separação de tipos e marcas diferentes de fiambre. Foram realizados diversos ensaios prévios utilizando o espectrómetro de FT-IR de forma a determinar quais os melhores métodos para analisar o produto em questão. Por fim, foram efectuados ensaios com fiambre fresco, fiambre embalado e diferentes colónias obtidas na superfície de fiambre, utilizando o FT-IR, sendo os resultados comparados com os obtidos por métodos microbiológicos tradicionais, sempre que possível. Concluiu-se que, apesar de ser necessário um estudo mais exaustivo, a técnica de FT-IR permite separar facilmente diferentes colónias de microrganismos obtidas em fiambre, marcas de fiambre e distinguir fiambre de porco, peru e frango. Este método de análise permite ainda separar, em diversos casos, amostras de fiambre consoante a sua carga microbiana total ou grau de contaminação.To face the increasing demands for food quality, particularly in microbiological terms, it is important to develop rapid and effective methods for detecting contaminations The present study evaluated the potential of Infrared Spectroscopy with Fourier Transform to detect changes in ham caused by microorganisms as function of time and to distinguish different microorganism colonies. We also tested the ability of this method of analysis to allow the separation of types and brands of ham. Several preliminary tests were conducted using the FT-IR spectrometer to determine the best method to analyze the product in question. Finally, tests were made with freshly cut and packed ham and different colonies obtained from the ham surface and the results were compared with those obtained by traditional microbiological methods, whenever possible. It was concluded that, despite of the necessity of further studies, FT-IR easily discriminates different colonies of microorganisms obtained in ham and distinguished different brands and types of ham (pork, turkey and chicken ham). This method of analysis also allows the separation, in several cases, of samples of ham according to their total microbial contamination

Desenvolvimento de espectroscopia de infravermelho para avaliar a qualidade bacteriana em alimentos

Doutoramento em BiologiaRapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.A deteção rápida e específica de bactérias que podem provocar deterioração de alimentos ou doenças associadas ao seu consumo é cada vez mais importante na indústria alimentar. A deteção, identificação e classificação de bactérias são geralmente realizadas utilizando métodos tradicionais baseados em testes bioquímicos e/ou serológicos e em métodos moleculares baseados em análise de DNA ou RNA. Contudo, estas metodologias são dispendiosas, demoradas e trabalhosas. A espectroscopia de infravermelho é uma técnica confiável, rápida e económica, que pode ser explorada como ferramenta para a indústria alimentar. Nesta tese foi avaliado o potencial da espectroscopia de infravermelho para estudar a qualidade bacteriana de alimentos. No capítulo 2, foi desenvolvido um modelo de calibração que permitiu prever com sucesso a concentração bacteriana de fiambre naturalmente contaminado, mantido em refrigeração durante 8 dias. Nesta parte, foi desenvolvida a metodologia que permitiu obter a melhor reprodutibilidade dos espectros das colónias de bactérias, com preparação mínima das amostras, que foi utilizada no trabalho subsequente. Foram realizadas várias tentativas para a obtenção de espectros de infravermelho, testando diferentes resoluções e número de scans. Os melhores resultados foram obtidos utilizando uma resolução espectral de 4 cm-1 e 32 varrimentos. De seguida, no capítulo 3, foi feita uma tentativa de identificar 22 bactérias provenientes de alimentos usando a espectroscopia de infravermelho associada a análise multivariada. A análise de componentes principais, utilizada como método exploratório, permitiu a formação de grupos distintos, cada um correspondendo a um género diferente, na grande maioria dos casos. Posteriormente, foi realizada uma análise hierárquica por clusters de forma a investigar a formação de grupos e a possibilidade de distinção de espécies dentro de um mesmo género de bactérias. Observou-se que a espectroscopia de infravermelho é adequada não só para a distinção de diferentes géneros, mas também para diferenciar espécies dentro de um mesmo género, com o uso simultâneo de análise de componentes principais e análise hierárquica por clusters. A utilização de espectroscopia de infravermelho e análise estatística multivariada foram também investigadas no capítulo 4 para confirmação da presença de Listeria monocytogenes e Salmonella spp., isoladas a partir de alimentos contaminados, após crescimento em meio selectivo. Isto permitiria a substituição dos métodos bioquímicos e serológicos que são usados para confirmar a presença destas bactérias patogénicas e que podem atrasar a obtenção de resultados por 2 dias. Os resultados obtidos permitiram a distinção de Salmonella spp. de outras bactérias que se possam confundir com elas. Por fim, no capítulo 5, o processamento por alta pressão, uma metodologia emergente que permite produzir alimentos microbiologicamente seguros e aumentar o seu tempo de prateleira, foi aplicada a 12 bactérias alimentares, de forma a determinar a sua resistência e os efeitos da pressão a nível das células. Foi aplicado um tratamento de 300 MPa, à temperatura ambiente e durante 15 minutos. As bactérias de Gram-negativo foram inativadas até níveis não detetáveis, enquanto as de Gram-positivo mostraram diferentes níveis de resistência. As espécies Bacillus cereus e Staphyloccus aureus decresceram apenas 2 unidades logarítmicas enquanto a espécie Listeria innocua diminuiu cerca de 5 unidades logarítmicas. A espectroscopia de infravermelho foi utilizada na análise das colónias bacterianas antes e após o tratamento por alta pressão, de forma a investigar as alterações que são provocadas nos componentes celulares com este tipo de processamento. Descobriu-se que a alta pressão altera bandas espectrais correspondentes a alguns componentes celulares, de entre os quais proteínas, lípidos, oligopolissacarídeos, grupos fosfato da parede celular e ácidos nucleicos, podendo indicar rutura da parede/membrana celular. Neste trabalho, a quantificação de bactérias e a sua classificação, bem como a análise de modificação nos componentes celulares após processamento por alta pressão foram realizados com sucesso. Assim, a espectroscopia de infravermelho demonstrou ser uma técnica bastante promissora para analisar bactérias provenientes de alimentos de uma forma simples e pouco dispendiosa

New insights on phage efficacy to control Aeromonas salmonicida in aquaculture systems: an in vitro preliminary study

A major source of financial loss for the fish-farming industry is the occurrence of bacterial infections. Phage therapy can be a useful alternative tool to conventional treatments to control bacterial infections in aquaculture. The promising results obtained with phage AS-A to control the agent of furunculosis, Aeromonas salmonicida, led us to isolate two new phages and evaluate their dynamics, in cocktails and individually, to control this pathogenic bacterium. Moreover, considering that in outdoor facilities aquaculture water is exposed to the natural variability of physical and chemical parameters, the influence of pH, salinity, temperature, solar radiation and UV radiation on phage AS-D stability was also evaluated in this study, in order to develop an effective phage therapy protocol. Phages were assigned to the family Myoviridae and revealed identical morphological characteristics. Phage AS-A presents a higher burst size and a shorter latent period, decreasing the concentration of A. salmonicida sooner than phages AS-D and AS-E. However, phage AS-D presented higher rate of bacterial reduction, inducing also less bacterial resistance. Bacterial control with the cocktails was higher, namely when phage AS-A was combined with one of the two new phages or with both, but the main difference in the bacterial control was in the treatment time. Phage cocktails decrease the concentration of A. salmonicida sooner than single suspensions. The use of phage cocktails, in general, decreased phage-resistant mutants. The survival of AS-D phage was mostly affected by sunlight exposure (decrease of 3 log PFU/mL after 12 h) and high temperatures (decrease of 3 PFU/mL after 21 days and of 7 log PFU/mL after 49 days at 37 °C, but no decrease after 21 and 49 days at 25 °C and decrease of only 1 and 2 log PFU/mL after 21 and 49 days, respectively, at ambient temperature). The high bacterial control and low development of phage-resistant bacterial clones suggest that these phages can be used to control the furunculosis in aquaculture. Nonetheless, the stability of the phages was affected by solar radiation, this can be overcome by the application of phages at the end of the day or at night.publishe

Antioxidant and antimicrobial films based on brewers spent grain arabinoxylans, nanocellulose and feruloylated compounds for active packaging

In this study, brewers spent grain (BSG) arabinoxylans-based nanocomposite films were prepared by solvent casting of arabinoxylans (AX) suspensions containing different amounts of nanofibrillated cellulose (NFC, 5, 10, 25, 50 and 75% mass fraction). The obtained nanocomposite films were homogeneous and presented thermal stability up to 230 °C and good mechanical properties (Young's modulus up to 7.5 GPa). Additionally, the films with 50% NFC were loaded with ferulic acid or feruloylated arabinoxylo-oligosaccharides enriched fraction from BSG (75 mg per g of film). This combination enhanced the UV–Vis barrier properties and imparted additional functionalities to the films, namely (i) antioxidant activity up to 90% (DPPH scavenging activity), (ii) antibacterial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, and (iii) antifungal activity towards the polymorphic fungus Candida albicans. Therefore, these fully biobased nanocomposite films show potential for application as active food packaging systems.publishe

The Health-Promoting Potential of Salix spp. Bark Polar Extracts: Key Insights on Phenolic Composition and In Vitro Bioactivity and Biocompatibility

Salix spp. have been exploited for energy generation, along with folk medicine use of bark extracts for antipyretic and analgesic benefits. Bark phenolic components, rather than salicin, have demonstrated interesting bioactivities, which may ensure the sustainable bioprospection of Salix bark. Therefore, this study highlights the detailed phenolic characterization, as well as the in vitro antioxidant, anti-hypertensive, Staphylococcus aureus growth inhibitory effects, and biocompatibility of Salix atrocinerea Brot., Salix fragilis L., and Salix viminalis L. bark polar extracts. Fifteen phenolic compounds were characterized by ultra-high-performance liquid chromatography-ultraviolet detection-mass spectrometry analysis, from which two flavan-3-ols, an acetophenone, five flavanones, and a flavonol were detected, for the first time, as their bark components. Salix bark extracts demonstrated strong free radical scavenging activity (5.58–23.62 µg mL−1 IC50 range), effective inhibition on angiotensin-I converting enzyme (58–84%), and S. aureus bactericidal action at 1250–2500 µg mL−1 (6–8 log CFU mL−1 reduction range). All tested Salix bark extracts did not show cytotoxic potential against Caco-2 cells, as well as S. atrocinerea Brot. and S. fragilis L. extracts at 625 and 1250 µg mL−1 against HaCaT and L929 cells. These valuable findings can pave innovative and safer food, nutraceutical, and/or cosmetic applications of Salix bark phenolic-containing fractions.info:eu-repo/semantics/publishedVersio

Effects of cadmium and phenanthrene mixtures on aquatic fungi and microbially mediated leaf litter decomposition

This version does not correspond to the published one. To access the final version go to: http://www.springerlink.com/content/t8t302617003m078/Urbanization and industrial activities have contributed to widespread contamination by metals and polycyclic aromatic hydrocarbons, but the combined effects of these toxics on aquatic biota and processes are poorly understood. We examined the effects of cadmium (Cd) and phenanthrene on the activity and diversity of fungi associated with decomposing leaf litter in streams. Leaves of Alnus glutinosa were immersed for 10 days in an unpolluted low-order stream in northwest Portugal to allow microbial colonization. Leaves were then exposed in microcosms for 14 days to Cd (0.06–4.5 mg L−1) and phenanthrene (0.2 mg L−1) either alone or in mixture. A total of 19 aquatic hyphomycete species were found sporulating on leaves during the whole study. The dominant species was Articulospora tetracladia, followed by Alatospora pulchella, Clavatospora longibrachiata, and Tetrachaetum elegans. Exposure to Cd and phenanthrene decreased the contribution of A. tetracladia to the total conidial production, whereas it increased that of A. pulchella. Fungal diversity, assessed as denaturing gradient gel electrophoresis fingerprinting or conidial morphology, was decreased by the exposure to Cd and/or phenanthrene. Moreover, increased Cd concentrations decreased leaf decomposition and fungal reproduction but did not inhibit fungal biomass production. Exposure to phenanthrene potentiated the negative effects of Cd on fungal diversity and activity, suggesting that the co-occurrence of these stressors may pose additional risk to aquatic biodiversity and stream ecosystem functioning.The Portuguese Foundation for the Science and Technology supported this work (POCI/MAR/56964/2004) and S. Duarte (SFRH/BPD/47574/2008

Towards the Understanding of the Function of Lanthipeptide and TOMM-Related Genes in Haloferax mediterranei

Research on secondary metabolites produced by Archaea such as ribosomally synthesized and post-translationally modified peptides (RiPPs) is limited. The genome of Haloferax mediterranei ATCC 33500 encodes lanthipeptide synthetases (medM1, medM2, and medM3) and a thiazole-forming cyclodehydratase (ycaO), possibly involved in the biosynthesis of lanthipeptides and the TOMMs haloazolisins, respectively. Lanthipeptides and TOMMs often have antimicrobial activity, and H. mediterranei has antagonistic activity towards haloarchaea shown to be independent of medM genes. This study investigated (i) the transcription of ycaO and medM genes, (ii) the involvement of YcaO in bioactivity, and (iii) the impact of YcaO and MedM-encoding genes’ absence in the biomolecular profile of H. mediterranei. The assays were performed with biomass grown in agar and included RT-qPCR, the generation of knockout mutants, bioassays, and FTIR analysis. Results suggest that ycaO and medM genes are transcriptionally active, with the highest number of transcripts observed for medM2. The deletion of ycaO gene had no effect on H. mediterranei antihaloarchaea activity. FTIR analysis of medM and ycaO knockout mutants suggest that MedMs and YcaO activity might be directly or indirectly related t lipids, a novel perspective that deserves further investigation

Towards the Understanding of the Function of Lanthipeptide and TOMM-Related Genes in <i>Haloferax mediterranei</i>

Research on secondary metabolites produced by Archaea such as ribosomally synthesized and post-translationally modified peptides (RiPPs) is limited. The genome of Haloferax mediterranei ATCC 33500 encodes lanthipeptide synthetases (medM1, medM2, and medM3) and a thiazole-forming cyclodehydratase (ycaO), possibly involved in the biosynthesis of lanthipeptides and the TOMMs haloazolisins, respectively. Lanthipeptides and TOMMs often have antimicrobial activity, and H. mediterranei has antagonistic activity towards haloarchaea shown to be independent of medM genes. This study investigated (i) the transcription of ycaO and medM genes, (ii) the involvement of YcaO in bioactivity, and (iii) the impact of YcaO and MedM-encoding genes’ absence in the biomolecular profile of H. mediterranei. The assays were performed with biomass grown in agar and included RT-qPCR, the generation of knockout mutants, bioassays, and FTIR analysis. Results suggest that ycaO and medM genes are transcriptionally active, with the highest number of transcripts observed for medM2. The deletion of ycaO gene had no effect on H. mediterranei antihaloarchaea activity. FTIR analysis of medM and ycaO knockout mutants suggest that MedMs and YcaO activity might be directly or indirectly related t lipids, a novel perspective that deserves further investigation

Evaluation of the potential of mid-infrared spectroscopy to assess the microbiological quality of ham

The accurate reliable detection and identification of microorganisms in food is critical to public safety. Consequently, it is extremely important to develop rapid and inexpensive methods for the detection of food microorganisms in order to minimize or even replace the traditional analysis methods that are expensive and time-consuming. In this study, the potential of mid-infrared spectroscopy was evaluated, for the first time, to detect changes in colony forming units of microorganisms in freshly cut ham along the time. A partial least squares regression model was performed and a good linear relationship was obtained between spectra information and microbial load. It was concluded that infrared spectroscopy easily and quickly allows the separation of ham samples according to their microbial content and could be used to predict the microbial concentration from the spectra, using the fingerprint region (1,200–950 cm−1), without sample preparation or handling. Practical Applications As it is essential to avoid infections caused by foodborne bacteria, it is important to develop a rapid, low cost and easy to perform technique to face the increasing demands of the food industry. Mid-infrared spectroscopy, coupled to multivariate analysis, has potential to be used as a first-screening approach and to assess the microbial concentration in ham samples, avoiding the traditional plating methods that are time-consuming

Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment

The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells