Antimicrobial activity of ethanolic extract of propolis in "Alheira", a fermented meat sausage

The objective of this study was to evaluate the efficacy of an ethanolic extract of propolis (EEP) in the control of Listeria innocua PHLS 2030c (as a surrogate for Listeria monocytogenes) during storage of Alheira at 4°C. Total phenolic content was evaluated to determine the minimal inhibitory concentration of EEP against the growth of L. innocua by the agar dilution method. Alheiras were manufactured by incorporating EEP (0.28 mg/mL) and pathogenic bacteria and storage during 62 days at 4°C. Growth of L. innocua was determined during storage. The behaviour of L. innocua in the food matrix was significantly affected (p < 0.01) by the addition of EEP. The ethanolic extract of propolis reduced the Listeria population to below the detection limit of the technique after 8 days of storage. These results suggest that incorporation of EEP in a food susceptible to Listeria contamination may be an interesting alternative to existing chemical preservatives and can extend the shelf life of these products

Non-thermal synergistic approach to Listeria monocytogenes inactivation in milk: the combined effect of high pressure and bacteriophage P100

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High hydrostatic pressure and pediocin PA-1 as a synergistic system to listeria monocytogenes inactivation in fermented meat sausage

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Innovative hurdle system towards Listeria monocytogenes inactivation in a fermented meat sausage model - high pressure processing assisted by bacteriophage P100 and bacteriocinogenic Pediococcus acidilactici

Consumers' quest for healthier, locally produced foods, renders the demand for these products increasingly prominent. The purpose of the present work was to evaluate the impact of a non-thermal multi-hurdle approach, which associated mild high hydrostatic pressure (HHP, 300 MPa), the bacteriophage Listex™ P100, and the pediocin PA-1 producing Pediococcus acidilactici HA 6111-2, as a novel minimal processing towards Listeria monocytogenes eradication in Alheira (a traditional fermented meat sausage from Northern Portugal). The combination of the three hurdles achieved the USDA-FSIS 5 log reduction (in accordance with the standard guidelines for ready-to-eat foods), being the only treatment to elicit the absence of L. monocytogenes immediately following processing (p  0.05) in the pH values were observed, and the semi-quantification of the in situ biosynthesized pediocin PA-1 was documented for the first time in a fermented meat sausage model.publishe

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB

Streptococcus canis Are a Single Population Infecting Multiple Animal Hosts Despite the Diversity of the Universally Present M-Like Protein SCM

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence

Efeito de tratamentos de pressão e temperatura na actividade e estabilidade da pectina metilesterase e na textura de pimento (Capsicum annuum)

Doutoramento em BioquímicaDe entre os diversos métodos de conservação de alimentos, o processamento térmico é o mais comum. No entanto, a alta pressão tem ganho interesse já que inactiva microorganismos e várias enzimas relacionadas com a qualidade alimentar, enquanto que outros atributos (cor, aroma, vitaminas) são mantidos. Do mesmo modo, a aplicação de pressão a baixas temperaturas (abaixo de 0oC) tem tido um interesse crescente na área do processamento por congelação, já que este provoca danos irreversíveis na textura de diversos frutos e vegetais. Industrialmente, os pimentos são congelados, após branqueamento térmico, para serem consumidos crus em saladas. Considerada como um importante parâmetro de qualidade no pimento, a textura é muitas vezes afectada por alterações que ocorrem durante o processamento, onde se incluem as modificações ao nível da pectina, devido a reacções químicas e/ou à acção de enzimas endógenas, tal como a pectina metilesterase (PME). Tratamentos de pressão de 100 e 200 MPa foram estudados como possíveis substitutos para o branqueamento térmico de pimentos. Os resultados revelaram que os pimentos tratados por pressão apresentam de um modo geral melhor qualidade em diversos parâmetros, nomeadamente ao nível da proteína solúvel, do conteúdo em ácido ascórbico e da textura (firmeza). Os pimentos tratados por pressão apresentam também uma textura melhor após congelação/descongelação que os pimentos branqueados termicamente. Como se verificou que apenas os pimentos verdes apresentavam actividade de PME (nos pimentos vermelhos não se detectou actividade de PME), usaram-se os pimentos verdes para estudar as características desta enzima sob diversas condições de pressão e temperatura. A enzima foi assim purificada através de um passo único por cromatografia de afinidade e caracterizada em termos bioquímicos. A enzima apresentou 2 bandas por SDS-PAGE, com 33 e 37 kDa, e um pH óptimo de 7,5 e uma temperatura óptima a pH neutro entre 52,5 e 55oC. A inactivação isotérmica da enzima PME purificada seguiu uma cinética de inactivação que pode ser descrita através de um modelo de inactivação fraccional (55-57oC, pH 7,5) e de um modelo bifásico (58-70oC, pH 7,5; 62- 76oC, pH 5,6). A pH ácido, a enzima PME purificada apresentou uma estabilidade térmica superior. No extracto não purificado, a pH 5.6, a inactivação isotérmica foi igualmente descrita por um modelo bifásico, entre 62oC e 76oC. Estes resultados de inactivação térmica da enzima PME purificada e no extracto não purificado indiciam a presença de várias isoformas com diferente estabilidade térmica. A enzima nestes dois sistemas apresentou ainda um comportamento relativamente estável aos dois pHs face a diversos tratamentos de pressão-temperatura. Quando se submeteu os pimentos, em pedaços e puré, a tratamentos de pressão a temperaturas moderadas (25oC, 40oC), verificou-se um aumento da actividade da PME, o que poderá estar relacionado com um aumento da extractibilidade da PME, provavelmente devido alterações na estrutura da parede celular. Em puré, a enzima revelou-se mais estável do que em pedaços às condições de temperatura e pressão estudadas. A estabilidade da enzima purificada a diversos tratamentos de pressão (0,1- 800MPa) e temperatura (10-64oC) foi também estudada em tampão a pH 5.6. Com os dados cinéticos obtidos para a fracção lábil da enzima, construiu-se um diagrama P-T de inactivação da enzima, onde se verificou um efeito antagonista na inactivação a P £ 300MPa e T > 54oC. A actividade da enzima purificada foi igualmente estudada sob pressão e temperatura (0,1-600MPa e 18-65oC), na presença de pectina a pH 5,6. Observou-se um óptimo de actividade a condições de pressão e temperatura moderadas (200MPa, 55oC). Adicionalmente, utilizou-se um modelo polinomial do 3º grau (derivado de um modelo termodinâmico) para descrever quer a dependência da constante cinética de inactivação da fracção lábil da enzima PME, quer a dependência da taxa de produção de metanol pela enzima, nas diferentes condições de pressão e temperatura estudadas. Por último, estudou-se o efeito de tratamentos de pressão e temperatura, isoladamente e em combinação, na textura do pimento. Entre 75oC e 95oC, a cinética de degradação da textura do pimento verde obedeceu a um modelo de conversão fraccional. A 90oC, a degradação da textura do pimento foi substancialmente retardada, quando o pimento foi submetido a prétratamentos moderados de temperatura (55oC, 60min) ou de pressão (200MPa, 25oC, 15min). A combinação destes tratamentos com imersão em cálcio (CaCl2, 0,50% (w/v)) retardou ainda mais a degradação de textura. A congelação desviada por pressão (“high pressure shift freezing” - HPSF) não alterou a firmeza dos pimentos pré-tratados (P > 0.05), enquanto que a congelação convencional à pressão atmosférica diminuiu significativamente (P 54oC. Purified pepper PME activity was also considered under combined high-pressure/temperature treatments (18-65oC, 0.1-600MPa) in the presence of pectin at pH 5.6. An optimum in the activity at mild pressure-temperature conditions (200MPa, 55oC) was observed. Moreover, a third-degree polynomial model (derived from a thermodynamic model) was successfully applied to describe the temperature/pressure dependence of the inactivation rate constants of the labile pepper PME fraction as well as the heat-pressure dependence of the initial rates of purified pepper PME-catalyzed methanol formation. Finally, the effect of several pressure and temperature treatments, separately and combined, on pepper texture was also evaluated. Between 75oC to 95oC, the texture degradation kinetics of green pepper could be accurately described by a fractional conversion model. At 90oC, texture degradation of pepper was substantially retarded when pepper was submitted to pre-treatments of temperature (55oC, 60min) and pressure (200MPa, 25oC, 15min). The combination of these treatments with calcium (CaCl2, 0.50% (w/v)) immersion retarded even more the texture degradation. High pressure shift freezing (HPSF) did not change the firmness of pretreated peppers (P > 0.05), while conventional freezing at atmospheric pressure decreased (P < 0.05) the firmness of peppers submitted to the same pre-treatments. From the peppers frozen by HPSF, those pre-treated by high pressure showed a firmness substantially better than the ones thermally treated, after freezing storage at -18oC during 2.5 months. These results clearly showed the advantage of pre-treatments to improve green pepper texture, and of HPSF to maintain the texture of green pepper during freezing and storage at -18oC, when compared to freezing at atmospheric pressure. The applied pressure/temperature treatments revealed to be insufficient to cause fully PME inactivation at the natural pH of peppers, while mild treatments, economically more advantageous, seemed to have beneficial effect in the activation of PME and, as a consequence, in the improvement of texture. HPSF also considerably improves the texture of pre-processed peppers, when compared to conventional freezing at atmospheric pressure. The results of this work clearly illustrate the potential of pressure and pressure combined with temperature treatments to (in)activate PME, giving to high pressure an addvalue as a (pre-)processing technology, in the improvement of the quality of processed green bell pepper, regarding its textural properties

Nuts and Dried Fruits Potential as Functional Foods

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Inactivation of pepper (Capsicum annuum) pectin methylesterase by combined high-pressure and temperature treatments

Pressure and/or temperature inactivation (at mild temperature, 10–64 °C, in combination with high-pressure, 0.1–800 MPa) of the labile fraction of purified pepper pectin methylesterase (PME) was studied in a model system at pH 5.6. Inactivation of the labile fraction under mild-heat and high-pressure conditions could be accurately described by a fractional conversion model, while a biphasic model was used to estimate the inactivation rate constant of both fractions at more drastic conditions of temperature/pressure. At lower pressures (P ⩽ 300 MPa) and high temperatures (>54 °C), an antagonistic effect of pressure and temperature was observed. Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the Eyring and Arrhenius relations, respectively. A third-degree polynomial model (derived from the thermodynamic model) was successfully applied to describe the temperature/pressure dependence of the inactivation rate constants of the labile pepper PME fraction

Identification of pressure/temperature combinations for optimal pepper (Capsicum annuum) pectin methylesterase activity

Pectin methylesterase (PME) was extracted from green bell peppers and purified by affinity chromatography. The optimal pectin and salt concentrations for the PME catalysed reaction were investigated. Purified pepper PME activity was studied during combined high-pressure/temperature treatments (18–65 °C, 0.1–600 MPa) in a model system of pectin at pH 5.6. The activity of purified pepper PME showed a maximum at 200 MPa and 55 °C. A third-degree polynomial model (derived from a thermodynamic model) was successfully used to describe the heat–pressure dependence of the initial rates of purified pepper PME-catalyzed methanol formation