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- Edible films as carrier for lactic acid bacteriaPublication . Pereira, Joana Odila; Soares, José; Sousa, Sérgio; Madureira, Ana Raquel; Gomes, Ana M. P.; Pintado, M. E.The use of edible coatings and films formulated with bioactive compounds in food products in order to convey new functionalities or extend shelf-life opens new possibilities as a carrier for functional lactic acid bacteria. In this work the main objective was to study the stability of probiotic microorganisms, viz. Bifidobacterium animalis Bb-12® and Lactobacillus casei-01, in edible film formulations based on whey protein isolate (WPI). The results demonstrated a loss of bacterial cell viability of ca. 3 log cycles (reaching 106 CFU/g film) until 60 d at both 23 and 4 C, noting that the most marked decrease was at 23 C for both strains. Bifidobacterium animalis Bb-12® remained viable for a longer period of time and with less decrease in its cell numbers (108 CFU/g film). Physical properties, namely color, water activity, thickness, young’s modulus, tensile strength, elongation at break and the molecular structure of WPI films were maintained stable throughout the storage period at both temperatures tested. Edible films incorporated with probiotics can be good carriers for these to be ingested together with food products.
- Features and performance of edible films, obtained from whey protein isolate formulated with antimicrobial compoundsPublication . Ramos, Óscar L.; Silva, Sara I.; Soares, José C.; Fernandes, João C.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThe goal of this research effort was to assess the efficacy of edible films produced fromwhey protein isolate (WPI) and glycerol, including incorporation of lactic acid (LA) and propionic acid (PRO), chitooligosaccharideswith nominal MW of 3 kDa (COS) and natamycin (NA) as antimicrobial agents. Their features were evaluated in vitro via agar diffusion and viable cell counting, against spoilage microflora often found contaminating cheese surfaces. The effect of incorporating the aforementioned compounds upon thickness, moisture content (MC), solubility (S), density (ρs),water activity (aw) and water vapor permeability (WVP), as well as upon tensile and optical properties of those films were also evaluated. Films formulated with LA, PRO or COS exhibited antimicrobial activity against all microorganisms tested, yet the viable cell count assay was more sensitive and reproducible. COS was the most active against Gram-negative bacteria, whereas LA was the most active against Gram-positive ones. NA was not active against bacteria, but displayed the strongest effect against yeasts. Incorporation of said antimicrobial compounds did not significantly (pN0.05) affect film thickness, yet it significantly (pb0.05) reduced tensile strength (TS). Incorporation of LA and NA in particular did not significantly (pb0.05) affect MC, S, ρs, WVP, elongation at break (EB) and Young's modulus (YM) values; however, a statistically significant increase (pb0.05) of MC, S andWVP, together with a statistically significant decrease (pb0.05) of ρs were attained upon incorporation of PRO or COS. Moreover, PRO produced the highest variation (pb0.05) in EB, TS and YM,whereas COS produced the highest change (pb0.05) in optical properties.
- Perfil de diversidade da comunidade estafilocócica da pele em doentes com dermatite atópicaPublication . Lopes, Cristina; Soares, José; Tavaria, Freni; Silva, Rosa; Oliveira, Vera; Morgado, José; Delgado, Luís; Pintado, ManuelaIntrodução: A dermatite atópica (DA) é uma doença cutânea crónica imunologicamente mediada em que a maioria dos doentes está colonizada por Staphylococcus aureus (S. aureus) capaz de produzir vários factores de virulência. O S. aureus pode ser cultivado em cerca de 90% das lesões cutâneas e pode colonizar a pele de aspecto morfologicamente normal. Os estafilococos coagulase negativos (SCN) geralmente não produzem toxinas com actividade superantigénica, mas o seu papel patogénico na DA não pode ser excluído. Neste estudo, pretende-se caracterizar a comunidade estafilocócica da pele de doentes com DA e indivíduos saudáveis, assim como identificar factores de virulência nas espécies identificadas. Métodos: Todos os isolados estafilocócicos foram submetidos a análise numérica de factores de virulência. As espécies isoladas da pele de doentes com DA e indivíduos saudáveis foram submetidas a técnicas de identificação molecular por Multiplex-PCR para identificação de bactérias pertencentes às espécies S. aureus, S. epidermidis, S. capitis, S. hominis e S. haemolyticus através de fragmentos específicos de ADN de 700, 124, 208, 806 e 271 bp, respectivamente. Identificação complementar de cada isolado, previamente identificado por Multiplex-PCR e de 22 isolados não identificados foram realizados por sequenciação do gene sodA. Resultados: Nos doentes com DA isolaram -se estirpes de S. aureus, com 71 (36,2%), S. epidermidis com 59 (30,1%) e S. hominis com 54 (27,6%) isolados. Foi analisada a pele de indivíduos -controlo saudáveis com prevalência para o S. warneri com 10 (23,8%) isolados e S. saprophyticus com 9 (21,4%) isolados juntamente com mais seis espécies identificadas, i.e., S. epidermidis, S. aureus, S. capitis, S. hominis, S. haemolyticus e S. lugdunensis. A maioria das espécies de estafilococos foi coagulase negativo (158/238 isolados) e desoxirribonuclease negativos (161/238). Verificou -se maior biodiversidade na pele de indivíduos saudáveis, com 8 espécies identificadas, do que na pele de doentes com DA, com 4 espécies identificadas. Conclusão: Existe uma maior diversidade de espécies estafilocócicas em indivíduos saudáveis comparativamente aos doentes com DA na presente amostra. A predominância de SA na pele de doentes com DA evidencia a sua maior adaptação. A caracterização detalhada e o perfil de virulência para cada doente poderão ser úteis numa terapêutica antimicrobiana individualizada. A relação simbiótica versus antagonista entre os estafilococos comensais e SA deverá ser melhor esclarecida.
- Chitosan: antimicrobial action upon staphylococci after impregnation onto cotton fabricPublication . Tavaria, F.K.; Soares, J.C.; Reis, I.L.; Paulo, M.H.; Malcata, F.X.; Pintado, M.E.Background: High levels of viable Staphylococcus aureus, which are often found on inflamed skin surfaces, are usually associated with atopic dermatitis. Textiles, owing to their high specific surface area and intrinsic hydrophilicity, retain moisture while also providing excellent environmental conditions for microbial growth and proliferation. Recently, a number of chemicals have been added to textiles, so as to confer antimicrobial activity. Aims: To evaluate the antimicrobial action of chitosan upon selected skin staphylococci. Methods and Results: We isolated staphylococci from normal skin of 24 volunteers and studied their survival upon contact with chitosan-impregnated cotton fabric. Low and high molecular weight chitosans were used at two concentrations; all four did effectively reduce the growth of some staphylococci (namely Staph. aureus), by up to 5 log cycles, thus unfolding a potential towards control and even prevention of related skin disorders. Conclusion: Our data suggest an effective, but selective antibacterial action of chitosans towards skin bacteria. Significance and Impact of the Study: The possibility to use a natural biopolymer incorporated in a textile to alleviate and even treat some of the symptoms associated with this skin condition may raise an alternative to existing medical treatments. The selectivity observed prevents full elimination of bacteria from the skin surface, which is an advantage.
- In vitro evaluation of “horchata” co-products as carbon source for probiotic bacteria growthPublication . Sánchez-Zapata, Elena; Fernández-López, Juana; Pérez-Alvarez, José A.; Soares, José; Sousa, Sérgio; Gomes, Ana M. P.; Pintado, Manuela M. E.Tiger nut milk ("horchata") liquid co-products (TNLC) were evaluated as carbon source for probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium animalis) growth by screening via microplate assay and determination of viable cells and metabolic activity. Based on MRS five different basal media were prepared ((i) without carbon source, (ii) with 2% (w/v) glucose, (iii) with 2% (w/v) FOS, (iv) with 2% (v/v) TNLC and (v) 3% (v/v) TNLC). Additionally, reconstituted TNLC was also used as basal medium. For determination of viable cells and metabolic activity skim milk powder was used. Glucose was found to be the best substrate to L. acidophilus grows, followed by FOS and TNLC (2% and 3%, respectively). TNLC (3%) was found to be the best substrate followed by TNLC (2%), glucose and FOS, in the promotion of growth of B. animalis. The growth of L. acidophilus and B. animalis in skim milk reconstituted TNLC + water was higher than samples inoculated in skim milk reconstituted only with water (p < 0.05). This is supported by the pH effect and by the faster organic acid production (mainly lactic acid, acetic acid, and butyric acid), confirming the property of TNLC as a carbon source for probiotic bacteria growth.
- Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in PortugalPublication . Soares, José C.; Marques, M. Rosário; Tavaria, Freni K.; Pereira, Joana O.; Xavier Malcata, F.; Pintado, Manuela M.The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.
- Effect of the incorporation of salted additives on probiotic whey cheesesPublication . Madureira, Ana Raquel; Soares, José C.; Pintado, Manuela E.; Gomes, Ana Maria; Freitas, Ana Cristina; Malcata, F. XavierThe research effort described here has focused on incorporation of Lactobacillus casei, in whey protein matrices, in the presence of selected salty additives. Those matrices were produced via thermal processing of a combination of either ovine or bovine whey (or a mixture thereof) with ovine milk, and were inoculated (at 10%) with L. casei strain LAFTI®L26; salt, salt and herbs, or salt and xanthan were further added to such matrices, which were then homogenized and stored at 7 °C for up to 21 d. In general, viable cell numbers maintained or even increased throughout the storage period, irrespective of the type of salty additive considered. Partial depletion of lactose was detected, and concomitant production of lactic acid throughout the 21 d-period of storage; lower lactic acid concentrations were found in matrices containing salty additives. In matrices with xanthan (SX), the probiotic strain exhibited the lowest metabolic activity. Matrices SX were less soft and firmer than the others, by the end of storage, and were similar to matrices with herbs (SH). The incorporation of salty additives affected bacterial metabolism, in terms of glycolysis and proteolysis, which in turn had a significant impact on the development of textural properties
- Bioactivity of probiotic whey cheese: characterization of the content of peptides and organic acidsPublication . Madureira, Ana R.; Soares, José C.; Amorim, Maria; Tavares, Tania; Gomes, Ana M.; Pintado, Maria M.; Malcata, F. XavierBackground Probiotic whey cheeses have been produced for several years. It is recognized that several bacterium-mediated metabolic activities contribute differently to the final sensory and nutritional profiles of dairy products. Hence the metabolic activity of probiotic strains in a whey cheese and their contribution to the bioactivity of such matrices were investigated here, including in particular Bifidobacterium animalis, Lactobacillus acidophilus and Lactobacillus casei. Results Both L. casei and B. animalis produce lactic and acetic acids, whereas L. acidophilus produce mainly lactic acid; these metabolites may be considered bioprotection factors. Water-soluble extracts (WSE) obtained from these cheese matrices were subjected to ultrafiltration through a 3 kDa cut-off membrane, and the eluted peptides were resolved by high-performance liquid chromatography. Different qualitative and quantitative profiles were obtained, depending on the strain. WSE were further assayed for their ability to inhibit angiotensin-converting enzyme; the <3 kDa fraction exhibited higher activities in the case of L. casei and B. animalis than the control and L. acidophilus. Conclusion Whey cheeses with higher nutritional value were those inoculated with L. casei.
- A diversity profile from the staphylococcal community on atopic dermatitis skin: a molecular approachPublication . Soares, J.; Lopes, C.; Tavaria, Freni; Delgado, L.; Pintado, M.AimsThe aim of this study was to determine the biodiversity of the skin staphylococcal community from patients with atopic dermatitis (AD) and superantigen (SAg) detection from Staphylococcus aureus isolates. Methods and ResultsIn this study, we developed a novel multiplex PCR that allows the identification and discrimination of bacteria belonging to the Staphylococcus genus both Staph.aureus and coagulase-negative Staphylococcus - Staph.capitis, Staph.epidermidis, Staph.haemolyticus and Staph.hominis isolated from the skin of patients with AD. In addition, a multiplex PCR assay that allows the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs), SE-like toxins and toxic shock syndrome toxin-1 was also performed and applied in Staph.aureus isolates. The microflora of the skin of patients with AD was dominated by Staph.aureus (69 isolates, 356%) followed by Staph.epidermidis (59 isolates, 304%) species. The SElM and SElN genes were the most frequently detected in our study (15 isolates, 714%), followed by SEG and SElO (14 isolates, 667%). Conclusions Our molecular-based approach successfully identified the staphylococcal microflora that was relatively specific to patients with AD. Considering skin colonization and expression of virulence factors, the Staph.aureus may play a relevant role in AD pathophysiology. Significance and Impact of the StudyThis ability to classify disease-related microbial species provides new insights into the relevance of those microbes in human disorders.
- Aqueous extracts of Vaccinium corymbosum as inhibitors of Staphylococcus aureusPublication . Silva, Sara; Costa, Eduardo M.; Costa, Maria Rosário; Pereira, Miguel F.; Pereira, Joana O.; Soares, José C.; Pintado, M. E.Staphylococcus aureus has been established has one of the most common pathogens causing nosocomial infections worldwide and also playing a role in for several outbreaks related to the consumption of contaminated foodstuffs. More recently, the discovery of methicillin resistant S. aureus (MRSA) in raw and processed foods increased the risk factor associated with S. aureus associated foodborne diseases and has led to the search for new sources of antimicrobial agents. Therefore the aim of this study is to evaluate, for the first time, the impact of Vaccinium corymbosum L, fruit and leaf, infusions and decoctions upon methicillin resistant (MRSA) and sensitive S. aureus (MSSA). In order to accomplish these objectives V. corymbosum extracts were characterized and inhibition halos, inhibitory concentrations, impact upon enzymatic activity and biofilm formation were assessed. The results obtained showed that major compounds present in V. corymbosum constitution were quercetin-3-glucoside, chlorogenic and cafeic acids. MRSA and MSSA growth was inhibited at 12.5 mg/mL, for leaf, and 50 mg/ml, for fruit, and sub-MIC concentrations presented inhibition percentages as high as 3 log of viable cells and 47% of biomass. Furthermore DNase and coagulase were also inhibited at sub-MIC concentrations of the extracts. The results obtained imply an effective antibacterial and antibiofilm activity of these extracts towards MRSA and MSSA, thus revealing an interest potential for application in the food industry either as a functional ingredient or a preservative.