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- Chitosan mouthwash: toxicity and in vivo validationPublication . Costa, E. M.; Silva, S.; Costa, M. R.; Pereira, M.; Campos, D. A.; Odila, J.; Madureira, A. R.; Cardelle-Cobas, A.; Tavaria, F. K.; Rodrigues, A. S.; Pintado, M. M.A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashesas it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes.As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, thebiological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT andV79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. Theresults showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercialmouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca.5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwashpossessed significantly higher antimicrobial activity. The conjunction of these results proves that thechitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.
- A novel direct contact method for the assessment of the antimicrobial activity of dental cementsPublication . Costa, E. M.; Silva, S.; Madureira, A. R.; Cardelle-Cobas, A.; Tavaria, F. K.; Pintado, M. M.Dental cements are a crucial part of the odontological treatment, however, due to the hazardous nature and reduced biological efficiency of some of the used materials, newer and safer alternatives are needed, particularly so those possessing higher antimicrobial activity than their traditional counterparts. The evaluation of the antimicrobial properties of solid and semi-solid antimicrobials, such as dental cements and gels, is challenging, particularly due to the low sensitivity of the current methods. Thus, the main aim of this study was the evaluation of the antimicrobial activity of a novel chitosan containing dental cement while simultaneous assessing/validating a new, more efficient, method for the evaluation of the antimicrobial activity of solid and gel like materials. The results obtained showed that the proposed method exhibited a higher sensitivity than the standard 96 well microtiter assay and allowed the determination of bactericidal activity. Additionally, it is interesting to note that the chitosan containing cement, which presented higher antimicrobial activity than the traditional zinc oxide/eugenol mix, was capable of inducing a viable count reduction above 5 log of CFU for all of the studied microorganisms.
- Development of oral strips containing chitosan as active ingredient: a product for buccal healthPublication . Cardelle-Cobas, Alejandra; Madureira, Ana Raquel; Costa, Eduardo; Barros, Rui; Tavaria, Freni K.; Pintado, Manuela E.In the last years, the number of products for oral care has been expanded to adapt to consumer needs. Thus, in addition to conventional products, new pocket products such as sugar-free chewing gums and oral strips (OS) have been developed for oral care. In the present study, OS were formulated using chitosan as the film-forming polymer in adequate concentrations to also be used as antimicrobial agent. Other strip components, such as the type of plasticizer, were also optimized. Mechanical properties of the optimal OS were evaluated and, due to chitosan's characteristic astringency, the strips were also sensorially evaluated.
- Detailed kinetic model describing new oligosaccharides synthesis using different β-galactosidasesPublication . Rodriguez-Fernandez, Maria; Cardelle-Cobas, Alejandra; Villamiel, Mar; Banga, Julio R.The production of prebiotic galactooligosaccharides (GOS) from lactose has been widely studied whereas the synthesis of new prebiotic oligosaccharides with improved properties as those derived from lactulose is receiving an increasing interest. Understanding the mechanism of enzymatic oligosaccharides synthesis from lactulose would help to improve the quality of the products in a rational way as well as to increase the production efficiency by optimally selecting the operating conditions. A detailed kinetic model describing the enzymatic transgalactosylation reaction during lactulose hydrolysis is presented here for the first time. The model was calibrated with the experimental data obtained in batch assays with two different B-galactosidases at various temperatures and concentrations of substrate. A complete system identification loop, including model selection, robust estimation of the parameters by means of a global optimization method and computation of confidence intervals was performed. The kinetic model showed a good agreement between experimental data and predictions for lactulose conversion and provided important insights into the mechanism of formation of new gligosaccharides with potential prebiotic properties.
- Synthesis, optimization and structural characterization of a chitosan-glucose derivative obtained by the Maillard reactionPublication . Gullón, Beatriz; Montenegro, María I.; Ruiz-Matute, Ana I.; Cardelle-Cobas, Alejandra; Corzo, Nieves; Pintado, Manuela E.Chitosan (Chit) was submitted to the Maillard reaction (MR) by co-heating a solution with glucose (Glc). Different reaction conditions as temperature (40, 60 and 80 °C), Glc concentration (0.5%, 1%, and 2%, w/v), and reaction time (72, 52 and 24h) were evaluated. Assessment of the reaction extent was monitored by measuring changes in UV absorbance, browning and fluorescence. Under the best conditions, 2% (w/v) of Chit, 2% (w/v) of Glc at 60°C and 32 h of reaction time, a chitosan-glucose (Chit-Glc) derivative was purified and submitted to structural characterization to confirm its formation. Analysis of its molecular weight (MW) and the degree of substitution (DS) was carried out by HPLC-Size Exclusion Chromatography (SEC) and a colloid titration method, respectively. FT-IR and (1)H NMR were also used to analyze the functional groups and evaluate the introduction of Glc into the Chit molecule. According to our objectives, the results obtained in this work allowed to better understand the key parameters influencing the MR with Chit as well as to confirm the successful introduction of Glc into the Chit molecule obtaining a Chit-Glc derivative with a DS of 64.76 ± 4.40% and a MW of 210.37 kDa.
- Goat sausages containing chitosan towards a healthier product: microbiological, physicochemical textural evaluationPublication . Amaral, Deborah S. do; Cardelle-Cobas, Alejandra; Nascimento, Bárbara M. S. do; Madruga, Marta S.; Pintado, M. E.Goat meat is extensively known for its interesting nutritional value and for being an important source of protein with high quality. Its food derivatives are, therefore, a good alternative to develop new products addressed to health conscious consumers. In this work, a healthier goat product, namely, a low fat fresh sausage, was produced with the objective of evaluating the effect of inclusion of chitosan on quality, stability and shelf life. Sausages containing 2% chitosan were formulated with different fat levels (5%, 12.5% and 20%, w/w) and stored at 4 °C for 15 days. Results indicated the incorporation of 2% (w/w) chitosan was technologically feasible, due to the reduction of microbial growth and lipid oxidation, as well as the enhancement of red color. Additionally, the treated samples improved all the characteristics associated with cooking, showing the ability to bind water and fat and acquiring a firmer texture compared with control samples. Additionally, the reduction of fat content is technologically feasible without negative influences on the final product.
- Effects of hemicellulose-derived saccharides on behavior of Lactobacilli under simulated gastrointestinal conditionsPublication . Gullón, Patricia; Gullón, Beatriz; Cardelle-Cobas, Alejandra; Alonso, José Luis; Pintado, Manuela; Gomes, Ana MariaFour types of purified xylooligosaccharides (XOS) from several lignocellulosic materials, showing different structural features (including chain length, branching, and linkage types), obtained from different feedstocks (Eucalyptus globuluswood, rice husks,wheat bran or barley wastes), were assessed (using fructooligosaccharides as reference substrate) for their effects on the growth of six different probiotic Lactobacillus strains in basal MRS media and survival in different simulated gastrointestinal tract (GIT) conditions. The hydrophobicity of bacterial cells, which measures their potential for epithelial adhesion, was also evaluated. Improved growth of Lactobacillus strains was observed for media containing XOS as the single source of carbohydrate, confirming their role as prebiotics. These oligosaccharides also led to improvement in the resistance of the target Lactobacillus to the simulated GIT environment. The enhancement of the survival rates depended on the type of oligosaccharide. This is the first comparative study of the effects in lactobacilli involving four types of XOS obtained from different lignocellulosic materials. The obtained results show that these oligosaccharides manufactured from alternative sources could be interesting prebiotic substrates with different structures in comparison with the available commercially and with interesting potential towards the development of synbiotic products.
- A comprehensive study into the impact of a chitosan mouthwash upon oral microorganism's biofilm formation in vitroPublication . Costa, E. M.; Silva, S.; Madureira, A. R.; Cardelle-Cobas, A.; Tavaria, F.K.; Pintado, M. E.Modern dentistry emphasizes the importance of dental plaque control to improve oral health. To that endthe development of oral care formulations has been geared toward the incorporation of antiplaque agentsthat may play a crucial role in oral health maintenance. In later years the research into antiplaque agentshas led to the discovery of compounds with significant capability to affect biofilm formation. Among thesecompounds was chitosan, a polysaccharide which showed great ability to interfere with Streptococcusmutans biofilm formation. As such the aim of this work was to incorporate chitosan into a mouthwashmatrix and assess its effect upon biofilm formation of oral microorganisms. This assessment was per-formed via study of the impact the mouthwash upon microbial adherence, biofilm formation and maturebiofilms. Additionally, the action of the chitosan mouthwash was compared with two commerciallyavailable mouthwashes. The results here obtained show that only the chitosan containing mouthwashwas capable of interfering with all microorganisms’ adherence, biofilm formation and mature biofilmswhile at the same time showing vastly superior activity than both commercial mouthwashes assayed.As such a chitosan mouthwash shows great potential as a natural and efficient alternative to traditionalmouthwashes.
- Chitosan-based silver nanoparticles: a study of the antibacterial, antileishmanial and cytotoxic effectsPublication . Lima, Douglas dos Santos; Gullon, Beatriz; Cardelle-Cobas, Alejandra; Brito, Lucas M.; Rodrigues, Klinger A. F.; Quelemes, Patrick V.; Ramos-Jesus, Joilson; Arcanjo, Daniel D. R.; Plácido, Alexandra; Batziou, Krystallenia; Quaresma, Pedro; Eaton, Peter; Delerue-Matos, Cristina; Carvalho, Fernando Aecio; Silva, Durcilene Alves da; Pintado, M. E.; Leite, José Roberto de SáSilver nanoparticles have been studied as an alternative for treatment of microbial infections and leishmaniasis, without promoting induction of microbial or parasite resistance. In this study, chitosan-based silver nanoparticles were synthesized from silver nitrate (AgNO3), sodium borohydride as a reducing agent, and the biopolymer chitosan as a capping agent. The chitosan-based silver nanoparticles were characterized by ultraviolet-visible, Fourier transform infrared, dynamic light scattering, zeta potential, atomic force microscopy, and transmission electron microscope. The antibacterial assay was performed by determination of the minimum inhibitory concentration. The antileishmanial and the cytotoxic effects induced by AgNO3, chitosan, and chitosan-based silver nanoparticles were analyzed by resazurin and MTT colorimetric assays, respectively. AgNO3, chitosan, and chitosan-based silver nanoparticles induced a marked activity against all bacterial strains and promastigote forms of Leishmania amazonensis at minimum inhibitory concentrations ranging from 1.69 to 3.38 μg Ag/mL. Interestingly, the chitosan-based silver nanoparticles presented less cytotoxicity than the AgNO3 alone and were more active against L. amazonensis than solely chitosan. Furthermore, the cytotoxic concentrations (CC50) of both chitosan and chitosan-based silver nanoparticles against macrophages were significantly higher than the IC50 against promastigotes. Thus, the chitosan-based silver nanoparticles represent a promising alternative for the treatment of microbial infections and leishmaniasis.
- Low fat goat meat sausage with chitosan-glucose Maillard reaction product: impact on quality and shelf lifePublication . Amaral, Deborah Silva do; Cardelle-Cobas, Alejandra; Dias, Celina de Castro Querino; Lima, Darlinne Amanda Soares; Pereira, Sérgio de Ferreira; Arcanjo, Naciza Maria de Oliveira; Dalmás, Paulo Sérgio; Madruga, Marta Suely; Pintado, Maria Manuela EstevezLow fat fresh goat sausages based on chitosan (LMWC) and derivative chitosan (LMWC-Glc) were developed aiming to produce a functional food product evaluating the effect of inclusion of these compounds on quality, stability and shelf life. Goat sausages (10% pork fat) were formulated with 2% (w/w) of LMWC or LMWC-Glc and stored at 4 °C during 21 days. Results indicated that incorporation of LMWC-Glc and LMWC is technologically feasible, originating a potential functional product with low fat content, besides the positive impact on microbial safety. The modified LMWC (towards better solubility and lower adstringency) incorporated at 2% as functional ingredient maintained positive effect on antimicrobial, nutrional, textural and sensory performance. The results also proved that derivative chitosan may also be a promising strategy for improving the quality and extending the shelf life of low fat goat sausage.