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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.
- Survival of probiotic bacteria in a whey cheese vector submitted to environmental conditions prevailing in the gastrointestinal tractPublication . Madureira, A. R.; Pereira, C. I.; Truszkowska, K.; Gomes, Ana M. P.; Pintado, M. E.; Malcata, F. X.Various foods may be used to deliver probiotic bacteria into the gastrointestinal tract; one such example is Requeijão, a Portuguese whey cheese. Survival and stability of Bifidobacterium animalis strains BLC-1, Bb-12, and Bo, Lactobacillus acidophilus strains LAC-1 and Ki, L. paracasei ssp. paracasei strain LCS-1 and L. brevis strain LMG 6906 inoculated into Requeijão, when exposed to simulated gastrointestinal tract conditions, were assessed. Homogenates of inoculated whey cheese in 0.85% (w/v) sterile saline water were exposed to a solution of hydrochloric acid (pH 2.5–3.0) and pepsin (1000 unitsmL–1) at 371C, and then to 0.3% (w/v) bile salts after 60 or 120 min of acid exposure. All bacterial strains retained their initial viable cell numbers. Bifidobacterium animalis strains Bb-12 and Bo, and L. brevis strain LMG 6906 exhibited the highest viable cell numbers when exposed to bile salts, whereas the other strains had variable death rates.
- Management of fruit industrial by-products - a case study on circular economy approachPublication . Campos, Débora; Gómez-García, Ricardo; Vilas-Boas, Ana A.; Madureira, Ana Raquel; Pintado, Maria ManuelaThe management of industrial fruit by-products is important not only to decrease the volume of food waste accumulated in the landfills but also to develop strategies through reuse with the purpose to valorise and add economic value. The disposal of food waste leads to di erent global issues in di erent sectors, such as social, environmental and economical. These by-products represent a rich source of valuable compounds (polyphenols) with high antioxidant activity, which can be extracted through biotechnological methodologies for future industrial applications. In this context, the management of fruit by-products is challenged to move from a linear economy to a circular economy. Therefore, the purpose of this review is to provide a critical view of an integrated valorisation of fruit by-products to overcome a global issue, via the production of antioxidant extracts with high economic value. A case study of pineapple processing industrialization in a circular economy is explored and discussed.
- Extraction and characterisation of cellulose nanocrystals from pineapple peelPublication . Madureira, Ana Raquel; Atatoprak, Tugba; Çabuk, Duygu; Sousa, Flávia; Pullar, Robert C.; Pintado, ManuelaThe potential of pineapple peel as a source of cellulose nanocrystals was evaluated. Peels skin from fresh-cut fruit was used as raw material. These residues were puri ed to remove pigments, lipids and hemicellulose, and a bleaching process for deligni cation was carried out for 4-6 h. All resulting products were characterised for their lignin, hemicellulose, cellulose and ash contents using standard techniques. Dry matter at the end was low (ca. 50%) compared with the raw material (ca. 90%). The process applied resulted in ca. 20% (m/m) of puri ed cellulose (ca. 80% purity), with ineligible levels of lignin and hemicellulose present, especially when using 6h of bleaching. The puri ed cellulose was subject to acid hydrolysis for nanocrystal extraction with two testing times, 30 and 60 minutes. These cellulose nanocrystals had small sizes (< 1000 nm), with high variability and negative zeta potential values. The time of extraction did not a ect the nanocrystals' chemical and physical properties. The use of 6 h of bleaching treatment during puri cation was shown to be more e ective than 4 h. Pineapple peel was demonstrated to be a good source of cellulose for the production of cellulose nanocrystals.
- Incorporation and survival of probiotic bacteria in whey cheese matricesPublication . Madureira, Ana R.; Gião, Maria S.; Pintado, Manuela E.; Gomes, Ana M. P.; Freitas, A. Cristina; Malcata, F. XavierThe viabilities of probiotic strains of Lactobacillus acidophilus, Lactobacillus paracasei, Bifidobacterium animalis, and Lactobacillus brevis were studied following incorporation in a whey cheese matrix. Experimental production of plain, as well as sugar-added or salt-added whey cheeses, was based on the traditional manufacture protocol of Requeijäo, a Portuguese whey cheese that essentially results from protein denaturation via heating of whey at about 85°C. After inoculation, the experimental whey cheeses were incubated at 7°C for 28 d. Our results have shown that all strains considered were able to maintain (or even increase) their initial viable numbers; L. paracasei ssp. paracasei strain LCS-1 and L. acidophilus strain Ki exhibited the highest cell viability in plain Requeijão by the end of the storage period—an increase of ca. 2 log cycles in their viable numbers was actually recorded. Among the parameters studied, bacterial species and matrix nature had the most important effect upon viable counts, whereas time of storage was the least important.
- Rheological, textural and microstructural features of probiotic whey cheesesPublication . Madureira, A. Raquel; Pintado, Ana I. E.; Gomes, Ana Maria; Pintado, Manuela E.; Malcata, F. XavierWhey cheeses have been manufactured with probiotic bacteria e viz. Bifidobacterium animalis Bo and Lactobacillus casei LAFTIrL26, from combinations of bovine whey and milk, following protein denaturation at 90 ºC; they were subsequently inoculated (at 10%) with those strains, and homogenized afterwards; additives such as salt and sugar were then incorporated; and the resulting solid matrices were stored at 7º C for up to 21 d. Oscillatory measurements and instrumental texture profile analyses were performed, and sensory analyses were carried out by a trained panel. Microstructural features were in addition ascertained by scanning electron microscopy. L. casei exhibited a higher acidifying activity than B. animalis, which produced distinct textures; higher firmness and viscoelasticity were indeed found in matrices inoculated with the former. Incorporation of sugar and L. casei favoured consumer acceptability, relative to plain matrices. Microstructural differences were detected between matrices at different times of storage and formulated with distinct additives.
- Optimization of two biopolymer-based oral films for the delivery of bioactive moleculesPublication . Castro, Pedro M.; Fonte, Pedro; Oliveira, Ana; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Manuela E.An experimental design was established in order to optimize the mechanical properties of two oral film formulations intended for oral delivery of bioactive compounds. Carboxymethylcellulose (CMC) and gelatin type A (GelTA) were selected as polymeric matrix. Scanning electron microscopy revealed that caffeine crystals were homogeneously dispersed onto oral film matrix. Fourier-transform infrared analysis did not indicate formation of new chemical entities. USP modified dissolution assay revealed that GelTA was more effective in controlling caffeine release since maximum caffeine release (97.4%±0.95) after 20min. On the other hand, CMC is better indicated for immediate release since maximum caffeine release (81.1%±2.14) occurred after 4min. Simulation of gastrointestinal tract with ex vivo permeability assay was in accordance with USP dissolution assay (42.0%±7.79 and 15.3%±4.0 of caffeine released from CMC and GelTA oral films (OF), respectively, permeated porcine intestinal mucosa after 120min). CMCOF and GelTAOF optimized formulations represent two suitable oral delivery systems for immediate and controlled release, respectively.
- 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.
- Technological stability of solid lipid nanoparticles loaded with phenolic compounds: drying process and stability along storagePublication . Campos, Débora A.; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Maria Manuela; Gomes, Ana MariaSolid lipid nanoparticles (SLNs) have been widely studied and tested as vehicles for natural compounds. Recently, Witepsol and Carnauba SLNs were shown to be effective systems for the entrapment of rosmarinic acid (RA) and herbal extracts. In the present work, the improvement of stability and bioactivity of these systems was studied. Thus, the freeze-drying of SLNs produced with Witepsol and Carnauba waxes loaded with RA and herbal extracts (sage and savory) were tested. The use of three different cryoprotectants (glucose, mannitol and trehalose) at two different concentrations (5 and 10%, w/v) were evaluated. Furthermore, the prepared SLNs were stored under different conditions (atmosphere, temperature, absence or presence of light) and in different packaging materials, over 365 days. The effect on the SLNs physical stability and bioactivity was assessed. The most suitable cryoprotectant was mannitol at 10% (w/v) for all formulations tested. The solid state of SLNs, with storage at room temperature, in glass flasks, protected from light and under N2 controlled atmosphere were the best storage conditions in which the SLNs bioactivity was maintained during 365 days.
- Current state on the development of nanoparticles for use against bacterial gastrointestinal pathogens: focus on chitosan nanoparticles loaded with phenolic compoundsPublication . Madureira, Ana Raquel; Pereira, Adriana; Pintado, ManuelaGastrointestinal diseases have a huge impact especially in third world countries, making it urgent to seek new effective antimicrobial therapies. Thus, the development of nanoparticles (NPs) with bioactive compounds having antimicrobial activity has been the target of research over the past years. The development of antimicrobial drug NPs may be promising to overcome the problems associated with antibiotic resistance caused by many pathogenic bacteria. Moreover, the NPs administration of antimicrobial agents has advantages associated therewith, as use of low cost materials, contribution to the improvement of the therapeutic index and a controlled release drug by increasing the pharmacokinetics. These systems can be used to specific strains of bacteria, and to release interesting antimicrobial compounds. The phenolic compounds (PC) are a class of such bioactive compounds for which their antimicrobial activity was already tested on the production of NPs. Polymeric or lipidic NPs systems have been investigated to deliver these compounds. Chitosan is a polymer widely known for their properties, especially the antimicrobial activity and its ability to adhere to intestinal epithelium. This review article aims to evaluate and discuss recent developments in PC new delivery systems with antimicrobial activity against gastrointestinal pathogens, their production processes, activities, focusing on NPs produced using chitosan as the main structural and functional material.