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- Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytesPublication . Fernandes, João C.; Borges, Margarida; Nascimento, Henrique; Bronze-da-Rocha, Elsa; Ramos, Oscar S.; Pintado, Manuela E.; Malcata, F. Xavier; Santos-Silva, AliceTwo COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07 mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10 mg/mL) and/or apoptosis (<0.10 mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.
- 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.
- Antimicrobial effects of chitosans and chitooligosaccharides, upon Staphylococcus aureus and Escherichia coli, in food model systemsPublication . Fernandes, João C.; Tavaria, Freni K.; Soares, José C.; Ramos, Óscar S.; Monteiro, M. João; Pintado, Manuela E.; Malcata, F. XavierThe objective of this study was to elucidate the controversial relationship between the molecular weight (MW) of chitosans and their antibacterial activity (upon different inoculum levels, at several concentrations). The influence of food components on the activity was also ascertained, as well as acceptance by a sensory panel. All the compounds tested exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. This activity was shown to be closely dependent on the inoculum level, MW and concentration used. Within 4 h at 10³ cells/mL, all five compounds, at every concentration (0.5%, 0.25% and 0.1%, w/v), proved to be bactericidal; for higher inocula, 0.1% (w/v) was only bacteriostatic; at 107 or 105 cells/mL, and independently of the inoculum level, 0.25% (w/v) of any chitooligosaccharide (COS) mixture was sufficient to reduce the E. coli initial population by at least 3 log cycles; COS never exhibited bactericidal action over S. aureus, unlike high and medium MW chitosans—which, at 0.5% (w/v), presented a bactericidal effect even against 107 cells/mL. When incorporated in liquid food matrices, medium and high MW chitosans maintained their activity, for both matrices and bacteria, although a slower activity was noticeable in milk; however, COS lost their activity upon both bacteria in milk after 4–8 h. Furthermore, addition of chitosans to apple juice led to several unpleasant off-flavors, such as astringency and after taste—which increased in magnitude with MW.
- Effect of composition of commercial whey protein preparations upon gelation at various pH valuesPublication . Ramos, Óscar S.; Pereira, Joana O.; Silva, Sara I.; Amorim, Maria M.; Fernandes, João C.; Lopes-da-Silva, José A.; Pintado, Manuela E.; Malcata, F. XavierThe major goal of this research effort was to comprehensively characterize various whey protein products available in the market — including one whey protein isolate (WPI) and three whey protein concentrates (two forms of WPC 80, and WPC 50), with regard to the effects of specific components (e.g. lecithin and minerals) and concentration of β-lactoglobulin (β-Lg) and α-lactalbumin upon thermal and gelation properties at various pH values (using micro differential scanning calorimetry, μDSC, and oscillatory rheometry). At pH values far from the isoelectric point of whey proteins, denaturation and aggregation appeared as one single endothermic peak in the corresponding μDSC heating thermograms, for WPI and both WPC 80; however, they appeared as separate transitions at pH 5. Acidic conditions increased the temperature of occurrence of the dominant endothermic transition associated to β-Lg, thus increasing the thermal stability of WPI, WPC 80A and WPC 80B. Gelation took place at the lowest temperature when pH was set at 5. WPI, WPC 80A and WPC 80B exhibited the highest G′ values at pH 5 — whereas WPI led to stronger gels than WPC, irrespective of pH. In the case of WPC 50, gelation did not occur at all.
- Effect of whey protein purity and glycerol content upon physical properties of edible films manufactured therefromPublication . Ramos, Óscar L.; Reinas, Isabel; Silva, Sara I.; Fernandes, João C.; Cerqueira, Miguel A.; Pereira, Ricardo N.; Vicente, António A.; Poças, M. Fátima; Pintado, Manuela E.; Malcata, F. XavierThis manuscript describes the detailed characterization of edible films made from two different protein products e whey protein isolate (WPI) and whey protein concentrate (WPC), added with three levels of glycerol (Gly) e i.e. 40, 50 and 60%(w/w). The molecular structure, as well as barrier, tensile, thermal, surface and optical properties of said films were determined, in attempts to provide a better understanding of the effects of proteinaceous purity and Gly content of the feedstock. WPI films exhibited statistically lower (p < 0.05) moisture content (MC), film solubility (S), water activity, water vapor permeability (WVP), oxygen and carbon dioxide permeabilities (O2P and CO2P, respectively) and color change values, as well as statistically higher (p < 0.05) density, surface hydrophobicity, mechanical resistance, elasticity, extensibility and transparency values than their WPC counterparts, for the same content of Gly. These results are consistent with data from thermal and FTIR analyses. Furthermore, a significant increase (p < 0.05) was observed in MC, S, WVP, O2P, CO2P, weight loss and extensibility of both protein films when the Gly content increased; whereas a significant decrease (p < 0.05) was observed in thermal features, as well as in mechanical resistance and elasticity e thus leading to weaker films. Therefore, fundamental elucidation was provided on the features of WPI and WPC germane to food packaging e along with suggestions to improve the most critical ones, i.e. extensibility and WVP.
- In vitro screening for antimicrobial activity of chitosans and Chitooligosaccharides, aiming at potential uses in functional textilesPublication . Fernandes, João C.; Tavaria, Freni K.; Fonseca, Susana C.; Ramos, Óscar S.; Pintado, Manuela E.; Malcata, F. XavierAntimicrobial finishing of textiles has been found to be an economical way to prevent (or treat) skin disorders. Hence, this research effort was aimed at elucidating the relationship between molecular weight (MW) of chitosan and its antimicrobial activity upon six dermal reference microorganisms, as well as the influence of the interactions with cotton fabrics on said activity. Using 3 chitosans with different MW, as well as two chitooligosaccharide (COS) mixtures, a relevant antimicrobial effect was observed by 24 h for the six microorganisms tested; it was apparent that the antimicrobial effect is strongly dependent on the type of target microorganism and on the MW of chitosan – being higher for lower MW in the case of E. coli, K. pneumoniae and P. aeruginosa, and the reverse in the case of both Gram-positive bacteria. Furthermore, a strong anti-fungal effect was detectable upon C. albicans, resembling the action over Gram-positive bacteria. Interactions with cotton fabric resulted in a loss of COS activity when compared with cultured media, relative to the effect over Gram-negative bacteria. However, no significant differences for the efficacy of all the 5 compounds were observed by 4 h. The three chitosans possessed a higher antimicrobial activity when impregnated onto the fabric, and presented a similar effect on both Gram-positive bacteria and yeast, in either matrix. Pseudomonas aeruginosa showed to be the most resistant microorganism to all five compounds.
- Edible films and coatings from whey proteins: a review on formulation, and on mechanical and bioactive propertiesPublication . Ramos, Óscar L.; Fernandes, João C.; Silva, Sara I.; Pintado, Manuela E.; Malcata, F. XavierThe latest decade has witnessed joint efforts by the packaging and the food industries to reduce the amount of residues and wastes associated with food consumption. The recent increase in environmental awareness has also contributed toward development of edible packaging materials. Viable edible films and coatings have been successfully produced from whey proteins; their ability to serve other functions, viz. carrier of antimicrobials, antioxidants, or other nutraceuticals, without significantly compromising the desirable primary barrier and mechanical properties as packaging films, will add value for eventual commercial applications. These points are tackled in this review, in a critical manner. The supply of whey protein-based films and coatings, formulated to specifically address end-user needs, is also considered.
- Antimicrobial activity of edible coatings prepared from whey protein isolate and formulated with various antimicrobial agentsPublication . Ramos, Óscar S.; Santos, Arménia C.; Leão, Mariana V.; Pereira, Joana O.; Silva, Sara I.; Fernandes, João C.; Franco, M. Isabel; Pintado, Manuela E.; Malcata, F. XavierUsing in vitro tests, the efficacy of edible coatings, made using whey protein isolates as a base material and glycerol as a plasticizer, and containing several antimicrobial compounds was assessed upon ubiquitous spoilage microflora in cheese. The formulation that showed the greatest activity was applied onto the surface of experimental cheeses, and their major microbiological and physicochemical features were compared with those of cheese added withcommercial coatings. Chitooligosaccharide (COS) and lactic acid exhibited the highest (P < 0.05) bacteriocidal effect against Gram-negative and Gram-positive bacteria, respectively, whereas sodium benzoate and COS exhibited the highest (P< 0.05) inhibitory effect against the yeast. Combinations of lactic acid and COS yielded the highest (P < 0.05) effect against all microorganisms tested; upon application on the cheese surface, they proved to be more active against bacteria, and less effective against yeasts and moulds (P < 0.05) than their commercial coating counterparts.
- Antioxidant activity of chitooligosaccharides upon two biological systems: erythrocytes and bacteriophagesPublication . Fernandes, João C.; Eaton, Peter; Nascimento, Henrique; Gião, Maria S.; Ramos, Oscar. L.; Belo, Luís; Santos-Silva, Alice; Pintado, Manuela E.; Malcata, F. XavierMost of the reports to date on the antioxidant capacity of chitosans and chitooligosaccharides (COS) are based on strictly chemical methods. When studying antioxidants with potential in vivo applications, the method used to evaluate the antioxidant activity should be representative of the conditions in which the antioxidant might have a protective effect. In this work we evaluate the antioxidant activity of two COS mixtures and a low MW chitosan (LMWC) upon two biological oxidizable substrates – erythrocytes and phages, subjected to accelerated oxidation conditions. Our results suggest that COS/LMWC can be used as antioxidants in biological systems. All the tested compounds reduced either the hemolytic and DNA damage, by inhibiting H2O2- and AAPH-radicals. However, the results obtained for these biological assays did not reveal a dose dependence, contrary to the chemical assay, suggesting that the protective concentrations should be established, in order to prevent enhancement of the oxidative damage – i.e. a prooxidant effect.