Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

The Effect of Molecular Weight on the Antimicrobial Activity of Chitosan from Loligo opalescens for Food Packaging Applications.

The growing requirement for sustainable processes has boosted the development of biodegradable plastic-based materials incorporating bioactive compounds obtained from waste, adding value to these products. Chitosan (Ch) is a biopolymer that can be obtained by deacetylation of chitin (found abundantly in waste from the fishery industry) and has valuable properties such as biocompatibility, biodegradability, antimicrobial activity, and easy film-forming ability. This study aimed to produce and characterize poly(lactic acid) (PLA) surfaces coated with ß-chitosan and ß-chitooligosaccharides from a Loligo opalescens pen with different molecular weights for application in the food industry. The PLA films with native and depolymerized Ch were functionalized through plasma oxygen treatment followed by dip-coating, and their physicochemical properties were assessed by Fourier-transform infrared spectroscopy, X-ray diffraction, water contact angle, and scanning electron microscopy. Their antimicrobial properties were assessed against Escherichia coli and Pseudomonas putida, where Ch-based surfaces reduced the number of biofilm viable, viable but nonculturable, and culturable cells by up to 73%, 74%, and 87%, respectively, compared to PLA. Biofilm growth inhibition was confirmed by confocal laser scanning microscopy. Results suggest that Ch films of higher molecular weight had higher antibiofilm activity under the food storage conditions mimicked in this work, contributing simultaneously to the reuse of marine waste.

Active Carboxymethylcellulose-Based Edible Films: Influence of Free and Encapsulated Curcumin on Films' Properties.

Carboxymethylcellulose (CMC)-based films can act as a protective barrier in food surfaces and a carrier of bioactive compounds, such as curcumin. However, incorporating curcumin in hydrophilic matrixes can be a challenge, and new strategies need to be explored. In this work, CMC-based films containing free curcumin and curcumin-loaded nanohydrogels (composed of lactoferrin and glycomacropeptide) were produced and characterized. The incorporation of curcumin-loaded nanohydrogels showed a significant decrease in films' thickness (from 0.0791 to 0.029 mm). Furthermore, the water vapor permeability of CMC-based films was significantly decreased (62%) by incorporating curcumin-loaded nanohydrogels in the films. The water affinity's properties (moisture, solubility, and contact angle) of films were also affected by incorporating encapsulated curcumin. The addition of nanohydrogels to CMC-based films reduced the tensile strength values from 16.46 to 9.87 MPa. Chemical interactions were analyzed using Fourier transform infrared spectroscopy. The release profile of curcumin from CMC-based films was evaluated at 25 °C using a hydrophilic food simulant and suggests that the release mechanism of the curcumin happens by Fick's diffusion and Case II transport. Results showed that protein-based nanohydrogels can be a good strategy for incorporating curcumin in edible films, highlighting their potential for use in food applications.

Development of Chitosan-Based Surfaces to Prevent Single- and Dual-Species Biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

Implantable medical devices (IMDs) are susceptible to microbial adhesion and biofilm formation, which lead to several clinical complications, including the occurrence of implant-associated infections. Polylactic acid (PLA) and its composites are currently used for the construction of IMDs. In addition, chitosan (CS) is a natural polymer that has been widely used in the medical field due to its antimicrobial and antibiofilm properties, which can be dependent on molecular weight (Mw). The present study aims to evaluate the performance of CS-based surfaces of different Mw to inhibit bacterial biofilm formation. For this purpose, CS-based surfaces were produced by dip-coating and the presence of CS and its derivatives onto PLA films, as well surface homogeneity were confirmed by contact angle measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antimicrobial activity of the functionalized surfaces was evaluated against single- and dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. Chitosan-based surfaces were able to inhibit the development of single- and dual-species biofilms by reducing the number of total, viable, culturable, and viable but nonculturable cells up to 79%, 90%, 81%, and 96%, respectively, being their activity dependent on chitosan Mw. The effect of CS-based surfaces on the inhibition of biofilm formation was corroborated by biofilm structure analysis using confocal laser scanning microscopy (CLSM), which revealed a decrease in the biovolume and thickness of the biofilm formed on CS-based surfaces compared to PLA. Overall, these results support the potential of low Mw CS for coating polymeric devices such as IMDs where the two bacteria tested are common colonizers and reduce their biofilm formation.

Lactoferrin-based nanoemulsions to improve the physical and chemical stability of omega-3 fatty acids.

Omega-3 (ω-3) polyunsaturated fatty acids are highly susceptible to oxidation and have an intense odour and poor water solubility, which make their direct applications in foods extremely difficult. In order to reduce their oxidation process and improve their stability in aqueous medium, protein-based nanoemulsions were produced and characterized. Lactoferrin (Lf) was used as an emulsifier at different concentrations (0.2% to 4% w/w). High energy methods (Ultra-Turrax and high-pressure homogenizer) were applied to produce Lf-based nanoemulsions with ω-3 PUFAs encapsulated. The nanoemulsions were characterized in terms of physical and chemical stability at 4 and 25 °C. The results obtained revealed that the Lf concentration influences the nanoemulsion size in a manner that higher Lf concentrations decrease the nanoemulsion size. It was also observed that the nanoemulsions are physically stable when stored at 4 °C for 69 days while at 25 °C they showed instability. The radical scavenging capacity of the nanoemulsions did not show significant changes over storage at 4 and 25 °C while a significant increase in oxidation was observed. The release profile at 37 °C showed that ω-3 PUFAs were slowly released at pH 2 but was rapidly released at pH 7.4 from Lf nanoemulsions. Moreover, MTT assay revealed that 2% (w/w) Lf nanoemulsions with 12.5 µg mL-1ω-3 PUFAs were not cytotoxic to Caco-2 cells. Nanoemulsions with high physical and chemical stability were selected and dried by two different methodologies: freeze-drying and nano spray-drying. ATR-FTIR spectroscopy, Raman spectroscopy and Circular Dichroism (CD) showed Lf structural changes after the drying processes. This work provides important information regarding nanoemulsions' design and drying technologies aiming at the encapsulation of lipophilic compounds for pharmaceutical and food applications.

Physicochemical characterisation and release behaviour of curcumin-loaded lactoferrin nanohydrogels into food simulants.

Whey protein nanostructures can be used as vehicles for the incorporation of nutraceuticals (e.g., antioxidants or vitamins) aimed at the development of functional foods, because nanostructures provide greater protection, stability and controlled release to such nutraceuticals. Fundamental knowledge is required regarding the behaviour of nanostructures when associated with nutraceuticals and their interactions with real food matrices. In this study, a lactoferrin (LF) nanohydrogel was developed to encapsulate curcumin (nutraceutical model) and its behaviour was evaluated in terms of the LF structure and the interaction with curcumin. The release kinetics of curcumin from LF nanohydrogels was also assessed using food simulants with a hydrophilic nature (10% ethanol) and lipophilic nature (50% ethanol). This system was able to encapsulate curcumin at 80 µg mL-1 with an efficiency of ca. 90% and loading capacity of ca. 3%. Through spectroscopic characterisation, it is suggested that LF and curcumin bind via hydrophobic interactions and the average binding distance between LF and curcumin was found to be 1.91 nm. Under refrigerated conditions (4 °C), this system showed stability for up to 35 days, while at room temperature (25 °C) it was shown to be stable for up to 14 days of storage. The LF nanohydrogel presented higher release rates of curcumin in a lipophilic food simulant (stable after ca. 7 h) as compared to a hydrophilic simulant (stable after ca. 4 h). LF nanohydrogels were successfully incorporated into a gelatine matrix and showed no degradation in this process. The behaviour of this system and the curcumin release kinetics in food stimulants make the LF nanohydrogel an interesting system to associate with lipophilic nutraceuticals and to incorporate in refrigerated food products of a hydrophilic nature.

Protein-Based Nanostructures for Food Applications.

Proteins are receiving significant attention for the production of structures for the encapsulation of active compounds, aimed at their use in food products. Proteins are one of the most used biomaterials in the food industry due to their nutritional value, non-toxicity, biodegradability, and ability to create new textures, in particular, their ability to form gel particles that can go from macro- to nanoscale. This review points out the different techniques to obtain protein-based nanostructures and their use to encapsulate and release bioactive compounds, while also presenting some examples of food grade proteins, the mechanism of formation of the nanostructures, and the behavior under different conditions, such as in the gastrointestinal tract.

Development of an immobilization system for in situ micronutrients release.

An immobilization system constituted by coated microcapsules was developed aiming at immobilizing probiotic bacteria capable of producing folate and release it in a sustained manner into the intestine. Despite no probiotic folate-producers have been immobilized so far, the system has been developed with this goal and this work reports its stability and ability to release folate under gastro-intestinal conditions. Microcapsules were made of alginate with three consecutive coatings of poly-l-lysine, sodium alginate and chitosan. Turbidity experiments showed a strong electrostatic interaction between these polymers. Fourier transform infrared spectroscopy (FTIR) and confocal analysis showed the stability of the coating materials when applied on the microcapsules, even after they were immersed in solutions simulating conditions in the stomach and small intestine (i.e. pH2, 60min and pH7.2, 120min, respectively). Coated microcapsules have an average diameter size ranged from 20 and 40µm, and swelled upon exposure to a neutral medium, without dissolution as showed by microscopy analyses. Release experiments proved the ability of the coated microcapsules to release folic acid, at different rates, depending on the applied coating. Release experiments showed that the first coating (Ɛ-PLL) is characterized by Fickian diffusion as the main release mechanism of folic acid. Fickian rate constant (kF) decreased with the number of consequent coatings, reflecting the decrease of predominance of Fick's behavior. Results showed that the developed coated microcapsules have suitable characteristics for encapsulation of folic acid aiming in situ release in the intestine.

Lactoferrin-based nanoparticles as a vehicle for iron in food applications - Development and release profile.

This study aims at developing and characterizing bovine lactoferrin (bLf) nanoparticles as an iron carrier. bLf nanoparticles were characterized in terms of size, polydispersity index (PdI), electric charge (ζ-potential), morphology, structure and stability over time. Subsequently, iron release experiments were performed at different pH values (2.0 and 7.0) at 37°C, in order to understand the release mechanism. bLf (0.2%, w/v) nanoparticles were successfully produced by thermal gelation (75°C for 20min). bLf nanoparticles with 35mM FeCl3 showed an iron binding efficiency value of approximately 20%. The nanoparticles were stable (i.e. no significant variation of size and PdI of the nanoparticles) for 76days at 4°C and showed to be stable between 4 and 60°C and pH2 and 11. Release experiments at pH2 showed that iron release could be described by the linear superposition model (explained by Fick and relaxation phenomenon). On the contrary, the release mechanism at pH7 cannot be described by either Fick or polymer relaxation behaviour. In general, results suggested that bLf nanoparticles could be used as an iron delivery system for future food applications.

Hollow chitosan/alginate nanocapsules for bioactive compound delivery.

This work aimed at the development of biodegradable nanocapsules as carriers of two bioactive compounds, 5-aminosalycilic acid and glycomacropeptide. Nanocapsules were produced through layer-by-layer (LbL) deposition of chitosan (CH) and alginate (ALG) layers on polystyrene nanoparticles. The bioactive compounds were incorporated on the third layer of the nanocapsules being its encapsulation efficiency and release behaviour evaluated. The LbL deposition process, stability, morphology and size of the multilayer nanocapsules were monitored by means of zeta potential and transmission electron microscopy (TEM). The bioactive compounds release from the CH/ALG nanocapsules was successfully described by a mathematical model (linear superimposition model - LSM), which allowed concluding that bioactive compounds release is due to both Brownian motion and the polymer relaxation of the CH/ALG layers. Final results demonstrated that the synthesized LbL hollow nanocapsules presented spherical morphology and a good capacity to encapsulate different bioactive compounds, being the best results obtained for the system containing 5-aminosalycilic acid (with an encapsulation efficiency of approximately 70%). CH/ALG multilayer nanocapsules could be a promising carrier of bioactive compounds for applications in food and pharmaceutical industries.

Chitosan/fucoidan multilayer nanocapsules as a vehicle for controlled release of bioactive compounds.

Hollow multilayer nanocapsules were successfully prepared through layer-by-layer assembly of two bioactive polysaccharides, chitosan and fucoidan. The stepwise adsorption of 10 chitosan/fucoidan layers and the consequent formation of a multilayer film on polystyrene nanoparticles (used as templates) were followed through ζ-potential measurement and the removal of the polystyrene core was confirmed by FTIR analysis. The chitosan/fucoidan nanocapsules morphology and size were evaluated by SEM and TEM, which showed that after the core removal, the nanocapsules maintained their spherical shape and a decrease of size occurred. A cationic bioactive compound, poly-L-lysine (PLL), was chosen to evaluate the loading and release behaviour of the nanocapsules. The chitosan/fucoidan nanocapsules showed a good capacity for the encapsulation and loading of PLL, which shows to be influenced by the initial PLL concentration and the method of encapsulation used. The results of fitting the linear superimposition model to the experimental data of PLL release suggest an anomalous behaviour, with one main polymer relaxation. The PLL release was found to be pH-dependent: at pH 2 relaxation is the governing phenomenon and at pH 7 Fick's diffusion is the main mechanism of PLL release. Chitosan/fucoidan nanocapsules is a promising delivery system for water soluble bioactive compounds, such as PLL, showing a great potential of application in food and pharmaceutical industries.

Development and characterization of hydrogels based on natural polysaccharides: policaju and chitosan.

The development of hydrogels based on natural polysaccharides was investigated by preparing mixtures of policaju/chitosan at weight ratios of 1:4 and 2:3. Utilizing dynamic light scattering (DLS) techniques for these mixtures, an increase on the hydrodynamic particle radius was observed varying their pH from 3.0 to 12.0. Furthermore, a reduction of ζ-potential was also observed for the same pH interval. Following rounds of drying/hydration cycles at a specific pH value, hydrogel matrices were formed. The pore size distribution of these formed hydrogels was examined using scanning electron microscopy. Further FT-IR analyses confirmed a physical interaction between the polysaccharides policaju and chitosan. Swelling experiments revealed water uptake values, after 24h of immersion in water, close to 270% for 1:4, and 320% for 2:3 hydrogels. Finally, rheological measurements were then conducted in order to confirm hydrogel viscoelastic features. These results indicate a promising road to biomaterials fabrication and biomedical applications.

Alginate/chitosan nanoparticles for encapsulation and controlled release of vitamin B2.

This work aims at evaluating encapsulation and controlled release of vitamin B2 from alginate/chitosan nanoparticles. Ionotropic polyelectrolyte pre-gelation was used as production method being chitosan and alginate used as main materials. Nanoparticles were characterized in terms of average size, polydispersity index (PDI), zeta potential and vitamin entrapment efficiency. The average size for alginate/chitosan nanoparticles was 119.5±49.9nm for samples without vitamin B2 and 104.0±67.2nm with the encapsulation of vitamin B2, presenting a PDI of 0.454±0.066 and 0.319±0.068, respectively. The nanoparticles showed encapsulation efficiency and loading capacity values of 55.9±5.6% and 2.2±0.6%, respectively. Release profiles were evaluated at different conditions showing that the polymeric relaxation was the most influent phenomenon in vitamin B2 release. In order to study their stability nanoparticles were stored at 4°C being particles sizes and PDI evaluated during 5 months showing the results that vitamin B2-loaded nanoparticles are more stable (in terms of size and PDI) than nanoparticles without vitamin B2.