RESUMEN
This work aimed to compare the performance of two relatively underexplored methods for the swollen micelles (SMs) production as nanocarriers for essential oils (EOs). Origanum vulgare and Thymus vulgaris EOs were examined. The first method (SMs-1), involved a self-assembly process, while the second one (SMs-2), employed titration operation of an emulsion into a surfactant solution for SMs formation. Tween 80 and ethanol were used as surfactant and co-surfactant, respectively. The solubilization kinetics and the saturation concentration of EOs were determined. Particle size (measured by DLS) and encapsulation efficiency (EE) were the control parameters assessed, along with the EOs-loaded SMs' stability during 30 days of storage. Additionally, the EOs-loaded SMs' morphology was analyzed using atomic force microscopy (AFM). Finally, the antioxidant activity through the ABTS+ radical scavenging and the reducing power of EOs encapsulated in SMs was determined. The results showed that the solubilization of EOs in SMs was a rapid process with high EE. EOs-loaded SMs-2 systems exhibited greater colloidal stability and higher EE compared to EOs-loaded SMs-1 systems, showing smaller and more homogeneous particle sizes. Moreover, EOs-loaded SMs-2 systems maintained constant EE throughout the storage period. AFM imaging confirmed the rounded and heterogeneous morphology of EOs-loaded SMs-1 and the smaller, more homogeneous, and spherical morphology of EOs-loaded SMs-2. EOs-loaded SMs-2 showed high ABTS+ radical scavenging and reducing power when encapsulated in SMs. In conclusion, the SMs-2 method emerged as an effective approach for producing efficient nanocarriers for EOs, signifying a promising path for future developments in antioxidant delivery systems.
Asunto(s)
Benzotiazoles , Aceites Volátiles , Surfactantes Pulmonares , Ácidos Sulfónicos , Antioxidantes , Micelas , TensoactivosRESUMEN
In the present work, squalene (SQ) was encapsulated by a conventional emulsion method using egg white protein nanoparticles (EWPn) as a high molecular weight surfactant, followed by a freeze-drying process to obtain an SQ powder ingredient. EWPn was produced by heat treatment at 85°C, 10 min, and pH 10.5. EWPn showed higher emulsifying activity regarding native egg white protein (EWP), highlighting their potential to be used for the SQ encapsulation by an emulsification process. First, we explored the encapsulation conditions using pure corn oil as an SQ carrier. Conditions were oil fraction (0.1-0.2), protein amount (2-5 wt.%), homogenization pressure (100 and 200 bar), and maltodextrin amount (10-20 wt.%). At 0.15 oil fraction, 5 wt.%. protein concentration, 200 bar homogenization pressure, and 20% maltodextrin, the highest encapsulation efficiency (EE) was reached. Then, according to these conditions, SQ was encapsulated to obtain a freeze-dried powder ingredient for bread formulation. The total and free oil of SQ freeze-dried powder were 24.4% ± 0.6% and 2.6% ± 0.1%, respectively, resulting in an EE value of 89.5% ± 0.5%. The physical, textural, and sensory properties of functional bread were not affected by the addition of 5.0% SQ freeze-dried powder. Finally, the bread loaves showed higher SQ stability than the one formulated with unencapsulated SQ. Hence, the encapsulation system developed was suitable for obtaining functional bread based on SQ fortification.
Asunto(s)
Pan , Escualeno , Polvos , Proteínas , Emulsiones , LiofilizaciónRESUMEN
This work is aimed to obtain nanocomplexes based on egg white protein nanoparticles (EWPn) and bioactive compounds (BC), carvacrol (CAR), thymol (THY) and trans-cinnamaldehyde (CIN), and evaluate their application as antifungal edible coatings on preservative-free breads. The nanocomplex formation was studied through stoichiometry, affinity, colloidal behavior, morphology, and encapsulation efficiency (EE, %). Rounded-shape nanocomplexes with particle sizes < 100 nm were obtained. The EE values were similar for all BC (>83%). Furthermore, the in vitro antifungal activity of the nanocomplexes was verified using the Aspergillus niger species. The nanocomplexes were applied as coatings onto the crust of preservative-free breads, which were stored for 7 days (at 25 °C). The coatings had no impact on the physicochemical properties of the bread loaves (moisture, aw, texture, and color). Finally, the coatings based on EWPn-THY and EWPn-CAR nanocomplexes showed higher antifungal efficacy, extending the bread shelf life after 7 days.
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Películas Comestibles , Nanopartículas , Antifúngicos/farmacología , Pan , Proteínas del Huevo , Conservantes de Alimentos/farmacologíaRESUMEN
The aim of this work was to examine the behavior of conjugated linoleic acid (CLA) delivery systems based on ovalbumin (OVA) and their derived nanoparticles (OVAn1 and OVAn2), under static in vitro gastrointestinal digestion model. In addition, potential cytotoxic effect of these inclusion complexes on a human colon cancer cell line (HT-29) was evaluated. OVA was resistant to gastric and intestinal digestion, while OVA nanoparticles were very susceptible to digestive enzymes hydrolysis. Particle size distribution (PDS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for OVA evidenced the presence of a protein fragment of similar size after simulated digestive process. Conversely, for nanoparticles, partial and total hydrolysis in gastric and intestinal phases, respectively, was evidenced. After in vitro gastrointestinal digestion, released CLA (RCLA) was assayed. In case of OVA, as CLA carrier, RCLA was 37%, while for OVA nanoparticles, lower RCLA values (~10-20%) were obtained. From cytotoxic assays, it was observed that CLA molecule was responsible for cell death, whereas OVA or their derived nanoparticles were not cytotoxic on HT-29 cells. On the other hand, flow cytometry analysis revealed that main death mechanism for CLA, and their inclusion complexes was apoptosis. OVA-CLA and OVAn1-CLA inclusion complexes displayed the highest potential cytotoxic activity and apoptotic index. Information derived from this work could be relevant for the design of CLA delivery systems as promising nanosupplements for production of new functional and excipient foods for both prevention and control of colon cancer.
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Ácidos Linoleicos Conjugados , Nanopartículas , Digestión , Células HT29 , Humanos , Nanopartículas/toxicidad , OvalbúminaRESUMEN
The objective of this work was to obtain and characterize conjugated linoleic acid (CLA) delivery systems based on ovalbumin nanoparticles (OVAn1 and OVAn2) and to study their behaviour under a standardised static in vitro digestion model. OVAn1 and OVAn2 were obtained by heat treatment (85 °C, 5 min, pH 11.35 and 7.5, respectively). OVAn1 and OVAn2 had hydrodynamic diameters of 24.63 ± 0.04 and 92.0 ± 0.2 nm, respectively, showing no significant differences in ζ potential values (p < 0.05) at pH 7.0. CLA nanocomplexes were examined in terms of size and ζ potential at pH 3.0 and 7.0, highlighting that binding of CLA caused an increase in size for OVA and both OVA nanoparticles. Morphological characterization was performed by confocal laser scanning microscopy (CSLM) finding that OVA and OVA nanoparticles had a circular shape. Also, the CLA encapsulation efficiency (EE) for OVA and OVA nanoparticles was studied, yielding EE values greater than 97% for all systems. Finally, systems were assayed for a standardized in vitro gastrointestinal digestion model considering gastric and intestinal steps. Macroscopic appearance, CSLM images and quantification of CLA retention by HPLC were evaluated after digestion. All the systems showed the formation of macroscopic aggregates both in gastric and intestinal phases, which generated a visible precipitate. In all systems, CSLM confirmed the presence of numerous undefined-form aggregates. Finally, high CLA retention (around 99%) was found for native protein and nanoparticles.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Nanopartículas/metabolismo , Ovalbúmina/metabolismo , Digestión , Ácidos Linoleicos Conjugados/química , Modelos Biológicos , Nanopartículas/química , Ovalbúmina/químicaRESUMEN
Chrysin (5,7-dihydroxyflavone) (Chrys) is a natural flavone extracted from many plants, and it has been proposed as a bioactive agent for cancer therapy. Nevertheless, its use is limited mainly due to its poor water solubility. Bovine serum albumin (BSA) is a water soluble, biocompatible and non-toxic protein with a promising application in lipophilic bioactive compound delivery. Moreover, BSA is heat sensitive, feature that could be used for producing self-assembled nanoparticle with tailor-made properties. In this contribution, we studied the formation of BSA nanoparticles (BSAnp) by thermal treatment at different conditions of temperature (70 °C/5 min and 85 °C/5 min), protein concentration (1.0-4.0%wt.) and aqueous medium pH values (9.0 and 11.0) in which it is known that BSA is found in different unfolded conformations. Binding of Chrys dissolved in dimethyl sulfoxide (DMSO) was studied by fluorescence titration experiments. Characterization of Chrys-loaded and unloaded BSAnp was performed in phosphate buffered saline (PBS) pH 7.4 by applying a set of complementary techniques: dynamic light scattering (DLS), size exclusion fast protein liquid chromatography (SEC-FPLC) and transmission electron microscopy (TEM). Different populations of BSAnp were obtained, which showed different diameters in the range of 1328 nm, ζ potentials around -10.0 mV, molecular weight in the range of 400-1000 kDa and spherical shape. Chrys encapsulation efficiency (EE. %) was also determined, and values between 44-84% were obtained, which mainly depended on the mode of Chrys binding and physicochemical BSAnp properties. Results highlight the ability of self-assembled BSAnp for Chrys vehiculization in an aqueous medium which could found potential application in antitumor therapies.
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Antineoplásicos Fitogénicos/química , Portadores de Fármacos , Flavonoides/química , Nanopartículas/química , Albúmina Sérica Bovina/química , Animales , Tampones (Química) , Bovinos , Calor , Concentración de Iones de Hidrógeno , Peso Molecular , Nanopartículas/ultraestructura , Tamaño de la Partícula , Solubilidad , Agua/químicaRESUMEN
Two polysaccharides (PS), gum arabic (GA) and sodium alginate (SA), and whey protein concentrate (WPC) were used to design bio-based films at two ratios (RPS:WPC, 1:2 and 1:3). The effects of PS, RPS:WPC and WPC thermal treatment (unheated vs. aggregate) were determined on films characteristics. Film-forming dispersions were tested using different complementary techniques: UV-Vis spectroscopy, electrophoretic mobility, bulk rheology and confocal microscopy. PS exhibited weak associations with proteins. However, this behavior was more significative in SA/WPC systems. Rheological and optical characteristics of filmogenic suspensions were influenced by PS, RPS:WPC and WPC heat treatment. Apparent viscosity values for SA/WPC systems were 80-250 times higher than the ones obtained for GA/WPC systems. Furthermore, thickness, moisture absorption, contact angle and mechanical properties were also affected by the film design factors. GA/WPC-aggregates films showed lesser moisture absorption; however, they have higher surface polarity than those made with SA/WPC-aggregates. Moreover, SA/WPC-aggregates systems provided stronger films in comparison with the GA/WPC-aggregates ones. In addition, mechanical properties were also affected by RPS:WPC and WPC treatment. It was observed that denatured WPC and 1:3 RPS:WPC produced weaker mechanical features. Results provide useful information for the design of bio-based mixed films with tailor-made properties.
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Alginatos/química , Plásticos Biodegradables/química , Goma Arábiga/química , Proteína de Suero de Leche/química , Almacenamiento de Alimentos/instrumentación , Humanos , Ensayo de Materiales , Membranas Artificiales , Reología , Viscosidad , HumectabilidadRESUMEN
Stoichiometric, kinetic and thermodynamic aspects of complex formation between heat-induced aggregates of ovalbumin (ovalbumin nanoparticles, OVAn) and linoleic acid (LA) were evaluated. Extrinsic fluorescence data were fitted to modified Scatchard model yielding the following results: n: 49±2 LA molecules bound per OVA monomer unit and Ka: 9.80±2.53×10(5)M. Kinetic and thermodynamic properties were analyzed by turbidity measurements at different LA/OVA monomer molar ratios (21.5-172) and temperatures (20-40°C). An adsorption approach was used and a pseudo-second-order kinetics was found for LA-OVAn complex formation. This adsorption process took place within 1h. Thermodynamic parameters indicated that LA adsorption on OVAn was a spontaneous, endothermic and entropically-driven process, highlighting the hydrophobic nature of the LA and OVAn interaction. Finally, Atomic Force Microscopy imaging revealed that both OVAn and LA-OVAn complexes have a roughly rounded form with size lower than 100nm.
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Ácido Linoleico/química , Nanopartículas/química , Ovalbúmina/química , Termodinámica , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Ácido Linoleico/metabolismo , Microscopía de Fuerza Atómica/métodos , Ovalbúmina/metabolismo , EstereoisomerismoRESUMEN
Information about the design of biopolymer nanoparticles (BNPs) for polyunsaturated fatty acid (PUFA) vehiculization is provided. Linoleic acid (LA) was used as a model PUFA. The binding ability of LA to ß-lactoglobulin (BLG) was applied for obtaining BLG-LA complexes. BLG-LA complex formation was monitored by fluorimetry and it was observed that a moderate heat treatment (60 °C, 10 min) enhanced BLG-LA complexation. Obtaining BNPs involved the electrostatic deposition of high methoxyl pectin (HMP) onto the BLG-LA complex surface. The phase behavior of biopolymer systems was discussed at different Prot:HMP ratio (RProt:HMP, wt.%) levels (1:1-6:1). Absorbance at 600 nm, particle size, and ζ potential were analyzed at pH 4.0. At 1:1-2:1 RProt:HMP, BNPs showed appreciable turbidity, a nanometric diameter (337-364 nm), and a negative ζ potential. Finally, intrinsic and extrinsic fluorimetry was used for examining the HMP protective role at the LA binding site. At 2:1 RProt:HMP, HMP cover could promote significant LA protection in BNPs.
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Biopolímeros/química , Ácidos Grasos Insaturados/química , Nanopartículas/química , Pectinas/química , Polisacáridos/química , Lactoglobulinas/químicaRESUMEN
In the present work, ovalbumin (OVA) solutions (10 g/L, 50 mM NaCl, pH 7.5) were heat-treated at 75, 80 and 85°C (namely, OVA-75, OVA-80 and OVA-85, respectively), from 0 to 25 min. OVA nanoparticles (OVAn) around 100 nm were obtained. For 3 min of heat treatment, OVAn sizes increased with temperature, but for a heating time longer than 10 min, OVA-75 showed the highest size values. OVAn surface hydrophobicity increased 6-8 folds in comparison with native OVA and wavelength blue shifts of 25-30 nm in maximum fluorescence intensity were registered. These results suggest that buried hydrophobic residues were exposed to the aqueous medium. Binding experiments with linoleic acid (LA) as polyunsaturated fatty acid (PUFA) model were carried out. Firstly, binding ability of OVAn was determined from LA titration curves of intrinsic fluorescence measurements. OVA-85 at 5 min presented the highest binding ability and it was used for further binding properties studies (turbidity, particle size distribution--PSD--analysis and ζ-potential measurements). Turbidity measurement and PSD analysis showed that OVAn-LA nanocomplexes were formed, avoiding LA supramolecular self-assembly formation. The union of LA to OVAn surface confers them significant lower ζ-potential and larger size. Hence, fluorescence and ζ-potential results showed that LA would bind to OVAn by mean of hydrophobic interactions. Information derived from this work could be important to potentially use OVAn as PUFA vehiculization with applications in several industrial sectors (food, pharmaceutical, cosmetics, etc.).
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Ácido Linoleico/química , Nanopartículas/química , Ovalbúmina/química , Animales , Sitios de Unión , Pollos , Fluorescencia , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Tamaño de la Partícula , Unión ProteicaRESUMEN
The aim of this work was to obtain heat-induced ß-lactoglobulin (BLG) aggregates in order to test them as carriers of a model polyunsaturated fatty acid (PUFA), linoleic acid (LA). BLG aggregates were obtained at 85 °C by varying the heating time (0-60 min) and pH of protein dispersion (6.5-7.5). Aggregates were characterised by intrinsic and extrinsic fluorescence and surface hydrophobicity (S0). Binding experiments were conducted by fluorescence spectroscopy. Results showed increased BLG aggregate S0 values which could strongly depend on the pH of aggregate formation. Aggregates obtained at pH 6.5 showed the greatest S0 values, so they could find application as LA carriers. Nevertheless, conjugation of LA to BLG aggregates showed complex behaviour depending on the aggregate producing conditions (pH, heating time and/or combination). The LA binding properties of BLG aggregates were not linked to their hydrophobic characteristics, suggesting that conjugation could require the structural preservation of the LA binding site.
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Ácidos Grasos Insaturados/química , Lactoglobulinas/química , Espectrometría de Fluorescencia/métodos , Calor , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
ß-Lactoglobulin (BLG) is a member of lipocalin family, proteins with ability to bind small hydrophobic ligands, such as retinol, vitamins and fatty acids. Moreover, BLG is susceptible to protease action producing a wide range of polypeptides depending on the hydrolysis degree (HD). In the present work, the effect of limited enzymatic hydrolysis on fatty acid binding properties of BLG was studied. Linoleic acid (LA) was used as a model fatty acid. Limited enzymatic hydrolysis was performed using α-chymotrypsin immobilised on agarose microparticles. BLG hydrolysates were produced at HD: 1%, 3% and 5%. In order to determine the influence of HD on BLG molecular weight SDS-PAGE was used. BLG structural modification and LA binding properties were monitored by means of fluorescence spectroscopic techniques. The increase in HD produced: (i) a BLG degradation and a molecular weight distribution of BLG hydrolysates and (ii) an increased exposition of buried hydrophobic residues, however it was observed a decrease in surface hydrophobicity possibly due to a deterioration of hydrophobic protein domains. It was observed that enzymatic hydrolysis treatment produced a decrease in BLG ability for binding LA. It was concluded that limited enzymatic hydrolysis could deteriorate the specific site on BLG structure necessary for binding LA.
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Quimotripsina/química , Lactoglobulinas/química , Ácido Linoleico/química , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Milk whey proteins (MWP) and pectins (Ps) are biopolymer ingredients commonly used in the manufacture of colloidal food products. Therefore, knowledge of the interfacial characteristics of these biopolymers and their mixtures is very important for the design of food dispersion formulations (foams and/or emulsions). In this paper, we examine the adsorption and surface dilatational behaviour of MWP/Ps systems under conditions in which biopolymers can saturate the air-water interface on their own. Experiments were performed at constant temperature (20 °C), pH 7 and ionic strength 0.05 M. Two MWP samples, ß-lactoglobulin (ß-LG) and whey protein concentrate (WPC), and two Ps samples, low-methoxyl pectin (LMP) and high-methoxyl pectin (HMP) were evaluated. The contribution of biopolymers (MWP and Ps) to the interfacial properties of mixed systems was evaluated on the basis of their individual surface molecular characteristics. Biopolymer bulk concentration capable of saturating the air-water interface was estimated from surface pressure isotherms. Under conditions of interfacial saturation, dynamic adsorption behaviour (surface pressure and dilatational rheological characteristics) of MWP/Ps systems was discussed from a kinetic point of view, in terms of molecular diffusion, penetration and configurational rearrangement at the air-water interface. The main adsorption mechanism in MWP/LMP mixtures might be the MWP interfacial segregation due to the thermodynamic incompatibility between MWP and LMP (synergistic mechanism); while the interfacial adsorption in MWP/HMP mixtures could be characterized by a competitive mechanism between MWP and HMP at the air-water interface (antagonistic mechanism). The magnitude of these phenomena could be closely related to differences in molecular composition and/or aggregation state of MWP (ß-LG and WPC).
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Proteínas de la Leche/química , Pectinas/química , Adsorción , Aire , Concentración de Iones de Hidrógeno , Cinética , Lactoglobulinas/química , Proteínas de la Leche/farmacocinética , Concentración Osmolar , Propiedades de Superficie , Temperatura , Termodinámica , Agua/química , Proteína de Suero de LecheRESUMEN
In this contribution, we present experimental information about the effect of xanthan gum (XG) on the adsorption behaviour of two milk whey protein samples (MWP), beta-lactoglobulin (beta-LG) and whey protein concentrate (WPC), at the air-water interface. The MWP concentration studied corresponded to the protein bulk concentration which is able to saturate the air-water interface (1.0 wt%). Temperature, pH and ionic strength of aqueous systems were kept constant at 20 degrees C, pH 7 and 0.05 M, respectively, while the XG bulk concentration varied in the range 0.00-0.25 wt%. Biopolymer interactions in solution were analyzed by extrinsic fluorescence spectroscopy using 1-anilino-8-naphtalene sulphonic acid (ANS) as a protein fluorescence probe. Interfacial biopolymer interactions were evaluated by dynamic tensiometry and surface dilatational rheology. Adsorption behaviour was discussed from a rheokinetic point of view in terms of molecular diffusion, penetration and conformational rearrangement of adsorbed protein residues at the air-water interface. Differences in the interaction magnitude, both in solution and at the interface vicinity, and in the adsorption rheokinetic parameters were observed in MWP/XG mixed systems depending on the protein type (beta-LG or WPC) and biopolymer relative concentration. beta-LG adsorption in XG presence could be promoted by mechanisms based on biopolymer segregative interactions and thermodynamic incompatibility in the interface vicinity, resulting in better surface and viscoelastic properties. The same mechanism could be responsible of WPC interfacial adsorption in the presence of XG. The interfacial functionality of WPC was improved by the synergistic interactions with XG, although WPC chemical complexity might complicate the elucidation of molecular events that govern adsorption dynamics of WPC/XG mixed systems at the air-water interface.