Liliana G. Santiago

β-Lactoglobulin heat-induced aggregates as carriers of polyunsaturated fatty acids

The aim of this work was to obtain heat-induced β-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.

Milk whey proteins and xanthan gum interactions in solution and at the air-water interface: A rheokinetic study

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.

Effect of limited enzymatic hydrolysis on linoleic acid binding properties of β-lactoglobulin.

β-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.

Biopolymer nanoparticles designed for polyunsaturated fatty acid vehiculization: Protein–polysaccharide ratio study

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 β-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.

Surface adsorption behaviour of milk whey protein and pectin mixtures under conditions of air-water interface saturation

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, β-lactoglobulin (β-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 (β-LG and WPC).

Self-assembly of myristic acid in the presence of choline hydroxide: effect of molar ratio and temperature.

Salt-free catanionic systems based on fatty acids exhibit a broad polymorphism by simply tuning the molar ratio between the two components. For fatty acid combined with organic amino counter-ions, very few data are available on the phase behavior obtained as a function of the molar ratio between the counter-ion and the fatty acid. We investigated the choline hydroxide/myristic acid system by varying the molar ratio, R=n(choline hydroxide)/n(myristic acid), and the temperature. Myristic acid ionization state was determined by coupling pH, conductivity and infra-red spectroscopy measurements. Self-assemblies were characterized by small angle neutron scattering and microscopy experiments. Self-assembly thermal behavior was investigated by differential scanning calorimetry, wide angle X-ray scattering and nuclear magnetic resonance. For R<1, ionized and protonated myristic acid molecules coexisted leading to the formation of facetted self-assemblies and lamellar phases. The melting process between the gel and the fluid state of these bilayers induced a structural change from facetted or lamellar objects to spherical vesicles. For R>1, myristic acid molecules were ionized and formed spherical micelles. Our study highlights that both R and temperature are two key parameters to finely control the self-assembly structure formed by myristic acid in the presence of choline hydroxide.

Chromatographic fractionation and molecular mass characterization of Cercidium praecox (Brea) gum.

BACKGROUND Brea gum (BG) is an exudate from the Cercidium praecox tree that grows in semi-arid regions of Argentina. Some previous studies on BG have shown physicochemical characteristics and functional features similar to those of gum arabic. However, there is a need to elucidate the molecular structure of BG to understand the functionality. In this sense, BG was fractionated using hydrophobic interaction chromatography and the obtained fractions were analyzed by size exclusion chromatography. RESULTS Analysis of the fractions showed that the bulk of the gum (approx. 84% of the polysaccharides) was a polysaccharide of 2.79 × 10(3)  kDa. The second major fraction (approx. 16% of the polysaccharides) was a polysaccharide-protein complex with a molecular mass of 1.92 × 10(5)  kDa. A third fraction consisted of protein species with a wide range of molecular weights. The molecular weight distribution of the protein fraction was analyzed by size exclusion chromatography. Comparison of the elution profiles of the exudates in native and reducing conditions revealed that some of the proteins were forming aggregates through disulfide bridges in native conditions. Further analysis of the protein fraction by SDS-PAGE showed proteins with molecular weight ranging from 6.5 to 66 kDa. CONCLUSIONS The findings showed that BG consists of several fractions with heterogeneous chemical composition and polydisperse molecular weight distributions.

Formation and characterization of self-assembled bovine serum albumin nanoparticles as chrysin delivery systems

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.

Emerging Technologies for Bioactive Applications in Foods

The fast development of micro- and nanotechnologies in recent decades is providing novel platforms for the development and production of modified foods. Micro- and nanoscale devices introduced in foods will allow to synthesize novel enriched foods for special purposes. Functional and/or fortified foods based on novel technologies are allowing specifically to deliver nutrients in adequate quantities and in the proper place without losing bioactivity. The design of matrices is a key issue that requires the biophysical and chemical analysis of matrix components, and the potential interactions with the load and the environment. The most accepted matrices for food applications include lipids and biopolymers, e.g., polysaccharides and proteins , also their hybrid systems. Novel lipidic structures for food encapsulation are the “nano” versions of liposomes and emulsions, but also novel devices as solid lipid nanoparticles and nanostructured lipid carriers useful for hydrophobic loads. Biopolymeric structures including polysaccharides and proteins, which are available in large scale as raw materials, are easy to chemically and enzymatically modify to specially tailor the matrices not only for the environmental conditions but also for the physicochemical properties of the load. Hybrid micro- and nanosystems, combining polysaccharides, proteins, and lipids, are providing a myriad of novel devices able to be employed for diverse food applications.