pH-response of protein-polysaccharide multilayers adsorbed on a flat gold surface: A surface plasmon resonance study.

Polysaccharide-protein multilayers (PPMLs) consisting of bovine serum albumin (BSA) and chondroitin sulfate (CS) are assembled in acidic solution (pH 4.2) via layer-by-layer deposition method. The formation of PPMLs on gold surface and their responsiveness to pH change from 4.2 to 7 is investigated by Surface Plasmon Resonance Spectroscopy. The buildup of the multilayer at pH 4.2 exhibits non-linear growth while the formation of the first layers is strongly affected by the physicochemical properties of the gold surface. Neutral solution (pH 7) affects the interactions between the biopolymers and results in a partially disassemble (disintegration) of the multilayer film. On one hand, the single pair of layers, BSA-CS and the double pair of layers, (BSA-CS)2, assemblies are stable in neutral pH, a result that will be of interest for biomedical applications. On the other hand, multilayer films consisting of more than four layers that is (BSA-CS)2

A sustainable bioprocess to produce bacterial cellulose (BC) using waste streams from wine distilleries and the biodiesel industry: evaluation of BC for adsorption of phenolic compounds, dyes and metals.

BACKGROUND: The main challenge for large-scale production of bacterial cellulose (BC) includes high production costs interlinked with raw materials, and low production rates. The valorization of renewable nutrient sources could improve the economic effectiveness of BC fermentation while their direct bioconversion into sustainable biopolymers addresses environmental pollution and/or resource depletion challenges. Herein a green bioprocess was developed to produce BC in high amounts with the rather unexplored bacterial strain Komagataeibacter rhaeticus, using waste streams such as wine distillery effluents (WDE) and biodiesel-derived glycerol. Also, BC was evaluated as a bio-adsorbent for phenolics, dyes and metals removal to enlarge its market diversification. RESULTS: BC production was significantly affected by the WDE mixing ratio (0-100%), glycerol concentration (20-45 g/L), type of glycerol and media-sterilization method. A maximum BC concentration of 9.0 g/L, with a productivity of 0.90 g/L/day and a water holding capacity of 60.1 g water/g dry BC, was achieved at 100% WDE and ≈30 g/L crude glycerol. BC samples showed typical cellulose vibration bands and average fiber diameters between 37.2 and 89.6 nm. The BC capacity to dephenolize WDE and adsorb phenolics during fermentation reached respectively, up to 50.7% and 26.96 mg gallic acid equivalents/g dry BC (in-situ process). The produced BC was also investigated for dye and metal removal. The highest removal of dye acid yellow 17 (54.3%) was recorded when 5% of BC was applied as the bio-adsorbent. Experiments performed in a multi-metal synthetic wastewater showed that BC could remove up to 96% of Zn and 97% of Cd. CONCLUSIONS: This work demonstrated a low-carbon approach to produce low-cost, green and biodegradable BC-based bio-adsorbents, without any chemical modification. Their potential in wastewater-treatment-applications was highlighted, promoting closed-loop systems within the circular economy era. This study may serve as an orientation for future research towards competitive or targeted adsorption technologies for wastewater treatment or resources recovery.

Advances in Small Angle Neutron Scattering on Polysaccharide Materials.

Polysaccharide materials and biomaterials gain the focus of intense research owing to their great versatility in chemical structures and modification possibilities, as well as their biocompatibility, degradability, and sustainability features. This review focuses on the recent advances in the application of SANS on polysaccharide systems covering a broad range of materials such as nanoparticulate assemblies, hydrogels, nanocomposites, and plant-originating nanostructured systems. It motivates the use of SANS in its full potential by demonstrating the features of contrast variation and contrast matching methods and by reporting the methodologies for data analysis and interpretation. As these soft matter systems may be organized in multiple length scales depending on the interactions and chemical bonds between their components, SANS offers exceptional and unique opportunities for advanced characterization and optimization of new nanostructured polysaccharide materials.

Hydration effects on thermal transitions and molecular mobility in Xanthan gum polysaccharides.

In this work, the xanthan gum (XG) polysaccharide is studied over a wide range of temperatures and water fractions 0 ≤ hw ≤ 0.70 (on a wet basis) by employing differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). The investigation reveals that the critical water fraction for ice formation is about 0.35. Glass transition temperature (Tg) was determined through calorimetry experiments for all the samples studied. Water acts as a strong plasticizer, i.e., decreasing Tg, for water fractions up to about 0.35. A secondary (local) relaxation process is recorded in both dry and hydrated samples, which is sensitive to the presence of water molecules. This fact indicates that this process originates due to the orientation of small polar groups of the side chain, or/and due to the local main chain dynamics. Two types of long-range charge transport processes were resolved. The first is related to the conductive paths being formed via bulk-like ice structures (at high hydration levels), whereas the second can be attributed to proton mobility via the hydrogen bond (HB) network of non-freezing water existing in XG. Interestingly, this process is exactly the same in all the hydrated samples with hw > 0.25. With respect to the sample with hw = 0.27, a Vogel-Tammann-Fulcher (VTF)-like polarization process has also been recorded which seems to be related to long-range charge mobility via interconnected water clusters. As far as we are aware, this is the first time that XG is studied in terms of glass transition and molecular mobility over a wide range of hydration levels combining DSC and BDS techniques.

Thermally stabilized chondroitin sulfate-hemoglobin nanoparticles and their interaction with bioactive compounds.

The preparation of nanoparticles (NPs) based on hemoglobin (Hb) with a fully biocompatible methodology is presented. The spontaneous formation of electrostatic complexes of Hb with chondroitin sulfate (CS) at pH 4 in the polysaccharide/protein mass ratio regime where charge neutrality is met leads to spherical nanostructures with monomodal hydrodynamic radii distribution in the range of 50-100 nm. The integrity of the electrostatic complexes is disturbed at pH 7 as the net electric charge of Hb is very low. Treating the NPs at mildly elevated temperature stabilizes them against the pH increase taking advantage of Hb's ability of unfolding and self-associating upon thermal treatment. The NPs surface charge is pH-tunable and changes from positive to strongly negative upon pH increase to 7 proving the presence of negative surface patches of Hb and CS segments in their exterior. The α-helix content of Hb does not change significantly by thermal treatment. The NPs are found to bind the bioactive compounds curcumin and ß-carotene and are stable in solutions with high salt content. This investigation introduces a straightforward method to formulate Hb in NPs with possibilities in the nanodelivery of nutrients and drugs.

Protein-induced transformation of unilamellar to multilamellar vesicles triggered by a polysaccharide.

We report on the morphological transitions of didodecyldimethylammonium bromide (DDAB) cationic vesicles and hybrid DDAB/hyaluronic acid (HA) vesicles upon addition of BSA at pH 7 where BSA is overall negatively charged. Small angle neutron scattering (SANS) is used to extract the size distributions of the nanovesicles, the thickness of the DDAB bilayers and their lamellarity. Although the HA-decorated DDAB vesicles contain the negatively charged polysaccharide the interaction with BSA appears to be more intense in comparison to bare vesicles. Characteristic peaks in the SANS patterns indicate the presence of multilamellar interfaces while the formation of multilamellar vesicles induced by BSA depends on the amount of added HA. Consequently, higher lamellarities are observed at higher BSA contents. This work demonstrates a simple methodology to tune the encapsulation of globular proteins in vesicular nanoassemblies by affecting their lamellarity and has direct implications on the application of vesicles and liposomes in protein delivery.

Physicochemical properties of electrostatically crosslinked carrageenan/chitosan hydrogels and carrageenan/chitosan/Laponite nanocomposite hydrogels.

In this work physical carrageenan/chitosan (Car/Chit) hydrogels are prepared by electrostatic complexation between the two oppositely charged polysaccharides. The hydrogels have storage moduli in the order of 5-10 kPa and swelling ratios in the order of 5000-6000 %. At conditions where both polysaccharides are highly charged (pH 5) the swelling ratios are lower than the ones at conditions of lower dissociation i.e., at pH 2 and 7 and the opposite trend is found for the storage modulus. Chit appears to act as a crosslinker for Car as increasing its concentration the swelling ratio decreases and the moduli increase. The hydrogels can incorporate the nanoclay Laponite (Lap) and form hybrid nanocomposites where the intercalation by the two biopolymers leads to exfoliation of the clay nanoplatelets in the presence of both Car and Chit. The composite hydrogels retain the mechanical properties of the Car/Chit hydrogels at the studied pH range (pH 2 to pH 7). This shows the prepared hydrogels can be potentially used as multifunctional biomaterials for drug delivery, tissue engineering and bone regeneration applications.

Sustainable Production of Novel Oleogels Valorizing Microbial Oil Rich in Carotenoids Derived from Spent Coffee Grounds.

Sustainable food systems that employ renewable resources without competition with the food chain are drivers for the bioeconomy era. This study reports the valorization of microwave-pretreated spent coffee grounds (SCGs) to produce oleogels rich in bioactive compounds. Microbial oil rich in carotenoids (MOC) was produced under batch fermentation of Rhodosporidium toruloides using SCG enzymatic hydrolysates. Candelilla wax (CLW) could structure MOC and sunflower oil at a 3.3-fold lower concentration than that of carnauba wax (CBW). MOC-based oleogels with 10% CBW and 3% CLW showed an elastic-dominant and gel-like structure (tan δ ≪ 1), providing gelation and oil binding capacity (>95%). Dendritic structures of CBW-based oleogels and evenly distributed rod-like crystals of CLW-based ones were observed via polarized light microscopy. MOC-based oleogels exhibited similar Fourier-transform infrared spectroscopy spectra. X-ray diffractograms of oleogels were distinguished by the oil type that presented ß'-type polymorphism. MOC-based oleogels could be applied in confectionary products and spreads as substitutes for trans fatty acids, reformulating fat-containing food products.

Development of biodegradable films using sunflower protein isolates and bacterial nanocellulose as innovative food packaging materials for fresh fruit preservation.

This study presents the valorization of side streams from the sunflower-based biodiesel industry for the production of bio-based and biodegradable food packaging following circular economy principles. Bacterial cellulose (BC) was produced via fermentation in 6 L static tray bioreactors using nutrient-rich supplements derived from the enzymatic hydrolysis of sunflower meal (SFM) combined with crude glycerol as carbon source. Novel biofilms were produced using either matrices of protein isolates extracted from sunflower meal (SFMPI) alone or SFMPI matrices reinforced with nanocellulose biofillers of commercial or bacterial origin. Acid hydrolysis was employed for ex-situ modification of BC to nanostructures (56 nm). The biofilms reinforced with bacterial nanocellulose structures (SFMPI-BNC) showed 64.5% higher tensile strength, 75.5% higher Young's modulus, 131.5% higher elongation at break, 32.5% lower water solubility and 14.1% lower water vapor permeability than the biofilms produced only with SFMPI. The biofilms were evaluated on fresh strawberries packaging showing that the SFMPI-BNC-based films lead to effective preservation at 10 °C considering microbial growth and physicochemical profile (weight loss, chemical characterization, color, firmness and respiration activity). The SFMPI-BNC-based films could be applied in fresh fruit packaging applications.

Preparation of trypsin-based nanoparticles, colloidal properties and ability to bind bioactive compounds.

Nanoparticles (NPs) based on the proteolytic enzyme trypsin (TRY) were prepared by a biocompatible methodology. TRY co-assembled with the anionic polysaccharide chondroitin sulfate (CS) in complexes with well-defined distributions of radii in the range of 100-200 nm by electrostatic complexation at acidic conditions. At pH 7 the complexes were unstable and lost their monomodal size distribution which is potentially related to TRY's weak positive net surface charge and a large negative charge patch that forms at neutral pH. Thermal treatment at conditions which were not expected to interfere with TRY's proteolytic activity was used to stabilize the complexes into NPs that resisted disintegration at pH 7 taking advantage of the ability of the TRY globules to thermally aggregate. The secondary conformation of TRY within the NPs was found fairly unperturbed even after thermal treatment which is crucial for its physiological function. The CS-TRY NPs could bind and encapsulate the bioactive substances curcumin (CUR) and ß-carotene (ß-C) owing to TRY's hydrophobic domains. The CS-TRY NPs may be considered as a platform for the immobilized active enzyme and multifunctional NPs for hydrophobic bioactive compounds.

Formation and physicochemical properties of glycogen phosphorylase in complex with a cationic polyelectrolyte.

The accumulation of rabbit muscle glycogen phosphorylase b (RMGPb) in electrostatic complexes with the cationic polyelectrolyte poly 2-(dimethylamino) ethyl methacrylate in its quenched form (QPDMAEMA) was studied in two buffer solutions. In the N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, large complexes of RMGPb-QPDMAEMA were formed which adopted smaller sizes as QPDMAEMA concentration increased. However, in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, the hydrodynamic radius of the formed complexes gradually increased as the polymer concentration increased. Zeta potential measurements (ζp) showed that RMGPb significantly changed the ζp of the QPDMAEMA aggregates. Fluorescence studies showed that the interaction between RMGPb and QPDMAEAMA was enhanced as polymer concentration increased. Specifically, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence indicated that in the BES buffer the aggregates became denser as more QPDMAEMA was added, while in the HEPES buffer the density of the formed structures decreased. RMGPb's secondary structure was examined by Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) and Circular Dichroism (CD) showing that QPDMAEMA interaction with RMGPb does not induce any changes to the secondary structure of the enzyme. These observations suggest that cationic polyelectrolytes may be utilized for the formulation of RMGPb in multifunctional nanostructures and be further exploited in innovative biotechnology applications and bioinspired materials development.

Current Advances of Polysaccharide-Based Nanogels and Microgels in Food and Biomedical Sciences.

Polysaccharides are natural polymers with hydrophilic, biocompatible and biodegradable characteristics and have many opportunities in the food and pharmaceutical sectors. This review focuses on the field of nano and microstructures whose internal structure is based on networked polysaccharide chains in 3D i.e., polysaccharide nanogels (NGs) and microgels (MGs). As it is observed the number of articles on NGs and MGs in peer reviewed scientific journals has been increasing over the last two decades. At the same time, the relative contribution of polysaccharides in this field is gaining place. This review focuses on the different applied methods for the fabrication of a variety of polysaccharide-based NGs and MGs and aims to highlight the recent advances on the subject and present their potentials and properties with regards to their integration in aspects of medicinal and food sciences. The presentation of the recent advances in the application of polysaccharide NGs and MGs is divided in materials with potential as emulsion stabilizers and materials with potential as carriers of bioactives. For applications in the medical sector the division is based on the fabrication processes and includes self-assembled, electrostatically complexed/ionically crosslinked and chemically crosslinked NGs and MGs. It is concluded that many advances are expected in the application of these polysaccharide-based materials and in particular as nutrient-loaded emulsion stabilizers, viscosity modifiers and co-assembled structures in combination with proteins.

Polysaccharide-Protein Multilayers Based on Chitosan-Fibrinogen Assemblies for Cardiac Cell Engineering.

The cell and tissue culture substrates play a pivotal role in the regulation of cell-matrix and cell-cell interactions. The surface properties of the materials control a wide variety of cell functions. Amongst various methods, layer-by-layer (LbL) assembly is a versatile surface coating technique for creating controllable bio-coatings. Here, polysaccharide/protein multilayers are proposed, which are fabricated by immersive LbL assembly and based on the chitosan/fibrinogen pair for improving the adhesion and spreading of cardiomyocytes. Two approaches in LbL assembly are employed for clarifying the effect of the bilayers order and their concentration on cardiomyocytes viability and morphology. Fourier transform infrared spectroscopy (FTIR) measurements show that the adsorption of the biopolymers is enhanced during the LbL deposition in a synergistic manner. Contact angle measurements indicate that the multilayers are alternating from less to more hydrophilic behavior depending on the biopolymer that is added last. Confocal microscopy with immunostained fibrinogen reveals that the amount of the protein is higher when the concentration of the immersion solution is increased, however, for low solution concentration it is speculated that interdigitation between the separate biopolymer layers takes place. This work motivates the use of fibrinogen in polysaccharide/protein multilayers for enhanced cytocompatibility in cardiac tissue engineering.

Lamellarity and size distributions in mixed DPPC/amphiphilic poly(2-oxazoline) gradient copolymer vesicles and their temperature response.

Mixed liposomes of dipalmitoylphosphatidylcholine (DPPC) and gradient (pseudodiblock) poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) (MPOx) copolymers are investigated by small angle neutron scattering (SANS). All experimental data, from different phospholipid-copolymer compositions, concentrations and temperatures are fitted with one model. This model allows the determination of the separate contributions from vesicular populations of different lamellarity and size. MPOx copolymers are proved to modify both the size and lamellarity of DPPC liposomes. The gradient copolymer with higher hydrophilic content induces shrinkage of the uni- and bi-lamellar DPPC vesicles. The copolymer with lower hydrophilic content causes dramatic changes on the lamellarity of DPPC vesicles by the formation of hexa-lamellar vesicles. The tendency of multi-lamellar vesicles to transform into uni-lamellar ones as temperature increases is more pronounced in the presence of the copolymers. These findings may have direct implications on the drug loading and release properties of liposomes and their interactions with cells.

Formation of Uni-Lamellar Vesicles in Mixtures of DPPC with PEO-b-PCL Amphiphilic Diblock Copolymers.

The ability of mixtures of 1.2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the amphiphilic diblock copolymers poly (ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) to stabilize uni-lamellar nano-vesicles is reported. Small angle neutron scattering (SANS) is used to define their size distribution and bilayer structure and resolve the copresence of aggregates and clusters in solution. The vesicles have a broad size distribution which is compatible with bilayer membranes of relatively low bending stiffness. Their mean diameter increases moderately with temperature and their number density and mass is higher in the case of the diblock copolymer with the larger hydrophobic block. Bayesian analysis is performed in order to justify the use of the particular SANS fitting model and confirm the reliability of the extracted parameters. This study shows that amphiphilic block copolymers can be effectively used to prepare mixed lipid-block copolymer vesicles with controlled lamellarity and a significant potential as nanocarriers for drug delivery.

Nanoformulation of fibrinogen by thermal stabilization of its electrostatic complexes with hyaluronic acid.

The formulation of well-defined and stable fibrinogen-based nanoparticles (NPs) without the use of any chemical reaction or any toxic organic solvent is reported. Electrostatic interaction between hyaluronic acid (HA) and fibrinogen (Fbg) leads to well-defined complexes at acidic pH which however readily dissolve at neutral pH. On the other hand, when thermal treatment is applied on the pre-formed complexes NPs keep their integrity. Circular dichroism indicates that the protein's native secondary conformation in the final NPs is not affected by the formulation. The tendency of the complexes to aggregate at elevated ionic strengths is greatly suppressed after the application of the temperature treatment protocol. This characteristic is even more pronounced at neutral pH and it is connected to the enhanced surface charge of the NPs. The encapsulation of the hydrophobic compound curcumin causes only weak secondary aggregation. This work shows that the ability of Fbg to self-assemble upon thermal treatment can be effectively used to stabilize Fbg nanoformulations inside complexes with polysaccharides.

Combining particle tracking microrheology and viscometry for the study of DNA aqueous solutions.

We use video particle tracking microrheology (VPTMR) in order to investigate the viscoelasticity of salmon DNA and correlate it to its steady-flow shear-thinning viscosity. Aqueous solutions of DNA are tested in a wide concentration range from the dilute to the semidilute unentangled concentration regime. The observed mean squared displacement shows power-law scaling with lag-time which is equivalent to power-law behavior of the complex modulus as a function of frequency that is, |G* (ω)| = S ∙ ω α . The relaxation exponent α changes abruptly with concentration in the semidilute regime from about 1 to about 0.5 which is the exponent predicted by the Rouse model. The quasi-property S follows the scaling of viscosity for uncharged polymers near θ-conditions in the semidilute regime that is, η ∼ c 1 / 3 ν eff - 1 with νeff = 0.50 - 0.51. The shear-thinning exponent observed by viscometry increases gradually towards the value of 0.5 which has been predicted for Rouse chains under flow. Our findings are in agreement with recent studies of DNA solutions where DNA is treated as a model polymer and addresses the low-molar mass regime of DNA viscoelasticity. This work demonstrates that the combination of passive particle tracking with viscometry can provide a complete picture on the viscoelasticity of DNA-based biopolymer materials.

Temperature-induced aggregation behavior in bovine pancreas trypsin solutions.

In this study, we investigate the effect of temperature treatment on Bovine Pancreas Trypsin (BPT) in aqueous solutions using dynamic, static and electrophoretic light scattering, fluorescence spectroscopy and circular dichroism. Static and dynamic light scattering at various solution conditions i.e. different salt content and pH, reveals that BPT aggregation is enhanced as temperature increases in a non-reversible manner. At acidic pH protein monomers are the dominant population over aggregates of globules, nevertheless the two populations co-exist at neutral and basic pH. The surface charge of the aggregates is intensified by aggregation and it is dominated by the negative residues of the protein at all pH conditions. Protein unfolding upon thermal treatment is probed by variation of the fluorescence spectrum which is caused by the exposure of tryptophan to the aqueous environment. The exposure of the hydrophobic interior of BPT upon heating may be considered as the reason of aggregation at the molecular level. Τhis study provides information that can be useful for utilizing thermal treatment protocols of BPT towards manufacturing protein-based nano formulated drugs.

Reorganizations inside thermally stabilized protein/polysaccharide nanocarriers investigated by small angle neutron scattering.

We use small angle neutron scattering (SANS) to characterize the internal morphology of nanoparticles (NPs) formed by the complexation between chondroitin sulfate (CS) and bovine serum albumin (BSA) and subsequent stabilization by thermal treatment. We demonstrate that SANS can quantify the internal rearrangements and protein morphology alterations upon thermal treatment and solution conditions variations. A three-level Guinier/power-law hierarchical model identifies BSA globules, interconnected clusters of globules and higher aggregates. The effect of temperature treatment which causes protein denaturation and ß-sheet inter-globular associations is captured by the model at the relevant length scales 1-100 nm. Furthermore, the nanogel behavior of the thermally treated CS/BSA NPs, which up to this moment had been characterized by light scattering, is elucidated. Loading of the nutraceutical compound ß-carotene (ß-C) is revealed to occur at the outer regions of the NPs. This study demonstrates that the combination of SANS and a hierarchical model for data analysis may provide very useful insights for nanocarriers which are based on polysaccharide/protein complexes and aim at biomedical applications and the food industry.

Association and Internal Morphology of Self-Assembled HPPhOx/BSA Hybrid Nanoparticles in Aqueous Solutions.

We investigate the formation of hybrid polyelectrolyte/protein nanoparticles by associations between aggregates of partially hydrolyzed poly(2-phenyl-2-oxazoline) (HPPhOx) and bovine serum albumin (BSA) in aqueous solutions. Light scattering experiments show that at conditions of low salt, BSA creates interaggregate bridges and increases the size of the HPPhOx nanoparticles. At high salt contents, breaking of aggregates leads to well-defined nanoparticles. The interior of the formed nanoparticles is probed by small-angle neutron scattering. At low salt, diffuse arrangements are observed, whereas at high salt concentration, scattering is dominated by well-defined hydrophobic domains enhanced by the incorporation of BSA. This system shows that the combination of hydrophobic and electrostatic interactions in random-amphiphilic-polyelectrolyte/protein complexes can be used to determine the properties of self-assembled hybrid multifunctional nanoparticles.