Freeze vs. Spray Drying for Dry Wild Thyme (Thymus serpyllum L.) Extract Formulations: The Impact of Gelatin as a Coating Material.

Freeze drying was compared with spray drying regarding feasibility to process wild thyme drugs in order to obtain dry formulations at laboratory scale starting from liquid extracts produced by different extraction methods: maceration and heat-, ultrasound-, and microwave-assisted extractions. Higher total powder yield (based on the dry weight prior to extraction) was achieved by freeze than spray drying and lower loss of total polyphenol content (TPC) and total flavonoid content (TFC) due to the drying process. Gelatin as a coating agent (5% w/w) provided better TPC recovery by 70% in case of lyophilization and higher total powder yield in case of spray drying by diminishing material deposition on the wall of the drying chamber. The resulting gelatin-free and gelatin-containing powders carried polyphenols in amount ~190 and 53-75 mg gallic acid equivalents GAE/g of powder, respectively. Microwave-assisted extract formulation was distinguished from the others by a higher content of polyphenols, proteins and sugars, higher bulk density and lower solubility. The type of the drying process mainly affected the position of the gelatin-derived -OH and amide bands in FTIR spectra. Spray-dried formulations compared to freeze-dried expressed higher thermal stability as confirmed by differential scanning calorimetry analysis and a higher diffusion coefficient; the last feature can be associated with the lower specific surface area of irregularly shaped freeze-dried particles (151-223 µm) compared to small microspheres (~8 µm) in spray-dried powder.

Design and characterization of alcalase-chitosan conjugates as potential biocatalysts.

In this study, alcalase (protease from Bacillus licheniformis) immobilization by adsorption, enzyme crosslinking and covalent enzyme binding to activated chitosan microbeads were examined. The biocatalysts highest activity was obtained by covalent immobilization of alcalase onto a solid support. The alcalase covalent immobilization onto different types of chitosan beads obtained by inverse emulsion technique and electrostatic extrusion was studied. Parameters examined under different conditions were beads diameter, enzyme loading, enzyme capacity yield, and biocatalyst activity. The highest activity and enzyme loading of 23.6 IU/mg protein and 340.2 mg/g, respectively, were achieved by the enzyme immobilized onto chitosan microbeads obtained by the electrostatic extrusion technique. FT-IR analysis was used to confirm formation of alcalase-chitosan conjugates. The activity of optimally produced alcalase-chitosan microbeads was then verified in the industrially feasible reaction systems of egg white and soy protein hydrolysis. The high degree of hydrolysis of 29.85 ± 0.967% after 180 min and five successive reuses obtained under real conditions (50 °C, pH 8) verified the covalently bound alcalase to chitosan beads a promising candidate for use in industrial egg white protein hydrolysis process.

Effects of different carrier materials on physicochemical properties of microencapsulated grape skin extract.

The goal of this study was to investigate the characteristics of grape skin extract (GSE) spray dried with different carriers: maltodextrin (MD), gum Arabic (GA) and skim milk powder (SMP). The grape skin extract was obtained from winery by-product of red grape variety Prokupac (Vitis vinifera L.). The morphology of the powders, their thermal, chemical and physical properties (water activity, bulk and tapped densities, solubility), as well as release studies in different pH conditions were analyzed. Total anthocyanin content and total phenolic content were determined by spectrophotometric methods. MD and GA-based microparticles were non-porous and spherical, while SMP-based ones were irregularly shaped. The process of spray drying Prokupac GSE using these three carriers produced powders with low water activity (0.24-0.28), good powder characteristics, high yields, and solubility higher than 90%. The obtained dissolution/release profiles indicated prolonged release of anthocyanins and phenolic compounds in different mediums, especially from GSE/GA microparticles. These results have shown that grape skin as the main by-product of wine production could be used as a source of natural colorants and bioactive compounds, and microencapsulation as a promising technique for the protection of these compounds, their stabilization in longer periods and prolonged release.

Native bovine hemoglobin reduces differentiation capacity of mesenchymal stromal cells in vitro.

We have tested in vitro effects of hemoglobin from bovine slaughterhouse blood (BHb) on stromal cells of mesodermal origin, with an aim to explore its use as a component of cell culture media. Human peripheral blood mesenchymal stromal cells (PB-MSCs) and three mouse cell lines (ATDC5, MC3T3-E1 and 3T3-L1) were employed to study BHb effects on their growth and migration. The cells multilineage differentiation capacity in the presence of BHb was evaluated after induced differentiation, by histochemical staining and by RT-PCR analysis of the expression of genes specific for chondrogenic, adipogenic and osteogenic lineages. The effects of BHb on the cell proliferation and motility were dependent on both, cell type and BHb concentration (0.1 µM, 1 µM and 10 µM). In the lowest concentration (0.1 µM) BHb showed the least prominent effect on the cell proliferation and migration. In this concentration BHb reduced the differentiation capacity of all tested cells and its effect was dependent of composition of induction medium and the culture period. Obtained data suggest that BHb has the potential to be used as a component of cell culture media through maintaining proliferation and reducing differentiation capacity of mesenchymal stromal cells.

Design of biopolymer carriers enriched with natural emulsifiers for improved controlled release of thyme essential oil.

This work aims to characterize a novel system for thyme essential oil delivery based on the combination of natural emulsifiers (soy protein and soy lecithin) and alginate, produced using the extrusion technique. The formulations are optimized concerning alginate and soy protein concentrations (both 1 to 1.5 wt.%), and consequently lecithin amount, in order to achieve spherical beads in the range 2.0 to 2.3 mm and 1.2 to 1.4 mm, wet and dry, respectively. Fourier-transform infrared analysis was performed, proving that there are interactions between all components. Lecithin-soy protein synergistic combination improved entrapment efficiency of total polyphenols (for nearly 12%) and decreased thymol release in a simulated gastric solution for nearly 35%, in comparison with beads without lecithin. The addition of lecithin enhances the thermal properties of the polysaccharide-protein systems at 50 °C after 3 hr of heating. The mechanical stability of the biopolymer carriers is improved with lecithin addition and the elastic modulus varied from 80.06 to 123.7 kPa, depending on the formulation. Alginate/soy protein/lecithin are effective carriers for the encapsulation, protection, and controlled release of thyme essential oil. PRACTICAL APPLICATION: There is unfortunately growing human resistance to antibiotics. This work offers a novel system for effective protection and controlled release of thyme essential oil in the small intestine. The mechanical and thermal properties of the carrier were estimated as they indicate how the beads will be able to resist stress during their incorporation into food (i.e. cookies-mixing, baking). The proposed approach offers ''green advantage'' as arises from all-natural materials.

Effect of gentisic acid on the structural-functional properties of liposomes incorporating ß-sitosterol.

Multifunctional liposomes incorporating ß-sitosterol were developed for delivery of gentisic acid (GA). The interactions of both compounds with phospholipid bilayer were interpreted viaeffects of different ß-sitosterol content (0, 20 and 50 mol %) and different gentisic acid to lipid ratio (nGA/nlip from 10-5 to 1) on membrane fluidity and thermotropic properties. Multilamellar vesicles of phosphatidylcholines (with size range between 1350 and 1900 nm) effectively encapsulated GA (54%) when nGA/nlip was higher than 0.01. Suppression of lipid peroxidation was directly related to concentration of GA. The resistance to diffusion of gentisic acid from liposomes increased for ˜50% in samples incorporating 50 mol % ß-sitosterol compared to sterol-free liposomes. Finally, simulated in vitro gastrointestinal conditions showed that the release was mainly affected by low pH of simulated gastric fluid and the presence of cholates in simulated intestinal fluid, rather than by enzymes activity.

Antioxidant edible films based on chitosan and starch containing polyphenols from thyme extracts.

The aim of this study was to analyse the antioxidant activity of different polymeric matrices based on chitosan and starch, incorporating a thyme extract (TE) rich in polyphenols. TE provided the films with remarkable antioxidant activity. When mixed with chitosan, the polyphenols interacted with the polymer chains, acting as crosslinkers and enhancing the tensile behaviour of films. The opposite effect was observed when incorporated into the starch matrix. All the films became darker, more reddish and less transparent when TE was incorporated. These colour changes were more marked in starch matrices, which suggests that TE compounds were poorly encapsulated. The use of chitosan-based matrices carrying TE polyphenols is recommended as a means of obtaining antioxidant films, on the basis of their tensile response and greater antioxidant activity, which could be associated with the development of polyphenol-chitosan interactions, contributing to a better protection of the functionality of polyphenols during film formation and conditioning.

Ultrasound Pretreatment as an Useful Tool to Enhance Egg White Protein Hydrolysis: Kinetics, Reaction Model, and Thermodinamics.

The impact of ultrasound waves generated by probe-type sonicator and ultrasound cleaning bath on egg white protein susceptibility to hydrolysis by alcalase compared to both thermal pretreatment and conventional enzymatic hydrolysis was quantitatively investigated. A series of hydrolytic reactions was carried out in a stirred tank reactor at different substrate concentrations, enzyme concentrations, and temperatures using untreated, and pretreated egg white proteins (EWPs). The kinetic model based on substrate inhibition and second-order enzyme deactivation successfully predicts the experimental behavior providing an effective tool for comparison and optimization. The ultrasound pretreatments appear to greatly improve the enzymatic hydrolysis of EWPs under different conditions when compare to other methods. The apparent reaction rate constants for proteolysis (k2 ) are 0.009, 0.011, 0.053, and 0.045 min-1 for untreated EWPs, and those pretreated with heat, probe-type sonicator, and ultrasound cleaning bath technologies, respectively. The ultrasound pretreatment also decreases hydrolysis activation (Ea ) and enzyme deactivation (Ed ) energy, enthalpy (ΔH), and entropy (ΔS) of activation and for the probe-type sonication this decrease is 61.7%, 61.6%, 63.6%, and 32.2%, respectively, but ultrasound has little change in Gibbs free energy value in the temperature range of 318 to 338 K. The content of sulfhydryl groups and ζ potential show a significant increase (P < 0.05) for both applied ultrasound pretreatments and the reduction of particle size distribution are achieved, providing some evidence that the ultrasound causes EWP structural changes affecting the proteolysis rate.

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate.

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio-derived materials for delivery of dexamethasone-sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/-HESI-MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts' surface morphology by field emission-scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046-1055, 2016.

Chitosan microbeads for encapsulation of thyme (Thymus serpyllum L.) polyphenols.

In this work chitosan microbeads were prepared by emulsion technique and loaded with thyme polyphenols by diffusion from an external aqueous solution of Thymus serpyllum L. The effects of concentrations of chitosan (1.5-3% (w/v)) and GA (glutaraldehyde) (0.1-0.4% (v/v)), as a crosslinking agent on the main properties of microbeads were assessed. The obtained microgel beads from ∼ 220 to ∼ 790 µm in diameter were exposed to controlled drying process at air (at 37 °C) after which they contracted to irregular shapes (∼ 70-230 µm). The loading of dried microbeads with polyphenols was achieved by swelling in the acidic medium. The swelling rate of microbeads decreased with the increase in GA concentration. Upon this rehydration, thyme polyphenols were effectively encapsulated (active load of 66-114 mg GAE g(beads)(-1)) and the microbeads recovered a spherical shape. Both, the increase in the amount of the crosslinking agent and the presence of polyphenols, contributed to a more pronounced surface roughness of microbeads. The release of encapsulated polyphenols in simulated gastrointestinal fluids was prolonged to 3h.

Erythrocyte membranes from slaughterhouse blood as potential drug vehicles: Isolation by gradual hypotonic hemolysis and biochemical and morphological characterization.

The present study was aimed at investigating the effect of isolation process-gradual hypotonic hemolysis on chosen parameters of the erythrocyte membranes (ghosts) originating from bovine and porcine slaughterhouse blood. The estimation of the gradual hypotonic hemolysis as a drug loading procedure for the erythrocyte ghosts was performed as well. Based on the results derived from analysis of the osmotic properties of the erythrocytes, the gradual hemolysis was performed with high volume of erythrocytes and 35mM hypotonic sodium-phosphate/NaCl, enabling >90% of hemolysis for both types of erythrocytes. Detailed insight into ghosts' morphology by field emission-scanning electron microscopy revealed a distortion from erythrocyte shape and an altered surface texture with increased bilayer curvature for both samples. Compared to erythrocytes, an average diameter of ghosts from both type of erythrocytes decreased for only about 10%. The reported unidispersity of the isolated ghosts is of great importance for their potential application as vehicles of active compounds. Gradual hemolysis did not lead to substantial loss of cholesterol and membrane/cytoskeleton proteins. This result indicated the ghosts' possibility to mimic the chemical and structural anisotropic environment of in vivo cell membranes, which is of significance for drug diffusion and partition coefficients. Induced shift of phosphatidylserine to external surface of the ghosts demonstrated their potential application as vehicles for targeted drug delivery to cells of reticuloendothelial system. Ultra high-performance liquid chromatography and Fourier transform infrared spectroscopy revealed the presence of a drug model - dexamethasone-sodium phosphate, and its interaction with structural components in both types of erythrocyte ghosts.