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.

Immobilization of horseradish peroxidase onto Purolite® A109 and its anthraquinone dye biodegradation and detoxification potential.

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite® A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 ± 1.41 U g-1 and 85.71 ± 1.62 U g-1 after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (~90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.

Influence of ultrasound probe treatment time and protease type on functional and physicochemical characteristics of egg white protein hydrolysates.

The objective of this study was to discover the relationship between the ultrasound probe treatment (UPT) on egg white proteins (EWPs) before EWPs hydrolysis by different proteases, and the functional properties of the obtained hydrolysates. To fulfill this goal, the protein solubility, foaming, and emulsifying properties were studied as a function of the UPT time and then related to the surface characteristics and structural properties. The changes in the hydrolysates microstructures and macromolecular conformation, induced by the UPT, were followed using scanning electron microscope analyzis (SEM) and Fourier transforms infrared spectroscopy (FTIR). The results showed that UPT influenced (P < 0.05) the proteolysis of egg white proteins for all examined treatment times. Alcalase hydrolysates (AHs) and papain hydrolysates (PHs) were found to have a higher solubility, as a consequence of their relatively higher foaming, and emulsifying properties compared to the untreated hydrolysates. The changes in surface hydrophobicity, sulfhydryl content and surface charge of AHs and PHs indicated unfolding of EWPs affected by ultrasound. SEM analyzis showed that UPT destroyed the microstructures of AHs and PHs, while FTIR spectra indicated remarkable changes in the macromolecular conformation of AHs and PHs after UPT. This study revealed that by combining ultrasound pre-hydrolysis treatment under controlled conditions with thoughtful proteases selection, hydrolysates with improved functional properties could be produced, enhancing utilization of EWPs in food products.

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.

Covalent Immobilization of Enzymes on Eupergit® Supports: Effect of the Immobilization Protocol.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

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.

Immobilization of horseradish peroxidase onto kaolin.

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer-Emmett-Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H2O2) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi-Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) as a potential industrial biocatalyst.

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low-cost, easy-to-prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA-co-EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA-co-EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA.

Decolorization of anthraquinonic dyes from textile effluent using horseradish peroxidase: optimization and kinetic study.

Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes.

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.

Synthesis of aliphatic esters of cinnamic acid as potential lipophilic antioxidants catalyzed by lipase B from Candida antarctica.

Immobilized lipase from Candida antarctica (Novozyme 435) was tested for the synthesis of various phenolic acid esters (ethyl and n-butyl cinnamate, ethyl p-coumarate and n-butyl p-methoxycinnamate). The second-order kinetic model was used to mathematically describe the reaction kinetics and to compare present processes quantitatively. It was found that the model agreed well with the experimental data. Further, the effect of alcohol type on the esterification of cinnamic acid was investigated. The immobilized lipase showed more ability to catalyze the synthesis of butyl cinnamate. Therefore, the process was optimized for the synthesis of butyl cinnamate as a function of solvent polarity (logP) and amount of biocatalyst. The highest ester yield of 60.7 % was obtained for the highest enzyme concentration tested (3 % w/w), but the productivity was for 34 % lower than the corresponding value obtained for the enzyme concentration of 1 % (w/w). The synthesized esters were purified, identified, and screened for antioxidant activities. Both DPPH assay and cyclic voltammetry measurement have shown that cinnamic acid esters have better antioxidant properties than cinnamic acid itself.

Improved covalent immobilization of horseradish peroxidase on macroporous glycidyl methacrylate-based copolymers.

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.

Immobilization of lipase from Candida rugosa on Sepabeads(®): the effect of lipase oxidation by periodates.

The objective of this paper was the investigation of a suitable Sepabeads(®) support and method for immobilization of lipase from Candida rugosa. Three different supports were used, two with amino groups, (Sepabeads(®) EC-EA and Sepabeads(®) EC-HA), differing in spacer length (two and six carbons, respectively) and one with epoxy group (Sepabeads(®) EC-EP). Lipase immobilization was carried out by two conventional methods (via epoxy groups and via glutaraldehyde), and with periodate method for modification of lipase. The results of activity assays showed that lipase retained 94.8% or 87.6% of activity after immobilization via epoxy groups or with periodate method, respectively, while glutaraldehyde method was inferior with only 12.7% of retention. The immobilization of lipase, previously modified by periodate oxidation, via amino groups has proven to be more efficient than direct immobilization of lipase via epoxy groups. In such a way immobilized enzyme exhibited higher activity at high reaction temperatures and higher thermal stability.

The immobilization of enzyme on Eupergit® supports by covalent attachment.

A selection of the best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on the industrial level. Eupergit) supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is a good recommendation for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient, considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads.