Incorporation Of Lysozyme Research Articles

Biocatalytic PEI-PSS membranes through aqueous phase separation: influence of casting solution pH and operational temperature.

Biocatalytic membranes combine the separation properties of membranes and the catalytic abilities of enzymes, holding great promise for industries where both purification and conversion are required. In this work, polyelectrolyte complex membranes incorporated with lysozyme were prepared using polyethyleneimine (PEI) and poly(sodium 4-styrenesulfonate) (PSS) through a one-step and mild pH shift aqueous phase separation (APS) approach. The effects of lysozyme addition and casting solution pH on the membrane properties were studied. All the membranes, both with and without added lysozyme, exhibited asymmetric structures with relatively dense top surfaces and porous cross-sections with finger-like macrovoids. The incorporation of lysozyme did not significantly influence the structure and permeability of the formed membranes. The PEI-PSS biocatalytic membranes exhibited temperature dependent enzymatic activity. The activity strongly increased with increased operational temperature, with the highest activity of 4.30 ± 0.15 U cm-2 at 45 °C. This indicates a responsive effect, where a higher temperature leads to some swelling of the polyelectrolyte complex membrane, making the enzyme more accessible to the used substrate. Moreover, the biocatalytic membranes demonstrate desirable enzymatic stability, maintaining 60% activity even after 60 days of storage. This study validates the potential of the water-based APS process as a straightforward approach for integrating enzymes into responsive biocatalytic membranes.

BSA-lysozyme coated NaCa2HSi3O9 nanorods on titanium for cytocompatibility and antibacterial activity

Extracellular supplement of Ca and Si accelerates osteogenisis and angiogenisis during bone repair. Ca-Si-based ceramics are biodegradable and can release Ca and Si ions efficiently. However, locally high alkalinity induced by excessively rapid degradation limits their application. In this paper, NaCa2HSi3O9 (NCS) nanorods were fabricated on Ti by micro-arc oxidation and hydrothermal treatment; protein layers consisted of different rations of bovine serum albumin (BSA) and lysozyme (LYS) were prepared on NCS nanorods by spinning coating and thermal stabilization. The microstructure, ion releasing and wettability of coatings were observed; osteoblast and endotheliocyte behaviors, as well as bacteria response in vitro, were evaluated. The protein layer did not change microstructure and phase composition of nanorods but acted as a barrier isolating nanorods from contacting medium, and it results in slowing down the degradation of nanorods. The reduced increase of pH value, released Ca and Si ions and incorporation of LYS in BSA protein layer gave NCS nanorods improved MC3T3-E1 and HUVECs response, compared with Ti. Meanwhile, the protein layers except pure LYS endowed nanorods with a highly antibacterial effect against staphylococcus aureus and escherichia coli simultaneously, indicating the key role of BSA in resisting antibacterial adhesion. NCS nanorods with BSA and LYS composited protein layers should be a potential coating applied on Ti implants for increasing osseointegration.

Preparation and characterization of genipin cross-linked and lysozyme incorporated antimicrobial sodium caseinate edible films

Sodium caseinate (NaCas) possesses good film forming capacity, but mechanical strength of NaCas films is declined by hydrophilic residues in NaCas molecules. Genipin (GP), a low-toxicity natural cross-linking agent, was used at the level at 1 mg/g NaCas to improve the mechnical properties and lysozyme (LYS) was used at the levels of 42, 84 and 126 mg/g NaCas to confer antimicrobial activity of NaCas films. While films without GP and LYS were colorless, light blueness was observed for all types of NaCas films containing GP. The whiteness index and transparency of films decreased as LYS amount increased. The moisture content of films was not significanly affected by the additon of GP and LYS, but the water vapor permeability slightly increased as the amount of LYS increased. Compared with films without GP, significanly higher tensile strength and Young’s modulus and significantly lower break elongation were detected in NaCas films containing GP. The cross-linking reaction between NaCas and GP was confirmed by FTIR-ATR analysis. Incorporation of LYS into NaCas films exerted antimicobial activities against Staphylococcus aureus and Eschericha coli. In conclusion, a novel type of active edible food packaging film with antimicrobial activity was developed by cross-linked with GP and incoporated with LYS.

Preparation of lysozyme loaded gelatin microcryogels and investigation of their antibacterial properties

Antibacterial micron-sized cryogels, so-called microcryogels, were prepared by cryogelation of gelatin and integration of lysozyme. Gelation yield, specific surface area, macro-porosity and swelling degree of the microcryogels were examined in order to characterize their physical properties. MTT method was utilized to measure cell viability of the gelatin microcryogels with a period of 24, 48, and 72 h and no significant decrease was observed at 72 h. Apoptotic staining assay also showed high viability at 24, 48, 72 h in parallel with the control group. The antibacterial performances of the gelatin microcryogels against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli were examined. The results showed that the incorporation of lysozyme into gelatin microcryogels exhibited the antibacterial activity against S. aureus, B. subtilis, and E. coli, that may provide great potential for various applications in the biomedical industry.

Incorporation of lysozyme into a mucoadhesive electrospun patch for rapid protein delivery to the oral mucosa.

The delivery of biopharmaceuticals to the oral mucosa offers a range of potential applications including antimicrobial peptides to treat resistant infections, growth factors for tissue regeneration, or as an alternative to injections for systemic delivery. Existing formulations targeting this site are typically non-specific and provide little control over dose. To address this, an electrospun dual-layer mucoadhesive patch was investigated for protein delivery to the oral mucosa. Lysozyme was used as a model antimicrobial protein and incorporated into poly(vinylpyrrolidone)/Eudragit RS100 polymer nanofibers using electrospinning from an ethanol/water mixture. The resulting fibrous membranes released the protein at a clinically desirable rate, reaching 90±13% cumulative release after 2h. Dual fluorescent fibre labelling and confocal microscopy demonstrated the homogeneity of lysozyme and polymer distribution. High encapsulation efficiency and preservation of enzyme activity were achieved (93.4±7.0% and 96.1±3.3% respectively). The released lysozyme inhibited the growth of the oral bacterium Streptococcus ratti, providing further evidence of retention of biological activity and illustrating a potential application for treating and preventing oral infections. An additional protective poly(caprolactone) backing layer was introduced to promote unidirectional delivery, without loss of enzyme activity, and the resulting dual-layer patches displayed long residence times using an in vitro test, showing that the adhesive properties were maintained. This study demonstrates that the drug delivery system has great potential for the delivery of therapeutic proteins to the oral mucosa.

Incorporation of lysozyme into cellulose nanocrystals stabilized β-chitosan nanoparticles with enhanced antibacterial activity

Cellulose nanocrystals (CNCs) were added to lysozyme (Lys) loaded β-chitosan (β-CS) nanoparticles (NPs) based on ionic gelation technique for enhancing their antibacterial stability. Particle size of CNCs stabilized Lys loaded low (L) and high (H) molecular weight (MW) β-CS NPs reached 140.93 and 284.03 nm with encapsulation efficacy (EE) of 59.38 and 51.23 %, respectively. CNCs stabilized Lys loaded LMW β-CS NPs with smaller particle size showed higher antibacterial activity against E. coli and L. innocua than that of LMW β-CS NPs and Lys loaded LMW β-CS NPs. Inhibition zones (IZ) of CNCs stabilized Lys loaded LMW β-CS NPs against E. coli and L. innocua were 13.29 and 15.29 mm, respectively. in vitro release study conducted at pH 4.5 and 7.4 showed that CNCs stabilized Lys loaded β-CS NPs sustained the release of Lys over 12 h. CNCs, as stabilizers and fillers, enhanced and extended antibacterial property of Lys loaded β-CS NPs through electrostatic interaction and colloidal stability. It was demonstrated that CNCs stabilized Lys loaded β-CS NPs could be used as packaging material for shelf life extension.

Chitosan-Lysozyme Conjugates for Enzyme-Triggered Hydrogel Degradation in Tissue Engineering Applications.

Tuning hydrogel degradation enables effective and successful tissue regeneration by modulating cellular behaviors and matrix formation. In this work, we develop a novel degradable hydrogel scaffold on the basis of a unique enzyme-substrate complex by photocrosslinking. Chitosan and lysozyme are chemically modified with methacrylate moieties to be tethered in hydrogels, and in the presence of riboflavin initiator, these hydrogels are cured by blue light irradiation. The incorporation of lysozyme to chitosan hydrogels accelerates the degradation rate of the crosslinked hydrogels in a dose-dependent manner, as evidenced by an increase in pore size and interconnectivity through cryogenic scanning electron microscopy over time. Those noncytotoxic materials significantly enhance cellular proliferation and migration, which contribute to osteogenic differentiation of encapsulated mesenchymal stem cells in vitro and bone formation in mouse calvarial defects. These findings suggest a promising strategy to modulate the degradation behavior of hydrogels for use in tissue engineering.

Immobilization of lysozyme on bacterial cellulose nanofibers: Characteristics, antimicrobial activity and morphological properties

The aim of this study was to immobilize of lysozyme onto bacterial cellulose nanofibers (BCNF) using physical absorption method. Antimicrobial activity and other properties of immobilized lysozyme and also morphological characteristics of BCNF were studied. Activity of lysozyme was decreased about 12% after immobilization. The lysozyme concentration of 1mg/ml was chosen for next studies on immobilized enzyme. The optimum pH and temperature were recorded as 7 and 45°C, respectively for immobilized enzyme. Storage stability was increased and immobilized lysozyme exhibited more than 70% of its initial activity after 9 cycles. Kinetic parameters of Km and Vmax were 31.18μg/ml and 112.35 ΔOD/min, respectively for immobilized enzyme. Antimicrobial activity of lysozyme against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, Yersinia entrocolitica, Aspergillus niger and Saccharomyces serviseae was increased after immobilization. The XRD analyses showed a decrease in diffraction intensities and crystallinity index of BCNF after immobilization of lysozyme. According to the results of scanning electron microscopy (SEM), the diameter of nanofibers was increased by lysozyme incorporation. This study demonstrates the potential for the use of BCNF as carrier for lysozyme immobilization.

Effect of the Incorporation of Lysozyme on the Properties of Jackfruit Starch Films

Jackfruit starch based biodegradable films containing lysozyme were characterized for their antimicrobial activity, thickness, solubility, water vapor permeability and mechanical properties. The biodegradable films had good appearance and antimicrobial activity against Micrococcus lysodeikticus. The thickness of the biodegradable films were not affected by the variation in pH, but the addition of lysozyme increased the thickness, the thickest films being those with the highest lysozyme concentrations. The variation in pH of the filmogenic solutions affected the solubility of the biodegradable films, water solubility being greatest at pH 7.0 and with the highest lysozyme concentration. The permeability of the biodegradable films was increased by incorporating lysozyme. The lysozyme concentration and pH variation caused changes in the mechanical properties. The addition of 8% lysozyme increased the tensile strength and Young’s modulus for all the pH values studied. With respect to the release of antimicrobial activity, the diffusion of lysozyme was shown to follow Fickian transport mechanism.

Physicochemical Properties and Antibacterial Effect of Lysozyme Incorporated in a Wheat-Based Active Packaging Film

In addition to offering a host of health benefits, antimicrobial (AM) wheat-based packaging is a promising form of active food packaging with great economic and environmental potential. The main objective of this study is to develop a formulation of AM wheat-based film in which the active compound, lysozyme, is incorporated into the polymeric material. A solution casting method was used in the film preparation, and lysozyme was incorporated prior to casting. The resultant film is slightly less opaque, more translucent and whitish in appearance, implying that the AM film could retain similar property with the initial opacity value of the wheat-based film. The water uptake of wheat-film is reduced with the incorporation of lysozyme. The reduction in moisture content of AM film indicates a relationship between lysozyme and water molecules in the diffusion mechanism throughout the film matrices. This film helps to reduce the growth of E. coli and B. subtilis to 1.74 and 3.48 log CFU/mL, respectively, for an incubation of 48 h. FTIR analysis implies the consistency of the chemical composition and structure of the AM film compared to the control film, which indicates that the addition of lysozyme into the wheat-based film did not affect or alter the carbonyl function groups of the wheat-based film. The study will help the researcher to discover and understand wheat-based films for incorporating antimicrobial properties and to explore new matrices for further development.

Enhancement of the antimicrobial activity of cinnamon essential oil-loaded electrospun nanofilm by the incorporation of lysozyme

The antimicrobial activity of cinnamon essential oil-based electrospun nanofilm is enhanced by the combination of lysozyme.

Development of a novel strategy for controlled release of lysozyme from whey protein isolate based active food packaging films

The purpose of this study is to develop a novel controlled release system based on pH-responsive polyacrylic acid (PAA)/lysozyme (LYS) complexes incorporated within a hydrophilic whey protein isolate (WPI) film matrix for active food packaging applications. Complex formation is simple under benign conditions that are suitable for preserving antimicrobial activity of the lysozyme. In addition, the pH-dependent charge density of complexes allowed a uniform distribution in the matrix. The properties of the complexes such as size, surface charge and hydrophilicity were varied by changing PAA/LYS ratio (0.1 and 0.3 w/w) and PAA molecular weight (2 kDa and 450 kDa). The effects of complex properties as well as mode of lysozyme incorporation into the films (100%-free, 50%-free+50%-PAA/LYS complex and 100%-PAA/LYS complex) on the LYS release rate, activity and antimicrobial efficacy of the films were investigated. The results have shown that ∼100% LYS loading into the complexes is possible regardless of PAA molecular weight or PAA/LYS ratio. Incorporating lysozyme into the film in complexed form extended its release time from less than 24 h up to 500 h and reduced its diffusivity from ∼10−9 to ∼10−13 cm2/s. The films including 50%-free-LYS+50%-PAA/LYS complex showed a 5.7 log reduction in bacterial population within 72 h whereas 100%-free-LYS containing film could not suppress Listeria innocua growth after 24 h. Overall, the results suggest that complexation of lysozyme with weak polyelectrolytes can be used as an effective strategy to achieve a long-lasting antimicrobial effect and that films prepared with such complexes have great potential as food packaging materials.

UV-curable enzymatic antibacterial waterborne polyurethane coating

Novel ultraviolet (UV)-curable enzymatic antibacterial coatings were designed and fabricated. The coatings were prepared by simply mixture of castor oil-based cationic waterborne polyurethane-acrylate dispersion (WPUA) and acryloyl chloride modified lysozyme followed by UV curing. The covalent incorporation of lysozyme into the coatings was proved by enzyme leakage and IR analysis of the coatings. As the lysozyme content increased from 0 to 2.5% (w/w), the tensile strength of the coating enhanced by 37% (1.1MPa vs 0.8MPa), and the contact angle increased by 10% (73.1° vs 80.6°). Those changes are possibly due to enhancement of crosslinking degree and hydrogen bonding induced by modified lysozyme. Based on live/dead staining and colonies number assay, the coatings containing lysozyme (2.5%, w/w) were highly efficient in killing Staphylococcus aureus (91.3%) and Escherichia coil (62.9%) via degradation of cell wall within 2h, and the colonies number for both strains was significantly decreased to less than 1% after incubation for 24h. In addition, the killing efficiency of the coatings remained 68.3% after six continuous uses. Such UV-curable waterborne polyurethane coatings containing lysozyme (UWPU-L) may be useful in anti-bacterial stains on various common surfaces in hospital settings and food packaging.

Effects of Alginate Oligosaccharide Mixture on the Bioavailability of Lysozyme as an Antimicrobial Agent.

In this study, we report an oral drug delivery system without any additional process using pH-sensitive biopolymer, alginate, and alginate oligosaccharide with lysozyme as a model drug. These biopolymers exhibited pH-sensitive characteristics such as shrinking at acidic pH and eroding with dissolution at alkaline pH. The incorporation of lysozyme and biopolymers was performed an artificial intestinal juice (pH 6.8). The immobilization efficiency and lysozyme stability in gastric juice (pH 1.2) has been tested by E coil antimicrobial activity. The lysozyme without biopolymer immobilization lost approximately 80-90% of antimicrobial activity than that of pure lysozyme. However, the pH-sensitive biopolymer-controlled lysozyme maintained similar antimicrobial activity compared to that of pure lysozyme (50-90% of cell mortality). Therefore, this simple, easy, and rapid system can be effectively and practically applied for pathogen treatment for in vivo oral drug delivery.

3D chemical characterization of frozen hydrated hydrogels using ToF-SIMS with argon cluster sputter depth profiling.

Hydrogels have been used extensively in bioengineering as artificial cell culture supports. Investigation of the interrelationship between cellular response to the hydrogel and its chemistry ideally requires methods that allow characterization without labels and can map species in three-dimensional to follow biomolecules adsorbed to, and absorbed into, the open structure before and during culture. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has the potential to be utilized for through thickness characterization of hydrogels. The authors have established a simple sample preparation procedure to successfully achieve analysis of frozen hydrated hydrogels using ToF-SIMS without the need for dry glove box entry equipment. They demonstrate this on a poly(2-hydroxyethyl methacrylate) (pHEMA) film where a model protein (lysozyme) is incorporated using two methods to demonstrate how protein distribution can be determined. A comparison of lysozyme incorporation is made between the situation where the protein is present in a polymer dip coating solution and where lysozyme is in an aqueous medium in which the film is incubated. It is shown that protonated water clusters H(H2O)n (+) where n = 5-11 that are indicative of ice are detected through the entire thickness of the pHEMA. The lysozyme distribution through the pHEMA hydrogel films can be determined using the intensity of a characteristic amino acid secondary ion fragment.

Antibacterial Activity and Physical Properties of Edible Chitosan Films Exposed to Low-pressure Plasma

The challenge for food industry is developing gentle processes concept, which will prevent food spoilage and leave a food natural, minimally processed, fresh-like and safe. A new technique of food preservation could be usage of combined methods of green process, such as cold gas plasma with bioactive substances, and protective coatings. The aim of this study was to determine the antibacterial activity of chitosan films incorporated with lysozyme exposed to helium plasma treatment as well as evaluate their physical properties. The edible films have been prepared basing on low molecular weight chitosan by casting from lactic acid solution with water solution of lysozyme in three various concentrations (0, 0.5 and 1 %). Dried films were then modified by exposition on cold helium plasma treatment for 0, 5 and 10 min. Obtained films were tested against growth of Listeria monocytogenes, Yersinia enterocolitica and Pseudomonas fluorescens. In order to characterize chitosan-based films, their mechanical properties, theromogravimetric analysis (TGA), dynamic mechanical thermal analysis (DMTA), contact angle measurement, water vapour permeability (WVP) and scanning electron microscopy (SEM) were evaluated. The films with 1 % lysozyme incorporation enhanced the inhibition efficiency of chitosan-based films against gram-positive (L. monocytogenes) and gram-negative (P. fluorescens) bacteria, where reduction zones were 42.5 and 69.8 mm, respectively. Besides that, hydrolytic changes of chitosan chain caused by lysozyme activity were confirmed by TGA and DMTA. Contact angles and WVP of tested films were not significantly affected by helium plasma exposition, nor lysozyme dosage. Hydrophilic natures of chitosan-based films were confirmed by both tests. Microscopy image of cross-section structure was smooth and continuous due to lysozyme addition in film composition. Application of chitosan films incorporated with lysozyme and low-pressure plasma treatment could be used as innovative preservation method in a wide range of food products.

Chemically modified gliadins as sustained release systems for lysozyme

The aim of this work has been to study the effectiveness of gliadin films cross-linked with cinnamaldehyde as systems for the release of the natural antimicrobial compound lysozyme. Prior to the incorporation of lysozyme, the gliadin film-forming solution was treated with different percentages of cinnamaldehyde as cross-linker, and glycerol was added as plasticizer. The effect of the concentration of the cross-linker on the swelling capacity and kinetics of release of the antimicrobial agent from the protein matrix was evaluated at pH 6.2. The antimicrobial activity of the films was assayed against Listeria innocua. The gliadin films cross-linked with cinnamaldehyde incorporating lysozyme preserved their integrity in water. The release rate of the antimicrobial agent was controlled by the reticulation of the protein matrix, thus a greater degree of cross-linking led to slower release of the active agent. Films with a loosely cross-linked structure released a greater amount of lysozyme, exhibiting greater antimicrobial activity.

Antimicrobial films containing lysozyme for active packaging obtained by sol–gel technique

Antimicrobial packaging is a promising form of active packaging that refers to the incorporation of antimicrobial agents into packaging systems with the aim to extend the product shelf life maintaining its quality and safety. In our work an antimicrobial active packaging based on poly(ethylene terephthalate) (PET) was prepared by sol–gel route, incorporating as antimicrobial agent lysozyme with the aim to obtain packaging films with controlled release properties. The fine tuning of the sol concentration and viscosity as well as the dip-coating extraction rate, allowed to deposit on the external surface of the PET support uniform coatings with controllable thickness at the sub-micron scale. FTIR microspectroscopic and tapping mode-Atomic Force Microscopy (TM-AFM) measurements were used to characterize the prepared coatings.The antimicrobial activity of the obtained surface modified films was tested on agar plates against Micrococcus lysodeikticus. Furthermore, the stability of the coating was evaluated in order to verify a possible migration of lysozyme to a liquid media (buffered solution at pH 6.2), due to a possible release from the plastic substrate. The quantitative determination of the released lysozyme in the buffered solution was performed by HPLC UV-DAD. Results show that the incorporation of lysozyme into the sol–gel modified PET do not lead to a loss of activity of the enzyme, and suggest a significant potential for the use of these packaging materials.

Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations

Lysozyme was attached through an amide linkage between some of the protein’s aspartate and glutamate residues to amino-glycine-cellulose, which was prepared by esterification of glycine to preparations of cotton nanocrystals. The nanocrystalline preparations were produced through acid hydrolysis and mechanical breakage of the cotton fibers from a scoured and bleached cotton fabric and a scoured and bleached, mercerized fabric, which was shown to produce cellulose I (NCI) and cellulose II (NCII) crystals respectively. A carbodiimide-activation coupling reaction was used to create the lysozyme-amino-glycine-cellulose conjugates using both NCI and NCII in a polar solvent and gave yields of covalently linked lysozyme at 604 mg/gram of cotton nanocrystal. The incorporation of lysozyme conjugated to the NCI and NCII preparations gave very high activity (1,500 U/mg cotton) when assessed using a fluorescence tag assay to measure antimicrobial activity against Micrococcus lysodeikticus. Scanning electron micrographs demonstrated an aggregation of nanoparticles corresponding to lysozyme bound on the surface of larger cotton nanocrystalline sheets. The approach of producing high enzyme activity on cotton nanocrystals is discussed in the context of selectively presenting robust hydrolase activity on nanocrystalline surfaces.

Particle engineering of materials for oral inhalation by dry powder inhalers. I—Particles of sugar excipients (trehalose and raffinose) for protein delivery

The pulmonary route of delivery offers a potential alternative to parenteral administration of peptides and proteins. Protection of protein structure is essential in both processing and storage of the final formulation. Sugars, such as trehalose and raffinose, have been employed to act as protein stabilisers. Optimisation of the aerodynamic characteristics of microparticles in dry powder inhaler formulations is critical to ensure optimum deposition of the formulation into the respiratory tract. In the present study we examine the adaptation to hydrophilic materials, specifically the disaccharide, trehalose and the trisaccharide, raffinose, of a previously reported spray drying process for producing nanoporous microparticles (NPMPs). We also investigate the feasibility of incorporating a model protein, lysozyme, into these sugar-based NPMPs. While spray drying raffinose or trehalose from aqueous solution or ethanol:water solutions resulted in non-porous microspheres, spray drying from a methanol:n-butyl acetate mixed solvent system resulted in microparticles which appeared to consist of an agglomeration of individual nanoparticles, i.e. nanoporous/nanoparticulate microparticles. NPMPs of trehalose and raffinose were amorphous, with glass transition temperatures (Tgs) that were sufficiently high (124 °C and ∼120 °C for trehalose and raffinose, respectively) to suggest good physical stability at room temperature and good potential to act as protein carriers and/or stabilisers. NPMPs demonstrated improved aerosolisation properties compared to spray dried non-porous particles. The successful incorporation of lysozyme into these NPMPs at a sugar to protein weight ratio of 1:4 demonstrated the potential of these systems to act as carriers for peptide or protein drugs which could be delivered via the pulmonary route.