Characterization of a water-in-oil-in-water multiple emulsion integrating biomimetic aqueous-core lipid nanoballoons housing protein entities (original) (raw)
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Drug Development and Industrial Pharmacy, 2016
Lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water enclose, at least in theory, a biomimetic aqueous-core suitable for housing hydrophilic biomolecules such as proteins, peptides and bacteriophage particles. The research effort entertained in this paper reports a full statistical 2 3 x3 1 factorial design study (three variables at two levels and one variable at three levels) to optimize biomimetic aqueous-core lipid nanoballoons for housing hydrophilic protein entities. The concentrations of protein, lipophilic and hydrophilic emulsifiers, and homogenization speed were set as the four independent variables, whereas the mean particle hydrodynamic size (HS), zeta potential (ZP) and polydispersity index (PI) were set as the dependent variables. The 2 3 x3 1 factorial design constructed led to optimization of the higher (+1) and lower (-1) levels, with triplicate testing for the central (0) level, thus producing thirty three experiments and leading to selection of the optimized processing parameters as 0.015% (w/w) protein entity, 0.75% (w/w) lipophilic emulsifier (soybean lecithin) and 0.50% (w/w) hydrophilic emulsifier (poloxamer 188). In the present research effort, statistical optimization and production of protein derivatives encompassing full stabilization of their three-dimensional structure, has been attempted via housing said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion.
Food Research International, 2012
Controlled aggregation of protein microspheres in water-in-oil (W/O) emulsions was used to form semi-solid lipid materials. The aqueous phase consisted of 10 wt% whey protein isolate (WPI) in buffer solution (pH 7.0, 100 mM NaCl). The oil phase consisted of a lipophilic nonionic surfactant (8 wt % polyglycerol polyricinoleate, PGPR) dispersed in a liquid oil (soybean oil). Lipid phases containing protein microspheres were formed by homogenization of the oil and aqueous phases to form a W/O emulsion followed by heating (90°C for 30 min) to promote gelation of the WPI in the aqueous phase. Temperature-scanning dynamic shear measurements showed that the W/O emulsions underwent an irreversible liquid-to-solid transition when heated above the thermal denaturation temperature of WPI, which was attributed to protein gelation and microsphere aggregation. Optical microscopy indicated that a three-dimensional network of aggregated protein microspheres was formed at high aqueous phase contents (>30 wt %). Shear rheology measurements (shear stress versus shear rate) indicated that these structured emulsions were non-ideal plastic-like materials. The apparent shear viscosity increased with thermal treatment, increasing aqueous phase content, and decreasing shear rate. The structured W/O emulsions developed in this study may be useful materials for the development of foods with highly viscous or gel-like lipid phases, but low saturated or trans-fat contents.
Journal of Food Engineering, 2013
The effect of heteroaggregation of oppositely charged protein microspheres dispersed within a liquid oil phase on the microstructure and rheological properties of water-in-oil (W/O) emulsions was evaluated. The aqueous phase of the initial W/O emulsions contained either 10% b-lactoglobulin or 10% lactoferrin (pH 7, 100 mM NaCl). At this pH, b-lactoglobulin (BLG) is negatively charged while lactoferrin (LF) is positively charged. The oil phase consisted of a lipophilic non-ionic surfactant (8% polyglycerol polyricinoleate, PGPR) dispersed within soybean oil. Three 40% W/O emulsions were formed containing different types of protein microspheres: (i) BLG: 100% BLG droplets; (ii) LF: 100% LF droplets; and (iii) Mixed: 50% BLG droplets and 50% LF droplets. Prior to heating, the mixed emulsions had a higher shear viscosity, yield stress, and shear modulus than the BLG or LF emulsions, which suggested that electrostatic attraction led to the formation of a three-dimensional network of aggregated droplets. All three W/O emulsions underwent an irreversible fluid-to-solid transition when they were heated above %70°C. This phenomenon was attributed to thermal denaturation of the globular BLG and LF molecules within the aqueous phase promoting aggregation and network formation of the protein microspheres. After heating, the mixed emulsions had a higher shear viscosity, yield stress and shear modulus than the BLG or LF emulsions, suggesting that a stronger droplet network was formed due to electrostatic attraction. Shear rheology measurements of the W/O emulsions showed that the lipid phases formed after heating were nonideal plastics characterized by a yield stress and shear thinning behavior. These results may facilitate the design of semi-solid or solid foods with reduced saturated-or trans-fat contents suitable for use in commercial products.
Pharmaceutics, 2021
Recently, we developed ultra-stable oil in water nano-emulsions (O/W NEs), able to carry both internal and external cargos (Somes), such as lipophilic compounds and hydrophilic coatings, respectively, that we call here NEsoSomes. O/W NEs are an excellent bioengineering tool for drug and molecules delivery, due to their ability to dissolve a large number of hydrophobic compounds and protect them from hydrolysis and degradation under biological conditions. At present, no report is available on the combination of cell membrane coatings with such nanocarriers, probably due to their typical instability feature. Since then, we have reported, for the first time, a new cell membrane (CM)-coated nanomaterial composed of membranes extracted from glioblastoma cancer cells (U87-MG) deposited on NEsoSomes, through a liquid–liquid interface method, to produce highly controllable membrane caked nano-capsules, namely CM-NEsoSomes. CM-NEsoSomes were physically characterized by dynamic light scatteri...
Food Biophysics, 2008
A stearin-rich milk fraction was used, alone or in combination with α-tocopherol, for the preparation of oilin-water sodium caseinate-stabilized nano-emulsions. Fat droplets in these two emulsions were characterized for their size distribution and physical stability against aggregationcoalescence, for their heat-induced structural behaviour, and for their ability to protect α-tocopherol during oxidation. Inclusion of α-tocopherol led to changes in the particle size distributions of fat droplets: in the presence of α-tocopherol, approximately 75% of fat droplets were lower than 1 μm, instead of 100% in the absence of α-tocopherol. On the other hand, thermal transitions observed by differential scanning calorimetry showed that supercooling (the increase in differences between temperature of initial crystallization and melting completion) was higher in the emulsified milk fat samples containing α-tocopherol. In addition to a decrease in the temperature of fat crystallization leading this change in the supercooling effect of α-tocopherol, small-and wide-angle X-ray diffraction patterns (SAXS and WAXS, respectively) observed under cooling and re-heating cycles showed that heat-induced polymorphic transitions from2L α ! 2L β 0 forms were more impeded in the emulsion containing α-tocopherol. Immobilisation of α-tocopherol in fat drop-lets composed by high melting temperature milk fat triglycerides seemed to lead to its protection against degradation.
2014
Based on oil-in-water template emulsion, lipid nanoemulsion has been developing with monitoring both the lipid-shell and lipid-core composition imparting outstanding long-term stability of nanoparticles. Taking advantage of its colloidal behavior and high tolerability, we modulated the surface droplet to tailor make relevant nanocarriers and also transport various molecules such as large drugs, biomacromolecules. Cationic and anionic nanoparticles were prepared by incorporation of either hydrophobic-tailed polysaccharides or cationic lipids within the shell lipids. Such cationic formulations are suitable for the complexation of nucleic acids such as DNA or RNA. Moreover, large biomacromolecules, such as proteins, peptides or antibodies are chemically grafted on nanoparticle surface via bioconjugation techniques using “home-made” PEGylated surfactants. Also, the fine-tuning of shell of lipid nanoemulsion offers great opportunity to adress new applications in targeted delivery field (...
Protein Formulations for Emulsions and Solid-in-Oil Dispersions
Needs from medical and cosmetic areas have led to the design of novel nanosized emulsions and solid-in-oil dispersions of proteins. Here, we describe the production of those emulsions and dispersions using high-energy methodologies such as high-pressure homogenization or ultrasound. Recent work has resulted in new mechanistic insights related to the formation of protein emulsions and dispersions. The production method and composition of these formulations can determine major parameters such as size, stability, and functionality, and therefore their final application. Aqueous nanoemulsions of proteins can be used for drug delivery, while solid-in-oil dispersions are often used in transdermal applications.
Food Hydrocolloids, 2016
Nanoemulsions are utilized within the food, pharmaceutical, and personal care industries because of their unique physicochemical properties and functional attributes: high optical clarity; prolonged stability; and, enhanced bioavailability. For many applications, it is desirable to utilize natural ingredients to formulate nanoemulsions so as to create "label-friendly" products. In this study, we compared the effectiveness of a number of natural emulsifiers at fabricating corn oil-in-water nanoemulsions using dualchannel microfluidization. These emulsifiers were either amphiphilic biopolymers (whey protein and gum arabic) or biosurfactants (quillaja saponin and soy lecithin). Differences in the surface activities of these emulsifiers were characterized using interfacial tension measurements. The influence of emulsifier type, concentration, and homogenization pressure on the efficiency of nanoemulsion formation was examined. The long-term stability of the fabricated nanoemulsions was also monitored during storage at ambient temperature. For all of the natural emulsifiers, nanoemulsions could be produced by dualchannel microfluidization, with the mean particle diameter decreasing with increasing emulsifier concentration and homogenization pressure. Whey protein isolate and quillaja saponin were more effective at forming nanoemulsions containing fine droplets than gum arabic and soy lecithin, with a lower amount of emulsifier required and smaller droplets being produced. This effect was attributed to faster emulsifier adsorption and a greater reduction in interfacial tension leading to more efficient droplet disruption within the homogenizer for saponins and whey proteins. This study highlights the potential of dualchannel microfluidization for efficiently producing label-friendly nanoemulsions from natural emulsifiers.
Influence of Emulsifier Structure on Lipid Bioaccessibility in Oil–Water Nanoemulsions
Journal of Agricultural and Food Chemistry, 2013
The influence of several nonionic surfactants and anionic surfactants (sodium lauryl sulfate, sodium stearoyl lactylate, and sodium stearyl fumarate) showed drastic differences in the rank order of lipase activity/lipid bioaccessibility. The biophysical composition of the oil and water interface has a clear impact on the bioaccessibility of fatty acids (FA) by altering the interactions of lipase at the oil−water interface. It was found that the bioaccessibility was positively correlated with the hydrophilic/lipophilic balance (HLB) of the surfactant and inversely correlated to the surfactant aliphatic chain length. Furthermore, the induction time in the jejunum increased as the HLB value increased and decreased with increasing aliphatic chain length. The rate of lipolysis slowed in the jejunum with increasing HLB and with increasing aliphatic chain length.
Rangsit University, 2011
Abstract Multi-stage membrane extrusion (ME), a process that extrudes a premixed emulsion prepared by a conventional method through a porous membrane for several repeated cycle, was introduced to prepare an oil-in-water (O/W) emulsion. A uniform-size and fine emulsion is produced using this method: this consumes less energy, a key point in maintaining the structure of the emulsifier employed in the emulsion system. In the present work, a hexadecane emulsion stabilized by bovine serum albumin (BSA) was initially produced by a sonicator before extrusion through a polycarbonate membrane. After that, the properties of the resulting emulsion were explored by measuring the oil content after extrusion, the mean diameter of oil droplets, and the adsorption of BSA at the oil-water interface. The multi-stage ME was processed at various permeate fluxes in the range of 0.07-3.8 Ls-1m-2 for 1 to 3 repeated cycles and it was demonstrated that a third extrusion at a permeate flux lower than 2 Ls-1m-2 was successful in producing an emulsion with a smaller and more uniform-sized of oil droplets compared to the emulsion before extrusion. Keywords: bovine serum albumin, multi-stage membrane emulsification, polycarbonate membrane, droplet size distribution, and protein adsorption.
Food Chemistry, 2016
Nanoemulsions have considerable potential for encapsulating and delivering -3 fatty acids, but they are typically fabricated from synthetic surfactants. This study shows that fish oil-inwater nanoemulsions can be formed from sunflower phospholipids, which have advantages for food applications because they have low allergenicity and do not come from genetically modified organisms. Nanoemulsions containing small droplets (d < 150 nm) could be produced using microfluidization, by optimizing phospholipid type and concentration, with the smallest droplets being formed at high phosphatidylcholine levels and at surfactant-to-oil ratios exceeding unity. The physical stability of the nanoemulsions was mainly attributed to electrostatic repulsion, with droplet aggregation occurring at low pH values (low charge magnitude) and at high ionic strengths (electrostatic screening). These results suggest that sunflower phospholipids may be a viable natural emulsifier to deliver ω-3 fatty acids into food and beverage products.
Food Chemistry, 2019
The physiological efficacy of nutraceuticals is dependent on their physicochemical nature and bioavailability across biological barriers. In the present work, effects of nano-sizing of emulsion-based delivery vehicle on the bioavailability of polyunsaturated fatty acids rich fish oil have been investigated via three-step experimental design; ex vivo rat everted intestinal sac model, cellular lipid uptake and the bioactivity in rat PBMCs. Nanoemulsion in comparison to the conventional emulsion has shown significant higher rate of uptake of polyunsaturated fatty acids in three segments of small intestine. The time-kinetics of such uptake was correlated with appearance of short-chain fatty acids in basal side of the everted sac. The bioavailability of the formulated fish oil and its inhibitory response against lipopolysaccharide-induced nitric oxide production in rat PBMCs were positively correlated. This formulation with nano-sized droplets can be utilized as smart delivery vehicles for designing oral therapies in future.
Polish Journal of Food and Nutrition Sciences, 2014
This review article demonstrates fundamentals regarding the manufacturing of multilayer oil-in-water (M-O/W) emulsions and factors affecting stability of these systems. Moreover, characteristics of major bioactive lipophilic components and ingredients mostly applied to form multilayered membranes as well analytical methods used to examine properties of M-O/W emulsions are specified. It has been shown that production of M-O/W systems is based on the layer-by-layer (LbL) electrostatic deposition technique which makes use of the electrostatic attraction of oppositely charged surfactants and biopolymers to form multicomposite protective layers around emulsion droplets. Finally, limitations regarding studies of M-O/W systems which should be developed are specified.
Food Biophysics, 2015
This study evaluates interactions between the lipid core and the phospholipid interface in oil in water emulsions and solid lipid nanoparticles. Interactions between the core and the interface are characterized based on changes in structural order and lateral mobility of the phospholipid interface as a function of physical state of the lipid core (solid vs. liquid) and composition of phospholipids and bile salts at the interface. Changes in structural order of the lipid core are also evaluated as a function of composition of the interface. Emulsions (liquid core) and solid lipid nanoparticles (solid core) are formulated using an eicosane lipid core. Phospholipid with long carbon chain (C16)-high melting phospholipids (41°C) and short carbon chain (C12)-low melting phospholipid (−1°C) are selected as emulsifiers. The results of fluorescence anisotropy measurements show that physical state of the lipid core does not significantly influence molecular order of the phospholipid interface. These measurements also demonstrate that molecular order of the lipid core is only marginally impacted by composition of the interface. Excimer formation measurements with pyrene labeled phospholipids illustrate that the composition of phospholipid and bile salts has a significant impact on lateral mobility of emulsifiers at the interface. Results also show that physical state of the lipid core has no significant influence on lateral mobility of emulsifiers at the interface. In summary, these results highlight that properties of phospholipid emulsion interface are a strong function of composition of emulsifiers and coemulsifiers and are independent of physical state of the lipid core.
Nanoemulsion-Based Delivery Systems to Improve Functionality of Lipophilic Components
Frontiers in Nutrition, 2014
The use of active lipophilic substances such as antimicrobials and health-related compounds in the food industry is still a challenge due to their poor water solubility and instability in food formulations. Nano-sized structures such as nanoemulsions of oil-in-water are regarded as useful tools with a great potential in the food sector to incorporate food ingredients. Reducing the size of the active compounds incorporated within a solution would increase the surface area per mass unit of nanoemulsions, thus enhancing solubility and stability in foods. In addition, the ability of the active lipids to penetrate across biological membranes is also enhanced, thus boosting their biological functionality. An overview of the most significant studies reporting data about the potential benefits of active lipid nanoemulsions over conventional emulsions is presented.
Stability of oil-in water emulsions containing protein
Food Colloids, 2001
This work is focused on the mechanisms for stabilisation of oil-in-water emulsion drops pressed against a homophase. With increasing concentration of a globular protein (βlactoglobulin) in the aqueous continuous phase the drop stability is improved considerably. Repulsive interactions (other than electrostatics) prevent rupture of the intervening o/w/o films at elevated concentrations. Then, the critical disjoining pressure for coalescence is higher than the equilibrium disjoining pressure which is established when the drop arrives at the surface. The latter fact correlates with very long lifetimes of drops. Conversely, at low concentrations (below ∼10-3 wt%) the films rupture soon after the process of thinning is completed. Faster formation of saturated adsorption layers is probably responsible for the more efficient stabilisation with rising concentration. The adsorption kinetics in the time scales of seconds and minutes is rather sensitive to the protein content. Interfacial rheological measurements give evidence that with increasing concentration of protein the layer is mechanically reinforced to some extent.
Structuring lipids by aggregation of acidic protein microspheres in W/O emulsions
LWT - Food Science and Technology, 2013
This study shows that controlled aggregation of acidic whey protein microspheres in water-in-oil (W/O) emulsions can be used to create semi-solid lipids. An acidic aqueous phase (10 g/100 g whey protein isolate, 100 mmol/L NaCl, pH 3.5) was homogenized with an oil phase (8 g/100 g polyglycerol polyricinoleate in soybean oil) to form a W/O emulsion. These emulsions were then heated (90 C, 30 min) to induce thermal denaturation and gelation of the globular proteins in the internal aqueous phase, which promoted irreversible gelation of the overall W/O emulsion. Rheology measurements indicated that the resulting materials had paste-like properties: they were solid-like below a critical yield stress and fluid-like (shear-thinning) above this value. The apparent shear viscosity of the W/O emulsions increased with thermal treatment, aqueous phase content, and decreasing shear rate. W/O emulsion gels containing acidic aqueous phases were considerably weaker than those containing neutral aqueous phases due to the pH dependence of whey protein gelation. Controlled aggregation of whey protein microspheres within W/O emulsions may be a useful means of producing highly viscous or semi-solid lipids with low saturated and trans-fat contents for use in the food and other industries.
Pharmaceutics, 2021
Nano-emulsions consist of stable suspensions of nano-scaled droplets that have huge loading capacities and are formulated with safe compounds. For these reasons, a large number of studies have described the potential uses of nano-emulsions, focusing on various aspects such as formulation processes, loading capabilities, and surface modifications. These studies typically concern direct nano-emulsions (i.e., oil-in-water), whereas studies on reverse nano-emulsions (i.e., water-in-oil) remain anecdotal. However, reverse nano-emulsion technology is very promising (e.g., as an alternative to liposome technology) for the development of drug delivery systems that encapsulate hydrophilic compounds within double droplets. The spontaneous emulsification process has the added advantages of optimization of the energetic yield, potential for industrial scale-up, improved loading capabilities, and preservation of fragile compounds targeted for encapsulation. In this study, we propose a detailed i...
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Malaria journal, 2018
Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the st...
Molecules, 2017
Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 • C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.
Immobilization of α-Amylase from Anoxybacillus sp. SK3-4 on ReliZyme and Immobead Supports
Molecules, 2016
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 • C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
Maltose Production Using Starch from Cassava Bagasse Catalyzed by Cross-Linked β-Amylase Aggregates
Catalysts
Barley β-amylase was immobilized using different techniques. The highest global yield was obtained using the cross-linked enzyme aggregates (CLEA) technique, employing bovine serum albumin (BSA) or soy protein isolate (SPI) as feeder proteins to reduce diffusion problems. The CLEAs produced using BSA or SPI showed 82.7 ± 5.8 and 53.3 ± 2.4% global yield, respectively, and a stabilization effect was observed upon immobilization at neutral pH value, e.g., after 12 h at 55 • C, the free β-amylase is fully inactivated, while CLEAs retained 25 and 15% of activity (using BSA and SPI, respectively). CLEA using SPI was selected because of its easier recovery, being chosen to convert the residual starch contained in cassava bagasse into maltose. This biocatalyst permitted to reach almost 70% of maltose conversion in 4 h using 30.0 g/L bagasse starch solution (Dextrose Equivalent of 15.88) and 1.2 U of biocatalyst per gram of starch at pH 7.0 and 40 • C. After 4 reuses (batches of 12 h) the CLEA using SPI maintained 25.50 ± 0.01% of conversion due to the difficulty of recovering.
Prospects for the Use of New Technologies to Combat Multidrug-Resistant Bacteria
Frontiers in Pharmacology
The increasing use of antibiotics is being driven by factors such as the aging of the population, increased occurrence of infections, and greater prevalence of chronic diseases that require antimicrobial treatment. The excessive and unnecessary use of antibiotics in humans has led to the emergence of bacteria resistant to the antibiotics currently available, as well as to the selective development of other microorganisms, hence contributing to the widespread dissemination of resistance genes at the environmental level. Due to this, attempts are being made to develop new techniques to combat resistant bacteria, among them the use of strictly lytic bacteriophage particles, CRISPR-Cas, and nanotechnology. The use of these technologies, alone or in combination, is promising for solving a problem that humanity faces today and that could lead to human extinction: the domination of pathogenic bacteria resistant to artificial drugs. This prospective paper discusses the potential of bacteriophage particles, CRISPR-Cas, and nanotechnology for use in combating human (bacterial) infections.
Molecules
There are many areas in medicine and industry where it would be advantageous to orally deliver bioactive proteins and peptides (BPPs), including ACE inhibitors, antimicrobials, antioxidants, hormones, enzymes, and vaccines. A major challenge in this area is that many BPPs degrade during storage of the product or during passage through the human gut, thereby losing their activity. Moreover, many BPPs have undesirable taste profiles (such as bitterness or astringency), which makes them unpleasant to consume. These challenges can often be overcome by encapsulating them within colloidal particles that protect them from any adverse conditions in their environment, but then release them at the desired site-of-action, which may be inside the gut or body. This article begins with a discussion of BPP characteristics and the hurdles involved in their delivery. It then highlights the characteristics of colloidal particles that can be manipulated to create effective BPP-delivery systems, includ...
Molecules
Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.
Pharmaceutics, 2021
Bromelain (Bro) is a multiprotein complex extracted from the pineapple plant Ananas comosus, composed of at least eight cysteine proteases. Bro has a wide range of applications in medicine and industry, where the stability of its active proteases is always a major concern. The present study describes the improvement of stability and gain of specific activity in the enzymatic content of Bro immobilized on gold nanoparticles (GNPs). GNPs were synthesized in situ using Bro as the reducing and stabilizing agents and characterized by surface plasmon resonance and transmission electron microscopy. Consistent with the structural changes observed by circular dichroism analysis, the association with GNPs affected enzyme activity. The active Bro immobilized on GNPs (NanoBro) remained stable under storage and gained thermal stability consistent with a thermophilic enzyme. Proteolytic assays were performed on type I collagen membranes using fluorescence spectroscopy of O-phthaldialdehyde (OPA),...
What Are the Potential Benefits of Using Bacteriophages in Periodontal Therapy?
Antibiotics, 2022
Periodontitis, which may result in tooth loss, constitutes both a serious medical and social problem. This pathology, if not treated, can contribute to the development of, among others, pancreatic cancer, cardiovascular diseases or Alzheimer’s disease. The available treatment methods are expensive but not always fully effective. For this reason, the search for and isolation of bacteriophages specific to bacterial strains causing periodontitis seems to be a great opportunity to target persistent colonization by bacterial pathogens and lower the use of antibiotics consequently limiting further development of antibiotic resistance. Furthermore, antimicrobial resistance (AMR) constitutes a growing challenge in periodontal therapy as resistant pathogens may be isolated from more than 70% of patients with periodontitis. The aim of this review is to present the perspective of phage application in the prevention and/or treatment of periodontitis alongside its complicated multifactorial aeti...
Biocatalysis and Biotransformation, 2017
Lipase B from Candida antarctica (CALB) immobilized on octyl-agarose (OC) was submitted to coating with polyethylenimine (PEI) and dextran sulfate (DS). Using lowly loaded enzyme preparations, the properties of OC-CALB preparations hardly improved, suggesting too large the distance between enzyme molecules. However, using OC-CALB preparations with maximum loading, CALB stability was greatly improved in different conditions after PEI coating. Moreover, the CALB release from the OC support in the presence of detergents, or during thermal or organic solvent inactivations was greatly reduced after this treatment (PEI plus DS coating). The results pointed that the main positive effect of this coating could be derived from the physical intermolecular crosslinking of the CALB molecules with the polymers that reduce the enzyme desorption from the support. The coating of OC-CALB-PEI with DS only produced a minimal improvement on enzyme performance. Even though the enzyme release was much more difficult after physical crosslinking, all enzyme molecules could be released from the OC support combining an ionic detergent (SDS), high buffer concentration, pH 3 and 45 C, while using the OC-CALB just 2% SDS at pH 7 and 25 C was enough to release all enzyme. The support could be reused several cycles. Thus, this strategy permitted to greatly reduce the enzyme desorption during operation and to improve enzyme stability while keeping the enzyme immobilization reversibility.
Journal of Colloid and Interface Science, 2021
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Pharmaceutics, 2021
Hemoglobin (Hb)-based oxygen carriers (HBOCs) display the excellent oxygen-carrying properties of red blood cells, while overcoming some of the limitations of donor blood. Various encapsulation platforms have been explored to prepare HBOCs which aim to avoid or minimize the adverse effects caused by the administration of free Hb. Herein, we entrapped Hb within a poly(lactide-co-glycolide) (PLGA) core, prepared by the double emulsion solvent evaporation method. We study the effect of the concentrations of Hb, PLGA, and emulsifier on the size, polydispersity (PDI), loading capacity (LC), and entrapment efficiency (EE) of the resulting Hb-loaded PLGA nanoparticles (HbNPs). Next, the ability of the HbNPs to reversibly bind and release oxygen was thoroughly evaluated. When needed, trehalose, a well-known protein stabilizer that has never been explored for the fabrication of HBOCs, was incorporated to preserve Hb’s functionality. The optimized formulation had a size of 344 nm, a PDI of 0....
Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose
Molecules, 2017
The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, L-arabinose (D-galactose) isomerase from Enterococcus faecium, and D-xylose (D-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing L-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and D-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 • C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 • C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system.
Cytokine-Mediated Inflammation in the Oral Cavity and Its Effect on Lipid Nanocarriers
Nanomaterials, 2021
Topical drug administration to the oral mucosa proves to be a promising treatment alternative for inflammatory diseases. However, disease-related changes in the cell barrier must be considered when developing such delivery systems. This study aimed at investigating the changes in the lining mucosa caused by inflammation and evaluating the consequences on drug delivery systems such as nanostructured lipid carriers (NLC). For this, TR146 cells were treated with inflammatory cytokines and bacterial components. Cell viability and integrity, reactive oxygen species (ROS), and interleukin (IL)-8 release were used as endpoints to assess inflammation. Translocation of phosphatidylserine, cytoskeletal arrangement, opening of desmosomes, and cell proliferation were examined. Transport studies with NLC were performed considering active and passive pathways. The results showed that IL-1ß and tumor necrosis factor α induced inflammation by increasing IL-8 and ROS production (22-fold and 2-fold)....
Practical insights on enzyme stabilization
Critical reviews in biotechnology, 2017
Enzymes are efficient catalysts designed by nature to work in physiological environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.
Biochemistry and Biophysics Reports, 2021
Background: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. Methods: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. Results: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. Conclusions: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. General significance: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.