Co-Encapsulation of Epigallocatechin-3-Gallate and Vitamin B12 in Zein Microstructures by Electrospinning/Electrospraying Technique.

EGCG is a catechin known for its antioxidant and anti-inflammatory characteristics. Vitamin B12 is an essential vitamin found in animal-derived products, and its deficiency may cause serious health problems such as anemia. The effectiveness of both catechin and vitamin B12 depends on their stability and bioavailability, which can be lost during industrial processes due to degradation when exposed to external factors. A potential solution to this issue is the microencapsulation, which protects the compounds from external agents. The current study aims to microencapsulate EGCG and vitamin B12 in a polymer matrix of biological origin, zein. Microencapsulation was performed using an electrospinning technique, and different concentrations of zein (1-30% w/v) and active compound (0.5-5% w/w) were tested, resulting in the production of micro/nanoparticles, fibers, or the mixture of both. The microstructures were analyzed and characterized in terms of morphology, release profile and kinetics, and encapsulation efficiency. High encapsulation efficiencies were obtained, and the highest were found in the samples with 1% w/w of active substance and 30% w/v of zein. Controlled release studies were conducted in deionized water and in an ethanolic solution, and five kinetic models were applied to the release profiles. The results indicated that the Weibull model was the best fit for the majority of results.

New Trends in the Microencapsulation of Functional Fatty Acid-Rich Oils Using Transglutaminase Catalyzed Crosslinking.

Preparing stable protein-based microcapsules containing functional fatty acids and oils for food applications has been a big challenge. However, recent advances with transglutaminase (TGase) enzyme as an effective protein cross-linker could provide workable solutions for the encapsulation of omega-3 and omega-6 fatty acids without compromising their targeted release and their biological and physicochemical characteristics. The recent and available literature related to the microencapsulation techniques, physical and oxidative properties, and core retention and release mechanisms of TGase-crosslinked microcapsules entrapping edible oils were reviewed. The effects of factors involved in microencapsulation processes, on the efficiency and quality of the produced innovative microcapsules were also discussed and highlighted. A brief focus has been finally addressed to new insights and additional knowledge on micro- and nanoencapsulation of lipophilic food-grade ingredients by TGase-induced gelation. Two dominant microencapsulation methods for fish, vegetable, and essential oils by TGase-crosslinking are complex coacervation and emulsion-based spray drying. The developed spherical particles (<100 µm) with some wrinkles and smooth surfaces showed an excellent encapsulation efficiency and yield. A negligible release rate and a substantial retention level can result for different lipid-based cores covered by TGase-crosslinked proteins during the oral digestion and storage. A significant structural, thermal and oxidative stability for edible oils-loaded microcapsules in the presence of TGase can be also obtained.

Valorization of Agricultural By-Products (Fragaria vesca) through the Production of Value-Added Micro/Nanostructures Using Electrohydrodynamic Techniques.

An innovative approach for the production of bio-micro/nanostructures with high-value compounds from agricultural by-products was studied. This research aimed to valorize bioactive compounds existing in the by-products of the plants of Fragaria vesca (wild strawberry). The particle characteristics, morphology, size, release properties, and antioxidant activity of micro/nanostructures containing the extract of by-products of the plants of Fragaria vesca or quercetin (one of the main polyphenols in the plant) were analyzed. The electrohydrodynamic (EHD) technique was utilized for encapsulation. The results showed that the morphology and size of the structures were influenced by the concentration of zein, with 10% w/v zein concentration leading to irregular and non-uniform nanostructures, while 20% w/v zein concentration resulted in a mixture of microparticles and thin fibers with an irregular surface. The type and concentration of the core material did not significantly affect the morphology of the micro/nanostructures. In vitro release studies demonstrated the controlled release of the core materials from the zein micro/nanostructures. The release profiles were analyzed using the Korsmeyer-Peppas and Weibull models, which provided insights into the release mechanisms and kinetics. The most relevant release mechanism is associated with "Fickian Diffusion". The antioxidant activity of the structures was evaluated using an ABTS radical-scavenging assay, indicating their potential as antioxidants. In conclusion, the EHD technique enabled the successful encapsulation of Fragaria vesca by-product extract and quercetin with zein, resulting in micro/nanostructures with different morphologies.

Statistical simplex centroid experimental design for evaluation of pectin, modified chitosan and modified starch as encapsulating agents on the development of vitamin E-loaded microparticles by spray-drying.

Vitamin E encapsulation into biopolymer-based microparticles, obtained by spray-drying technology, was proposed to improve the encapsulation efficiency and the controlled release of fat-soluble vitamin. Binary and ternary blends of pectin, modified chitosan and modified starch, modified starch + modified chitosan, modified starch + pectin, modified chitosan + pectin and modified starch + modified chitosan + pectin ((0.33, 0.33, 0.33), (0.70, 0.15, 0.15), (0.15, 0.70, 0.15) and (0.15, 0.15, 0.70)) were proposed to produce and evaluate different carrier-based delivery systems. Vitamin E-loaded microparticles and empty microparticles were created with a product yield between 9 and 49 %. The mean diameter among all microparticles varied between 3.74 ± 0.02 and 421 ± 21 µm (differential volume distribution). Oval, spherical or irregular microparticles, with a variable morphology from a smooth to a high rough surface structure, with concavities, were produced. All vitamin E-loaded microparticles exhibited an encapsulation efficiency higher than 70 %. The slower vitamin E controlled release was observed from microparticles composed by modified chitosan (>36 h), while the faster release was achieved from microparticles individually composed by pectin (39 min). In general, the Fickian diffusion is the main release mechanism involved in the microparticles produced with modified chitosan, other formulations combine also other mechanisms such as swelling.

In vitro bioaccessibility and intestinal transport of retinoic acid in ethyl cellulose-based microparticles and impact of meal co-ingestion.

The development of carrier-based delivery systems for oral administration of retinoic acid (RA), that provides its release and absorption at intestinal level, is of major relevance in the treatment of acute promyelocytic leukemia. The aim of this work was to evaluate RA bioaccessibility and intestinal transport on ethyl cellulose (EC)- and EC + polyethylene glycol (ECP)-based microparticles and to understand the impact of meal co-ingestion by applying in vitro assays. RA-loaded microparticles were produced by spray-drying with an encapsulation efficiency higher than 90 % for both formulations. The gastric bioaccessibility of RA (after in vitro static digestion of RA-loaded particles) was lower than 3 % for both types of microparticles, with and without meal co-ingestion. Whereas after intestinal digestion, RA bioaccessibility was significantly higher and affected by the type of microparticles and the presence of meal. The digestion of EC- and ECP-based microparticles without diet enabled a significantly higher bioaccessibility of RA when compared to the one recorded for the co-digestion of these microparticles with diet. Herein, RA bioaccessibility decreased from 84 ± 1 to 24 ± 6 % (p < 0.0001) for microparticles EC and 54 ± 4 to 25 ± 5 % (p < 0.001) for microparticles ECP. Moreover, comparing both types of microparticles, RA bioaccessibility was significantly higher for EC-based microparticles digested without diet (p < 0.0001). At last, the bioaccessibility of RA was similar among EC- and ECP-based microparticles when co-digested with diet. Intestinal transport experiments performed in Caco-2 monolayers evidenced that after 2 h of transport the amount of RA retained in the apical compartment was higher than the amount that reached the basolateral compartment evidencing a slow transport at intestinal level that was higher when RA is spiked in the blank of digestion and the meal digestion samples compared to RA dissolved in HBSS (44 ± 6 (p < 0.01) and 38 ± 1 (p < 0.05) vs 26 ± 2 %, respectively).

Pharmaceutical/Clinical Strategies in the Treatment of Acute Promyelocytic Leukemia: All-Trans Retinoic Acid Encapsulation by Spray-Drying Technology as an Innovative Approach-Comprehensive Overview.

Acute promyelocytic leukemia (APL) is phenotypically characterized by the accumulation of dysplastic promyelocytes, resulting from a cytogenetic condition due to the balanced chromosomal translocation t(15;17)(q22;q21). Current first-line treatment of APL includes all-trans retinoic acid (all-trans RA), with or without arsenic trioxide, combined with chemotherapy, and a chemotherapy-free approach wherein arsenic trioxide is used alone or in combination with all-trans RA. The usage of all-trans RA revolutionized the treatment of APL, with survival rates of 80 to 90% being achieved. The mechanism of action of all-trans RA is based on regulation of gene transcription, promoting the differentiation of leukemic promyelocytes. Encapsulation technology has been explored as an innovative strategy to overcome the major drawbacks related to the all-trans RA oral administration in the APL treatment. The most recently published works on this subject highlight the development and optimization of carrier-based delivery systems based in microparticle formulations obtained by spray-drying to be used in the treatment of APL. The ultimate goal is to obtain a controlled delivery system for RA oral administration capable of providing a slow release of this bioactive compound in the intestinal lumen.

Co-encapsulation of retinoic acid, curcumin and resveratrol by spray-drying of alginic acid sodium-based emulsions and ethyl cellulose-based solutions: Impact on the co-delivery profiles.

Co-encapsulation of retinoic acid (RA), curcumin and/or resveratrol into microparticles composed by alginic acid sodium and the ethyl cellulose + polyethylene glycol (EC + PEG) blend was proposed for the protection and co-delivery of these bioactive compounds. The final aim is to take benefit of combined therapeutic potential related to these molecules and use loaded microparticles obtained by spray-drying to improve the treatment of acute promyelocytic leukemia (APL). Alginic acid sodium-based emulsions were characterized regarding rheological properties (i.e. viscosity), stability and droplet size distribution. Biopolymer- and synthetic polymer-based microparticles loaded with RA, RA + curcumin, RA + resveratrol and RA + curcumin + resveratrol were produced with a product yield between 10 and 35 %. The obtained microparticles exhibited a variable form, a morphology that varied between a slightly and high rough surface and a mean diameter that ranged from 2.97 ± 0.04 and 88 ± 3 µm. Encapsulation efficiency was significantly influenced by the encapsulating agent(s) used in the microparticles formulations. The bioactive compounds that were co-encapsulated showed a similar release profile.

Application of Ethyl Cellulose and Ethyl Cellulose + Polyethylene Glycol for the Development of Polymer-Based Formulations using Spray-Drying Technology for Retinoic Acid Encapsulation.

Ethyl cellulose (EC)-based microparticles, with and without the incorporation of polyethylene glycol (PEG) as a second encapsulating agent, were prepared using the spray-drying process for the encapsulation of retinoic acid (RA). The production of a suitable controlled delivery system for this retinoid will promote its antitumor efficiency against acute promyelocytic leukemia (APL) due to the possibility of increasing the bioavailability of RA. Product yield ranged from 12 to 28% in all the microparticle formulations, including unloaded microparticles and RA-loaded microparticles. Microparticles with a mean diameter between 0.090 ± 0.002 and 0.54 ± 0.02 µm (number size distribution) and with an irregular form and rough surface were obtained. Furthermore, regarding RA-loaded microparticles, both polymer-based formulations exhibited an encapsulation efficiency of around 100%. A rapid and complete RA release was reached in 40 min from EC- and EC + PEG-based microparticles.

Food-Grade Microencapsulation Systems to Improve Protection of the Epigallocatechin Gallate.

Epigallocatechin gallate (EGCG) is a catechin and one of the most abundant polyphenols in green tea, and it is under research for its potential benefit to human health and for its potential to be used in disease treatments, such as for cancer. However, the effectiveness of polyphenols depends on preserving their bioactivity, stability, and bioavailability. The EGCG was microencapsulated by a spray-drying process, using different biopolymers as encapsulating agents (gum arabic, modified chitosan and sodium alginate), in order to overcome some of the limitations of this compound. The microparticles showed a diameter around 4.22 to 41.55 µm (distribution in volume) and different morphologies and surfaces, depending on the encapsulating agent used. The EGCG release was total, and it was achieved in less than 21 min for all the formulations tested. The EGCG encapsulation efficiency ranged between 78.5 and 100.0%. The release profiles were simulated and evaluated using three kinetic models: Korsmeyer-Peppas (R2: 0.739-0.990), Weibull (R2: 0.963-0.994) and Baker-Lonsdale (R2: 0.746-0.993). The Weibull model was the model that better adjusted to the experimental EGCG release values. This study proves the success of the EGCG microencapsulation, using the spray-drying technique, opening the possibility to insert dried EGCG microparticles in different food and nutraceutical products.

Optimization of electrospinning parameters for the production of zein microstructures for food and biomedical applications.

Electrohydrodynamic techniques have been focus for the development of structures for encapsulation purposes. Their physico-chemical characteristics confer them significant benefits for food and nutraceutical applications. The study reports the optimization of zein microstructures (electrosprayed beads/electrospun fibers/films). The effect of zein polymer properties (viscosity and conductivity), flow rate, applied voltage and distance tip-collector were investigated. Results by scanning electron microscopy revealed the morphology observed with zein. The importance of chain entanglement for fibers/beads/films formation in the optimum conditions system was evaluated. Compact electrosprayed microbeads with diameters ranging from 0.9 µm to 2.0 µm were obtained for 5 wt.% zein solution. For 30 wt.% zein, uniform smooth electrospun fibers with diameters of approximately 0.60 to 0.75 µm were produced. Films with different characteristics (with more or less homogeneous matrix and more or less bubbles) were also obtained. The developed zein microstructures are potential vectors that might encapsulate bioactive ingredients for functional food, nutraceutical and medical applications.

Grafting MSI-78A onto chitosan microspheres enhances its antimicrobial activity.

MSI-78A (Pexiganan A) is one of the few antimicrobial peptides (AMPs) able to kill Helicobacter pylori, a pathogenic bacterium that colonizes the gastric mucosa of half of the world's population. Antibiotics fail in 20-40% of H. pylori-infected patients, reinforcing the need for alternative treatments. Herein, a bioengineered approach was developed. MSI-78A with a C-terminal cysteine was grafted onto chitosan microspheres (AMP-ChMic) by thiol-maleimide (Michael-addition) chemistry using a long heterobifunctional spacer (NHS-PEG113-MAL). Microspheres with ∼4 µm diameter (near H. pylori length) and stable at low pH were produced by spray drying using a chitosan solution with an incomplete genipin crosslinking. A 3 × 10-5 µg AMP/microsphere grafting was estimated/confirmed by UV/Vis and FTIR spectroscopies. AMP-ChMic were bactericidal against H. pylori J99 (highly pathogenic human strain) at lower concentrations than the free peptide (∼277 µg grafted MSI-78A-SH/mL vs 512 µg free MSI-78A-SH/mL), even after pre-incubation in simulated gastric conditions with pepsin. AMP-ChMic killed H. pylori by membrane destabilization and cytoplasm release in a ratio of ∼10 bacteria/microsphere. This can be attributed to H. pylori attraction to chitosan, facilitating the interaction of grafted AMP with bacterium membrane. Overall, it was demonstrated that the peptide-microsphere conjugation chemistry did not compromise the MSI-78A antimicrobial activity, instead it boosted its bactericidal performance against H. pylori. STATEMENT OF SIGNIFICANCE: Half of the world's population is infected with Helicobacter pylori, a gastric bacterium that is responsible for 90% of non-cardia gastric cancers. Therefore, H. pylori eradication is now advocated in all infected individuals. However, available antibiotic therapies fail in up to 40% patients. Antimicrobial peptides (AMPs) are appealing alternatives to antibiotics, but their high susceptibility in vivo limits their clinical translation. AMP immobilization onto biomaterials surface will overcome this problem. Herein, we demonstrate that immobilization of MSI-78A (one of the few AMPs with activity against H. pylori) onto chitosan microspheres (AMP-ChMic) enhances its anti-H. pylori activity even at acidic pH (gastric settings). These results highlight the strong potential of AMP-ChMic as an antibiotic alternative for H. pylori eradication.

Formulation approaches for improved retinoids delivery in the treatment of several pathologies.

Retinoid acid (RA) and other retinoids are extensively used as therapeutic agents in the treatment of several types of cancer and skin disorders. However, the efficiency of these medical agents is compromised due to the unsatisfactory concentration of retinoids in the target cells/tissues. Furthermore, severe side-effects are related to retinoids administration. Incorporation of retinoids into carrier-based delivery systems using encapsulation technology has been proposed in order to overcome the limitations of using free retinoids in the treatment of several pathologies. The present work starts exploring the competences and the difficulties of using retinoids in health care. The metabolism and the main considerations about the mechanism of action of retinoids are also discussed. The final sections are focused on the most recent studies about RA controlled delivery systems to be used in the medical field.

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered ß-galactosidase.

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a ß-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the ß-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C.

Removal of 2,4-dichlorophenol and pentachlorophenol from waters by sorption using coal fly ash from a Portuguese thermal power plant.

Chlorophenols are one of the most important groups of priority pollutants, due to their high toxicity, mutagenicity and carcinogenicity. Although activated carbon has been the preferred choice for the removal of such pollutants from wastewaters, the search for cheaper alternative sorbents became common in the last years. Fly ash, a by-product from coal burning power plants, has a surface composition that may enable the sorption of specific organic compounds. Therefore, this feasibility study presents the optimization of the operating parameters of a fixed-bed column containing fly ash particles, percolated by aqueous solutions of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) with concentrations of 1 and 100 microg/ml. Both chlorophenols were analysed by gas chromatography with electron capture detection (GC-ECD), after solid-phase microextraction (SPME), with limits of detection (LODs) of 7.28 microg/l for 2,4-DCP and 1.76 microg/l for PCP. Removal efficiencies above 99% were obtained for an initial concentration of 10 microg/ml of chlorophenols. Column saturation was achieved after 7h of continuous operation for 2,4-DCP and 10h for the PCP for feed levels of 10 microg/ml. Fly ash exhibited more affinity towards the sorption of PCP, in comparison to 2,4-DCP.

Microencapsulation of Gulosibacter molinativorax ON4T cells by a spray-drying process using different biopolymers.

Molinate is a thiocarbamate herbicide used in rice crop protection. As other pesticides, molinate is a recognized environmental pollutant and bio-accumulated by some wildlife forms. Gulosibacter molinativorax ON4T is able to hydrolyse molinate into metabolites which are further degraded by other un-related bacteria. Hence, it can be used in molinate bioremediation processes. The aim of this work was to investigate the possibility of producing G. molinativorax ON4T microparticles, using different non-toxic biopolymers (arabic gum, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. Several formulations of microparticles were prepared, and their physicochemical structures were analyzed by scanning electron microscopy (SEM), laser granulometry analysis and zeta potential analysis. The obtained microparticles were evaluated considering their ability to degrade molinate, the metabolic activity (by colour development of the tetrazolium violet redox), and also the survival rate and shelf-life/storage stability of microparticles. Based on their molinate degrading activity, the biopolymers calcium alginate and modified chitosan cross-linked with tripolyphosphate appear to be the best options for the microencapsulation of the G. molinativorax ON4T. However, the microparticles produced with modified chitosan cross-linked with tripolyphosphate present the best combination of physical properties and activity degradation of molinate.

Production of microparticles of molinate degrading biocatalysts using the spray drying technique.

Previous studies demonstrated the capability of mixed culture DC1 to mineralize the thiocarbamate herbicide molinate through the activity of molinate hydrolase (MolA). Because liquid suspensions are not compatible with long-term storage and are not easy to handle when bioremediation strategies are envisaged, in this study spray drying was evaluated as a cost-effective method to store and transport these molinate biocatalysts. Microparticles of mixed culture DC1 (DC1) and of cell free crude extracts containing MolA (MA) were obtained without any carrier polymer, and with calcium alginate (CA) or modified chitosan (MCt) as immobilizing agents. All the DC1 microparticles showed high molinate degrading activity upon storage for 6 months, or after 9 additions of ∼0.4 mM molinate over 1 month. The DC1-MCt microparticles were those with the highest survival rate and lowest heterogeneity. For MA microparticles, only MA-MCt degraded molinate. However, its Vmax was only 1.4% of that of the fresh cell free extract (non spray dried). The feasibility of using the DC1-MCt and MA-MCt microparticles in bioaugmentation processes was assessed in river water microcosms, using mass (g):volume (L) ratios of 1:13 and 1:0.25, respectively. Both type of microparticles removed ∼65-75% of the initial 1.5 mg L(-1) molinate, after 7 days of incubation. However, only DC1-MCt microparticles were able to degrade this environmental concentration of molinate without disturbing the native bacterial community. These results suggest that spray drying can be successfully used to produce DC1-MCt microparticles to remediate molinate polluted sites through a bioaugmentation strategy.

Effect of the pH in the formation of ß-galactosidase microparticles produced by a spray-drying process.

The objective of this work was to investigate the influence of pH in the microencapsulation process, using a modified chitosan to microencapsulate the enzyme ß-galactosidase, by a spray-drying technique. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM), showing that the obtained microparticles have an average diameter smaller than 3.5 µm and in general a regular shape. The activity of the enzyme was studied by spectrophotometric methods using the substrate O-nitrophenyl-ß,D-galactopyranoside (ONPG). The parameters of Michaelis-Menten were calculated. The value of Km decreases with the decrease of the pH, which can be associated to an increase of the affinity between the enzyme and substrate to smaller pH's. The highest value of the parameter Vmax, representing the maximum reaction rate at a given enzyme concentration, was obtained at pH 6.

Enzyme kinetics: the whole picture reveals hidden meanings.

The methodology adopted by Michaelis and Menten in 1913 is still routinely used to characterize the catalytic power and selectivity of enzymes. These kinetic measurements must be performed soon after the purified enzyme is mixed with a large excess of substrate. Other time scales and solution compositions are no less physiologically relevant, but fall outside the range of applicability of the classical formalism. Here we show that the complete picture of an enzyme's mode of function is critically obscured by the limited scope of conventional kinetic analysis, even in the simplest case of a single active site without inhibition. This picture is now unveiled in a mathematically closed form that remains valid over the reaction time for all combinations of enzyme/substrate concentrations and rate constants. Algebraic simplicity is maintained in the new formalism when stationary reaction phases are considered. By achieving this century-old objective, the otherwise hidden role of the reversible binding step is revealed and atypical kinetic profiles are explained. Most singular kinetic behaviors are identified in a critical region of conditions that coincide with typical cell conditions. Because it is not covered by the Michaelis-Menten model, the critical region has been missed until now by low- and high-throughput screenings of new drugs. New possibilities are therefore raised for novel and once-promising inhibitors to therapeutically target enzymes.

Microencapsulation of ß-galactosidase with different biopolymers by a spray-drying process.

The aim of this work was to investigate the possibility of producing microparticles containing ß-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme ß-galactosidase, due to its importance in health and in food processing. Encapsulation of ß-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of ß-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3µm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-ß,d-galactopyranoside). The microencapsulated ß-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.