New aspects of the formation of physical hydrogels of chitosan in a hydroalcoholic medium

New aspects concerning the mechanism of formation of chitosan physical hydrogels without any cross-linking agent were studied. The gelation took place during the evaporation of a hydroalcoholic solution of chitosan. We first demonstrated that it was possible to form a physical hydrogel from a hydrochloride form of chitosan. Chromatographic methods showed that during the gel formation, when the initial concentration is over C, the critical concentration of chain entanglement, the water and acid used for the solubilization of the polymer were both eliminated. This particular situation contributed to decrease the dielectric constant of the medium and the apparent charge density of chitosan chains, thus inducing the formation of a three-dimensional network through hydrophobic interactions and hydrogen bonding. In the gelation process, this step was kinetically determining. The speed of evaporation of water and acid were determined and different initial conditions were compared. Thus, we investigated the influence of: the initial polymer concentration, the nature of the counterion and the alcohol, the temperature and the geometry of the reactor. Our results allowed us to confirm the existence of a second critical initial concentration C, from which the evaporation of water became more difficult. We suggested that C corresponded to a reorganization of the solution involving the presence of gel precursors. Then, a mechanism of formation of physical hydrogels of chitosan in a hydroalcoholic medium could be proposed. For the first time, we demonstrated that it was possible to generate physical hydrogels in the presence of various diols, which size of the carbonated chain appeared as a limiting factor for the gelation process. These physical hydrogels of chitosan are currently used in our laboratory for tissue engineering in the treatment of third degree burns with the possibility to adapt their mechanical properties from the choice of both the acid or the alcohol used.     

Conformational Behavior of Chitosan in the Acetate Salt: An X-Ray Study

A well-defined X-ray fiber pattern of chitosan acetate was obtained by immersing a tendon chitosan, prepared from a crab tendon chitin by a solid-state N-deacetylation, in an aqueous acetic acid-isopropanol solution at 110°C. This pattern was very similar to that of chitosan salts with some inorganic acids, such as HF, HCl, and H₂SO₄, in which chitosan chains form an 8/5 helix, indicating that chitosan acetate also take up this conformation. This information may give an influential clue to the chitosan conformation in the aqueous acetic acid solution, the most popular solvent for chitosan. However, after one month of storage of the chitosan acetate, the fiber pattern, the density and its IR spectrum changed to those of the anhydrous polymorph of chitosan, suggesting that the acetic acid was removed accompanied with water molecules from the crystal during storage and that the polymorph can be obtained not only by annealing chitosan, but also through the chitosan acetate.     

Tuning the Hydrophilic/Hydrophobic Balance to Control the Structure of Chitosan Films and Their Protein Release Behavior

The control over the crystallinity of chitosan and chitosan/ovalbumin films can be achieved via an appropriate balance of the hydrophilic/hydrophobic interactions during the film formation process, which then controls the release kinetics of ovalbumin. Chitosan films were prepared by solvent casting. The presence of the anhydrous allomorph can be viewed as a probe of the hydrophobic conditions at the neutralization step. The semicrystalline structure, the swelling behavior of the films, the protein/chitosan interactions, and the release behavior of the films were impacted by the DA and the film processing parameters. At low DAs, the chitosan films neutralized in the solid state corresponded to the most hydrophobic environment, inducing the crystallization of the anhydrous allomorph with and without protein. The most hydrophilic conditions, leading to the hydrated allomorph, corresponded to non-neutralized films for the highest DAs. For the non-neutralized chitosan acetate (amorphous) films, the swelling increased when the DA decreased, whereas for the neutralized chitosan films, the swelling decreased. The in vitro release of ovalbumin (model protein) from chitosan films was controlled by their swelling behavior. For fast swelling films (DA?=?45%), a burst effect was observed. On the contrary, a lag time was evidenced for DA?=?2.5% with a limited release of the protein. Furthermore, by blending chitosans (DA?=?2.5% and 45%), the release behavior was improved by reducing the burst effect and the lag time. The secondary structure of ovalbumin was partially maintained in the solid state, and the ovalbumin was released under its native form.     

Crystalline behavior of chitosan organic acid salts.

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous "Annealed" polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated "Tendon" polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Crystalline Behavior of Chitosan Organic Acid Salts

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous “Annealed” polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated “Tendon” polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

Gel Permeation Chromatography of Polysaccharides: Universal Calibration Curve

Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv = 1,720–12,600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan’s functionality, by heating.     

Dependence on the Preparation Procedure of the Polymorphism and Crystallinity of Chitosan Membranes

Differences in the polymorphism and crystallinity of chitosan were found in membranes prepared by different procedures when examined by X-ray diffraction measurements for four samples of chitosan differing in the degree of polymerization. When an acetic acid solution of chitosan was dried in air and then soaked in an alkaline solution (method A), both hydrated and anhydrous polymorphs of chitosan were present in the resulting membranes; the latter polymorph made chitosan insoluble in common solvents of chitosan, and its crystallinity increased with decreasing chitosan molecular weight. When a highly concentrated chitosan solution in aqueous acetic acid was neutralized with an alkaline solution (method B), no anhydrous polymorphs were detected in the membrane because of incomplete drying. When aqueous formic acid was used as the solvent, behavior basically similar to that in aqueous acetic acid was observed. In contrast, even with method A, aqueous hydrochloric acid gave a chitosan membrane having very little anhydrous crystallinity. The crystalline polymorph called “1–2”, which has been proposed to be one of four chitosan polymorphs, is considered to be a mixture of hydrated and anhydrous crystals.     

X‐ray crystal structure of anhydrous chitosan at atomic resolution

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P2₁2₁2₁] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016.     

One-pot synthesis in aqueous system for water-soluble chitosan-graft-poly(ethylene glycol) methyl ether

Chitosan is functionalized with poly(ethylene glycol) methyl ether (mPEG) at the amino and hydroxyl groups via a single step reaction in a homogeneous aqueous system. A chitosan aqueous solution obtained from the mixture of chitosan and hydroxybenzotriazole (HOBt) in water is a key factor in providing mild conditions to conjugate mPEG by using a carbodiimide conjugating agent. The reaction at ambient temperature for 24 h gives chitosan-g-mPEG with water solubility with mPEG content as high as 42%. This work demonstrates that a water-soluble chitosan-HOBt complex is an effective system for the preparation of chitosan derivatives via the aqueous system without the use of acids or organic solvents.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.     

Decoloration of chitosan by UV irradiation

Decoloration of chitosan by UV irradiation, which was used to replace a bleaching step during chitosan preparation, was evaluated under four separate treatments (effect of irradiation time, chitosan/water ratio, stirring speed, and UV light source). The optimal decoloration condition was defined as that producing white chitosan with higher viscosity. Decoloration of chitosan could be achieved effectively using a UV-C light by stirring unbleached chitosan in water (1:8, w/v) for 5 min at 120 rpm. UV irradiation applied under the optimal conditions could be used to produce chitosan with desirable white color (L^* = 76.95, a^* = −0.37, and b^* = 14.04) and high viscosity (1301.7 mPa s at 0.5% w/v in 1.0% v/v acetic acid).     

Studies on chitosan: 3. Evidence for the presence of random and block copolymer structures in partially N-acetylated chitosans

The chemical structures of moderately N-deacetylated chitosans (MDC) derived from chitin under heterogeneous reaction conditions and partially N-acetylated chitosans (PAC) derived from highly N-deacetylated chitosans (HDC) under homogeneous reaction conditions were deduced from the data of the stability of their solutions in alkaline media, the swelling behaviour and X-ray diffraction patterns of their films in connection with the degree of N-acetylation of them. The solutions of PAC with more than 51% acetyl content, which were prepared from HDC by N-acetylation, were stable and remained clear and homogeneous by adding 1.2 equivalents of NaOH. On the contrary the solutions of PAC with more than 52% acetyl content, which were prepared from MDC, became turbid by neutralization with less than 1.15 equivalents of NaOH. The films of PAC prepared from HDC were highly swollen in water. The degree of swelling of the chitosan film with 51% acetyl content, prepared from the 6% acetyl content chitosan, was 121% while that of the 53% acetyl content chitosan, prepared from the 30% acetyl content chitosan, was 28%. From these data it was possible to set up a hypothesis that PAC prepared from HDC were considered as random-type copolymers of N-acetyl-glucosamine and glucosamine units whereas MDC were considered as block-type copolymers.     

Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process

A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30–70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40–120 °C) for 48–144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1 g-ammonia + 1.0 g-water per g biomass, 80 °C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan + xylan in corn stover).     

Preparation and properties of alginate/carboxymethyl chitosan blend fibers

Alginate/carboxymethyl chitosan blend fibers, prepared by spinning their mixture solution through a viscose-type spinneret into a coagulating bath containing aqueous CaCl₂, were studied for structure and properties with the aid of infrared spectroscopy (IR), X-ray diffraction (XRD) and scanning electron micrography (SEM). The analyses indicated a good miscibility between alginate and carboxymethyl chitosan, because of the strong interaction from the intermolecular hydrogen bonds. The best values of the dry tensile strength and breaking elongation were obtained when carboxymethyl chitosan content was 30 and 10 wt%, respectively. The wet tensile strength and breaking elongation decreased with the increase of carboxymethyl chitosan content. Introduction of CM-chitosan in the blend fiber improved water-retention properties of blend fiber compared to pure alginate fiber. Antibacterial fibers, obtained by treating the fibres with aqueous solution of N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride and silver nitrate, respectively, exhibited good antibacterial activity to Staphylococcus aureus.     

Cross-linking chitosan into UV-irradiated cellulose fibers for the preparation of antimicrobial-finished textiles

Chitosan cross-linked cellulose fibers were prepared using non-toxic procedures in order to confer antimicrobial properties to cellulose fibers. Citric acid was used as the cross-linker and NaH₂PO₄ as catalyst in previously UV-irradiated cellulose fibers. Further heat dried-cure process and washing with detergent, water and acetic acid (0.1 M) gave a maximum incorporation of chitosan of 27 mg per gram of functionalized textile. The thermogravimetric analysis of the material with the highest chitosan content showed an increased thermal stability compared to cellulose and chitosan. The UV-irradiation induced morphological changes, such as less entangled cellulose fibers, as observed by scanning electron microscopy, which was prompted to enhance the chitosan incorporation. The biomass and spore germination percentage of Penicillium chrysogenum and colony forming units per millilitre for Escherichia coli decreased significantly on the composed materials as compared to raw cellulose fiber and it was similar to that obtained with a commercial antimicrobial cellulose fiber.     

Schedules for Sectioning Maize Kernels in Paraffin

For paraffin sectioning of maize kernels, the following technic is recommended: Use fresh, turgid kernels and utmost care in removing kernels from the cob and in subdividing into properly oriented slices. Kill and harden in a chrome-acetic-formalin formula. Rinse in water and dehydrate in four grades of dioxane to anhydrous; evacuate with an aspirator and infiltrate with paraffin. If anhydrous dioxane is excessively costly, dehydrate as above to the commercial grade and transfer by intermediate steps to one of the following two-solvent mixtures, using anhydrous ingredients, (A) dioxane and normal butyl alcohol, (B) dioxane and tertiary butyl alcohol, (C) normal butyl alcohol and chloroform, (D) tertiary butyl alcohol and chloroform. Evacuate in the final solvent. (Melted parowax floats on any of the above mixtures, affording gradual, progressive infiltration to pure parowax by periodic decantation and addition of wax.) Finally, transfer to compounded casting wax and cast in paper boats. To prepare a kernel segment for sectioning, fasten to a plastic block, shave the wax from the cutting plane and soak for 12-24 hours, at 35° C, in water containing a trace of safranin or other dye. Mordant starch grains in 1% tannic acid + 1/2% potassium metabisulphite. A wide choice of simple or multiple stains can be used. These methods are also applicable to tough old stems of corn and hemp, and possibly to many caryopses and seeds.     

两种不同方法制备的黑木耳多糖性质比较

本研究以黑木耳子实体为材料,对比了壳聚糖絮凝法制备的絮凝多糖HJD-1和传统水提醇沉法制备的醇沉多糖HJD-2的表观结构、α-葡萄糖甘酶抑制活性以及体外抑制肿瘤细胞增殖的活性。结果表明：（1）壳聚糖絮凝法制备粗多糖得率均值为4.76%,是醇沉法的2.17倍;醇沉法制备多糖的损失率为33.87%,是絮凝法的1.36倍;（2）絮凝多糖HJD-1和醇沉多糖HJD-2的表观评估及复溶性结果分析显示：絮凝多糖HJD-1为亮白色透明晶体,色泽均匀,颗粒规整;醇沉多糖HJD-2为棕褐色,颗粒状,有砂质感,前者相较后者的复溶性更好;（3）对α-葡萄糖甘酶抑制活性以及体外抗肿瘤能力结果分析表明：在相同浓度下,壳聚糖絮凝法制备黑木耳多糖对α-葡萄糖苷酶活性的抑制效果优于醇提法;絮凝多糖HJD-1对HepG2细胞的增殖抑制作用强于醇沉多糖HJD-2。     

Physical characteristics of decolorized chitosan as affected by sun drying during chitosan preparation

Some physical characteristics of decolorized chitosan as affected by sun drying, which was used to replace a bleaching step during chitosan preparation, were evaluated. One bleached and four unbleached chitosans were prepared and dried for 4 h by heat treatment at 60 °C or sun drying. The moisture content of chitosans dried by heat treatment was lower than that of chitosans dried by sun drying. Decoloration of the chitosan could be achieved more effectively by sun drying after deacetylation than by using a bleaching agent in the chitin preparation. Use of a bleaching agent significantly reduced the viscosity of the chitosan solution. A sequence of heat drying and sun drying in chitin and chitosan production (without using a bleaching agent) generally produced a whiter chitosan with higher viscosity without affecting water- and fat-binding capacities, compared to the bleached chitosan.     

Effect of solvent-dependent viscoelastic properties of chitosan membranes on the permeation of 2-phenylethanol

The viscoelastic behaviour of chitosan was followed by dynamic mechanical analysis (DMA) while the sample was immersed in gradient compositions of water/ethanol mixtures. The swelling equilibrium of chitosan membranes, both crosslinked with genipin or not, increased linearly with the water content. Increasing the water content, it was simultaneously observed a peak in the loss factor (around 25 vol.%) and a reduction of the storage modulus, which was attributed to the α-relaxation of chitosan. This was the first time that the glass transition dynamics in a polymer was monitored in immersion conditions where the composition of the plasticizer in the bath is changed in a controlled way. The water content at which tan δ presented a maximum increased with both increasing frequency and increasing crosslinking density. The permeability decreased steadily with the ethanol content, reaching very low values around the glass transition. Therefore we hypothesize that conformational mobility of the polymeric chains may play an important role in the diffusion properties of molecules trough polymeric matrices.     

Morphological and surface properties of electrospun chitosan nanofibers

Nonwoven fiber mats of chitosan with potential applications in air and water filtration were successfully made by electrospinning of chitosan and poly(ethyleneoxide) (PEO) blend solutions. Electrospinning of pure chitosan was hindered by its limited solubility in aqueous acids and high degree of inter- and intrachain hydrogen bonding. Nanometer-sized fibers with fiber diameter as low as 80 +/- 35 nm without bead defects were made by electrospinning high molecular weight chitosan/PEO (95:5) blends. Fiber formation was characterized by fiber shape and size and was found to be strongly governed by the polymer molecular weight, blend ratios, polymer concentration, choice of solvent, and degree of deacetylation of chitosan. Weight fractions of polymers in the electrospun nonwoven fibers mats were determined by thermal gravimetric analysis and were similar to ratio of polymers in the blend solution. Surface properties of fiber mats were determined by measuring the binding efficiency of toxic heavy metal ions like chromium, and they were found to be related with fiber composition and structure.