N-Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin

The deacetylation and depolymerization reactions of chitin/chitosan from three crustacean species (Paralomis granulosa, Lithodes antarcticus and Palinurus vulgaris) were evaluated under the same conditions. The average molecular weight and the mole fraction of N-acetylated units were the parameters studied in the resulting chitosans. During the N-deacetylation process P. granulosa, L. antarcticus and P. vulgaris follow a pseudo-first order kinetics and their apparent rate constants are very similar. However, the degradation rate of chitosan in the first 45 min of this process is higher for P. vulgaris. The depolymerization process follows a pseudo-first order kinetics for the three species, but in the first 9 min P. vulgaris shows a slightly lower depolymerization rate. Hence, depending on the ash contents, crystallinity and the physicochemical characteristics of chitin from these sources, the obtained chitosans show different qualities.     

Osteogenesis promoted by calcium phosphate N,N-dicarboxymethyl chitosan

The effects of N,N-dicarboxymethyl chitosan (DCMC) on the precipitation of insoluble calcium salts, namely phosphate, sulfate, oxalate, carbonate, bicarbonate and fluoride, and magnesium salts, namely phosphate and carbonate, were studied. Results indicated that the chelating ability of DCMC interfered effectively with the well-known physico-chemical behaviour of magnesium and calcium salts. Dicarboxymethyl chitosan formed self-sustaining gels upon mixing with calcium acetate, as a consequence of calcium chelation. DCMC mixed with calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) yielded a clear solution, from which, after dialysis and freeze-drying, an amorphous material was isolated containing an inorganic component about one half its weight. This compound was used for the treatment of bone lesions in experimental surgery and in dentistry. Bone tissue regeneration was promoted in sheep, leading to complete healing of otherwise non-healing surgical defects. Radiographic evidence of bone regeneration was observed in human patients undergoing apicectomies and avulsions. The DCMC–CaP chelate favoured osteogenesis while promoting bone mineralization.     

Effect of temperature modulations on TEMPO-mediated regioselective oxidation of unprotected carbohydrates and nucleosides

Regioselective oxidation of unprotected and partially protected oligosaccharides is a much sought-after goal. Herein, we report a notable improvement in the efficiency of TEMPO-catalyzed oxidation by modulating the temperature of the reaction. Mono-, di-, and tri-saccharides are oxidized regioselectively in yields of 75 to 92%. The present method is simple to implement and is also applicable for selective oxidations of other mono- and poly-hydroxy compounds including unprotected and partially protected nucleosides.     

Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous slurries of cotton linters. The water-insoluble fibrous fractions thus obtained in the yields of more than 78% were characterized by solid-state ¹³C-NMR, X-ray diffraction and scanning electron microscopic analyses for evaluation of distribution of carboxylate groups formed in the TEMPO-oxidized celluloses. The patterns of solid-state ¹³C-NMR spectra revealed that the oxidation occurred at the C6 primary hydroxyl groups of cellulose. X-ray diffraction and scanning electron microscopic analyses showed that such C6 oxidation took place at the surfaces of cellulose I crystallites without any oxidation at the C6 of inside cellulose I crystallites. Thus, carboxylate and aldehyde groups introduced into the TEMPO-oxidized celluloses are densely present on the surfaces of cellulose I crystallites. In addition, the obtained results revealed that the shoulder signal due to non-crystalline C6 carbons at about 63 ppm in solid-state ¹³C-NMR spectra of native celluloses is ascribed to those of surfaces of cellulose I crystallites or those of cellulose microfibrils.     

Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: A tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial

Two hundred years ago, Henri Braconnot described a polysaccharide containing a substantial percent of nitrogen, later to be called chitin: that discovery stemmed from investigations on the composition of edible mushrooms and their nutritional value. The present interdisciplinary article reviews the major research topics explored by Braconnot, and assesses their importance in the light of our most advanced knowledge. Thus, the value of fungi, seafoods and insects is described in connection with the significance of the presence of chitin itself in foods, and chitinases in the human digestive system. The capacity of chitin/chitosan to depress the development of microbial pathogens, is discussed in terms of crop protection and food preservation. Other topics cherished by Braconnot, such as the isolation of pectin from a large number of plants, and inulin from the Helianthus tubers, are presented in up-to-date terms. Acids isolated from plants at that early time, led to enormous scientific advancements, in particular the glyoxylic acid and levulinic acid used for the preparation of soluble chitosan derivatives that paved the way to a number of applications. An opportunity to trace the origins of the carbohydrate polymers science, and to appreciate the European scientific heritage.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

TEMPO-mediated oxidation of native cellulose: Microscopic analysis of fibrous fractions in the oxidized products

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous suspensions of cotton linters, ramie and spruce holocellulose at pH 10.5, and water-insoluble fractions of the TEMPO-oxidized celluloses collected by filtration with water were analyzed by optical and transmission electron microscopy and others. The results showed that both fibrous forms and microfibrillar nature of the original native celluloses were maintained after the TEMPO-mediated oxidation, even though carboxylate and aldehyde groups of 0.67–1.16 and 0.09–0.21 mmol/g, respectively, were introduced into the water-insoluble fractions. Neither crystallinity nor crystal size of cellulose I of the original native celluloses was changed under the conditions adopted in this study. Carboxylate groups in the TEMPO-oxidized ramie were mapped by labeling with lead ions as their counter ions. The transmission electron micrographs indicated that some heterogeneous distribution of carboxylate groups along each cellulose microfibril or each bundle of cellulose microfibrils seemed to be present in the TEMPO-oxidized celluloses.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.     

Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction

A new method to determine the degree of deacetylation (DD) of alpha-chitin and chitosan in the range of 17-94% DD using X-ray powder diffraction (XRD) is proposed. The results were calibrated using (1)H NMR spectroscopy for chitosan and FTIR for chitin, in comparison with the potentiometric titration method. The results showed a good linear correlation between the CrI020 from XRD and the calibrated DD value. This method is found to be simple, rapid and nondestructive to the sample.     

Novel chitin and chitosan nanofibers in biomedical applications

Chitin and its deacetylated derivative, chitosan, are non-toxic, antibacterial, biodegradable and biocompatible biopolymers. Due to these properties, they are widely used for biomedical applications such as tissue engineering scaffolds, drug delivery, wound dressings, separation membranes and antibacterial coatings, stent coatings, and sensors. In the recent years, electrospinning has been found to be a novel technique to produce chitin and chitosan nanofibers. These nanofibers find novel applications in biomedical fields due to their high surface area and porosity. This article reviews the recent reports on the preparation, properties and biomedical applications of chitin and chitosan based nanofibers in detail.     

Biomaterials based on chitin and chitosan in wound dressing applications

Wound dressing is one of the most promising medical applications for chitin and chitosan. The adhesive nature of chitin and chitosan, together with their antifungal and bactericidal character, and their permeability to oxygen, is a very important property associated with the treatment of wounds and burns. Different derivatives of chitin and chitosan have been prepared for this purpose in the form of hydrogels, fibers, membranes, scaffolds and sponges. The purpose of this review is to take a closer look on the wound dressing applications of biomaterials based on chitin, chitosan and their derivatives in various forms in detail.     

New flourinated chitin derivatives: synthesis, characterization and cytotoxicity assessment

New fluorinated chitin derivatives have been synthesized and characterized. Fluorination of chitin was achieved by facile homogenous reaction of chitin solution with diethyl amino sulfur trifluoride (C₄H₁₀NSF₃). The degree of substitution of the C6-hydroxyl functionality of N-acetyl-glucosamine repeat unit ranged from 50 to 98%, achieved by varying the reaction time from 1 to 144 h at room temperature. The use of pentafluoropropionic anhydride, trifluoromethylbenzoyl chloride and pentafluorobenzoyl chloride gave fluoro-chitin derivatives with 40, 10 and 5% substitution, respectively. Solid-state nuclear magnetic resonance and Fourier-transform infrared spectroscopy, powder X-ray diffraction, and elemental analysis support the identity of all fluorinated chitin derivatives. The fluorinated chitin derivatives were subjected to MTT assay using human (ATCC CCL-186) and mouse (ATCC CCL-1) fibroblast cell lines. Fluorinated chitin derivatives prepared from C₄H₁₀NSF₃ at 1, 6, 12, 72 and 96 h showed good cell viability of 80–100% for human fibroblast and 60–70% for mouse fibroblast. The % cell viability for the other fluorinated chitin derivatives were above 60% for both cell lines.     

Physiology of microbial degradation of chitin and chitosan

Chitin is produced in enormous quantities in the biosphere, chiefly as the major structural component of most fungi and invertebrates. Its degradation is chiefly by bacteria and fungi, by chitinolysis via chitinases, but also via deacetylation to chitosan, which is hydrolysed by chitosanases. Chitinases and chitosanases have a range of roles in the organisms producing them: autolytic, morphogenetic or nutritional. There are increasing examples of their roles in pathogenesis and symbiosis. A range of chitinase genes have been cloned, and the potential use for genetically manipulated organisms over-producing chitinases is being investigated. Chitinases also have a range of uses in processing chitinous material and producing defined oligosaccharides.     

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes

In this laboratory-scale study, we examined the potential of chitin and chitosan flakes obtained from shrimp wastes as carrier material for a hydrocarbon-degrading bacterial strain. Flakes decontamination, immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated. The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms. In terms of removal percentage of crude oil after 15 days, the microcosms treated with the immobilized inoculants proved to be the most successful. The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain. It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.     

从黑曲霉提取甲壳素和壳聚糖

采用电解法从培养的黑曲霉湿菌体制甲壳素 ;采用碱提取法从培养的黑曲霉湿菌体制壳聚糖。甲壳素提取的得率干菌重量的 2 0 6 %。所得干燥壳聚糖产品的游离胺基为 93 76 % ,0 5%壳聚糖的 0 5%醋酸的运动粘度为 5 4 4 8× 10 6 m2 /s ,粘均分子量为 8 2 75× 10 4 ,含水量为 9 16 % ,产品得率为 12 11%。     

Synthesis, characterization, cytotoxicity and antibacterial studies of chitosan, O-carboxymethyl and N,O-carboxymethyl chitosan nanoparticles

Chitosan (CS) is a naturally occurring biopolymer. It has important biological properties such as biocompatibility, antifungal and antibacterial activity, wound healing ability, anticancerous property, anticholesteremic properties, and immunoenhancing effect. Recently, CS nanoparticles have been used for biomedical applications. However, due to the limited solubility of CS in water its water-soluble derivatives are preferred for the above said applications. In this work, the nanoparticles of CS and its water-soluble derivatives such as O-carboxymethyl chitosan (O-CMC) and N,O-carboxymethyl chitosan (N,O-CMC) was synthesized and characterized. In addition, cytotoxicity and antibacterial activity of the prepared nanoparticles was also evaluated for biomedical applications.     

Bromination of regenerated chitin with N-bromosuccinimide and triphenylphosphine under homogeneous conditions in lithium bromide-N,N-dimethylacetamide

Chitin regenerated from LiCl-N,N-dimethylacetamide (DMA) was found to dissolve in 10 g/dL LiBr-DMA. The bromination of the regenerated chitin proceeded to a large extent (DS by bromine up to 0.94) with equimolar amounts of N-bromosuccinimide and triphenylphosphine under homogeneous conditions in LiBr-DMA at 50–90°C. ¹³C NMR spectroscopy of brominated products and GLC-MS analysis of their hydrolyzates showed that the bromine substitution took place regioselectively at C-6 of the chitin repeating units. Polymer chain scission occurred to some extent during the bromination, more extensively at higher temperatures with higher concentrations of reagents.     

Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state C NMR

Chitin, an important constituent of the exoskeleton of many organisms such as crustacea and insects, and its derivates promote the ordered healing of tissues and are therefore very suitable for use in wound dressings. The degree of substitution (DS) is an important parameter when assessing the conversion of chitin into one of its derivates. The degree of acetylation of chitin and chitosan and the degree of butyrylation of dibutyrylchitin was evaluated. It is found that FT-IR spectroscopy is a relatively easy but indirect way of determining the DS. FT-IR spectroscopy proved to be very useful for comparing the degrees of conversion and -substitution, as well as for differentiating between different chitin types. Absolute DS determinations by FT-IR however are only reliable when a calibration, using a direct technique such as ¹³C-NMR, is made.     

Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone