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.     

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.     

Chitin and chitinase: A kinetic model

A model is described for the action of insect molting chitinase on chitin microfibrils in cuticle. The model reconciles the disparate structures proposed for chitin in the literature. It also accounts for the kinetic characteristics of molting fluid chitinase insofar as known from in vitro studies, viz. positive co-operativity of possibly three catalytic sites, complexity, and processivity. These have hitherto been difficult to account for in vivo, given the arrangement of chitin in anhydrous microfibrils in arthropod cuticle.     

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid (M. L. Bade and A. Stinson, 1978, Biochem. Biophys. Res. Commun.84, 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N-acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.     

An enzymic method for the determination of chitin and chitosan in fungal cell walls

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain ofNeurospora crassa. Chitinase, obtained from cultures ofSerratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.     

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state ¹³C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40–100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state ¹³C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The ¹³C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of α-chitin than β-chitin.     

Chitin biosynthesis during pupal development of Drosophila melanogaster and the effect of the lethalcryptocephal mutation

Estimation of chitin deposition in the pupal and adult cuticles of adult Drosophila melanogaster during the pupal period is described. The timing of the periods of chitin deposition is compared with that deduced by previous workers using electron microscopy. The hypothesis that lethalcryptocephal mutant homozygotes are unable to evert their cephalic complexes at pupation because of excess chitin deposition is examined. The data obtained show no evidence that the mutation has any effect on chitin deposition.     

Preparation and evaluation of trimethylsilylated chitin as a versatile precursor for facile chemical modifications

Trimethylsilylation of chitin was studied in detail to establish a reliable method, and the properties of the resulting product were elucidated. Chitin was successfully trimethylsilylated with a mixture of hexamethyldisilazane and trimethylsilyl chloride in pyridine. Compared to alpha-chitin, beta-chitin was much more reactive and advantageous as a starting material to prepare fully substituted chitin in a simple manner, though alpha-chitin also underwent full silylation under appropriate conditions. The resulting silylated chitin was characterized by marked solubility in common organic solvents and by easy desilylation to regenerate hydroxy groups, which enabled clean preparation of chitin films. The reactivity of the silylated chitin was examined by treating with triphenylmethyl chloride and acetic anhydride as typical alkylating and acylating reagents, and complete substitutions were readily accomplished. The silylated chitin has thus proved to be a superb precursor for modification reactions.     

An alternative sputum processing method using chitin for the isolation of Mycobacterium tuberculosis

An alternative bio-friendly sputum processing method is the need of the hour to augment the rate of detection of TB cases and to improve the sensitivity of rapid growth based diagnostic methods. Chitin, mucolytic in nature and present ubiquitously in animal kingdom, was found to have decontaminating activity when used for processing sputum specimens. The aim of the present study is to develop an alternative bio friendly sputum processing method using chitin. Smear microscopy was done on direct sputum samples and on the deposits obtained after processing with modified Petroff’s method as well as Chitin method. Two direct smears were made from each of the sputum samples and stained by Ziehl Neelsen and Auramine phenol (AP) method. The samples were divided in to two aliquots and processed by chitin and modified Petroff’s method. Smears were made from each of the deposits and stained by both methods. The deposits were inoculated on to two Lowenstein Jensen slopes. AP method showed a sensitivity of 95% in direct smear. Samples processed by chitin and the deposit smears stained by AP method showed a sensitivity of 80% and a specificity of 89% compared to that of modified Petroff’s method. The sensitivity of chitin culture is 87% and the specificity is 85%. Chitin–H₂So₄ solution took less time compared to 4% NaOH to homogenize the mucopurulent sputum specimens. Chitin–H₂So₄ can be used as an alternative method of sputum processing for the detection of M. tuberculosis.     

The control of chitin deposition by ecdysterone in larvae of Bombyx mori

The effect of ecdysterone on the deposition of chitin in Bombyx larvae was examined. The chitin content in the abdomen decreased following hormone treatment to a value half that of the controls. Studies with ¹⁴C-glucose revealed that whereas controls exhibited a gradual decrease in the rate of ¹⁴C-glucose incorporation into chitin, the administration of ecdysterone resulted in a rapid fall in the rate of incorporation followed by a rise until ecdysis occurred. Chitin catabolism was estimated at 0·11 mg chitin/hr, based on the chitin content and incorporation rates. A dual rôle is indicated for ecdysterone in chitin metabolism, namely the activation of both synthetic and lytic systems.     

Preparation of chitin nanofibers from squid pen beta-chitin by simple mechanical treatment under acid conditions

A procedure for preparing individualized chitin nanofibers 3-4 nm in cross-sectional width and at least a few microns in length was developed. The key factors to prepare the chitin nanofibers with such high aspect ratios are as follows: (1) squid pen beta-chitin is used as the starting material and (2) ultrasonication of the beta-chitin in water at pH 3-4 and 0.1-0.3% consistency for a few minutes. Transparent and highly viscous dispersions of squid pen beta-chitin nanofibers in water can be obtained by this method. No N-deacetylation occurs on the chitin molecules during the nanofiber conversion procedure. Moreover, the original crystal structure of beta-chitin is maintained, although crystallinity index decreases from 0.51 to 0.37 as a result of the nanofiber conversion. Cationization of the C2 amino groups present on the crystallite surfaces of the squid pen beta-chitin under acid conditions is necessary for preparing the nanofibers.     

Optimization of the demineralization process for the extraction of chitin from Omani Portunidae segnis

Chitin is an organic polymer and it is the most frequent marine natural polysaccharide after cellulose. The main natural sources of chitin are exoskeletons of insects, mollusks, the cell walls of certain fungi and crustaceans such as crabs, shrimps and lobsters. The waste of these marine exoskeletons are pollutant for the environment, but these waste raw materials could be useful for production of commercial products like chitin. Chitin is an important raw material used for water treatment, agricultural, biomedical, biotechnological purposes, food and paper industry and cosmetics. Based on the variety of importance, the present targets of this study are to optimize the demineralization process for the removal of calcium and phosphate contents from the waste of Portunidae segnis (P. segnis) by using acid at ambient temperature and to characterize the isolated demineralized sample as well as the percentage of remaining calcium and phosphorus contents by using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). The prepared waste carbs coarse powder samples of P. segnis were demineralized with seven different concentrations of hydrochloric acid at ambient temperature for 1 h. All the demineralization samples by the different concentrations were analyzed by using sensitive ICP-OES. The results based on ICP-OES showed that among the seven different concentrations used in the demineralization process for the isolation of chitin, the best was 2 M of HCl concentration for the production of chitin. The results also showed that the optimized concentration 2 M HCl gave the minimum concentration of calcium and phosphorus compared to other concentrations applied in this experiment. In conclusion, the optimized concentration for demineralization process could be used commercially for the isolation or commercial production of chitin for agricultural, biomedical and biotechnological purposes.     

Determination of the degree of N-acetylation (DA) of chitin and chitosan in the presence of water by first derivative ATR FTIR spectroscopy

A new method for the determination of the degree of N-acetylation (DA) of chitin and chitosan is described using first derivative diamond ATR FTIR spectroscopy. Applying the derivative values of the amide III band at 1327 cm⁻¹ and the CH deformation band of the N-acetyl group at 1383 cm⁻¹ as measure of the N-acetyl content of the sample in relation to the derivative value of the bridge oxygen vibration at 1163 cm⁻¹ as internal standard, a linear correlation to the results of first derivative UV spectroscopy was obtained and confirmed by elemental analysis and Raman spectroscopy. The described method allows the determination of the degree of N-acetylation of chitosan and chitin in the presence of water thus making drying procedures unnecessary.     

Implications of molecular diversity of chitin and its derivatives

Chitin is a long unbranched polysaccharide, made up of β-1,4-linked N-acetylglucosamine which forms crystalline fiber-like structure. It is present in the fungal cell walls, insect and crustacean cuticles, nematode eggshells, and protozoa cyst. We provide a critical appraisal on the chemical modifications of chitin and its derivatives in the context of their improved efficacy in medical applications without any side effect. Recent advancement in nanobiotechnology has helped to synthesize several chitin derivatives having significant biological applications. Here, we discuss the molecular diversity of chitin and its applications in enzyme immobilization, wound healing, packaging material, controlled drug release, biomedical imaging, gene therapy, agriculture, biosensor, and cosmetics. Also, we highlighted chitin and its derivatives as an antioxidant, antimicrobial agent, anticoagulant material, food additive, and hypocholesterolemic agent. We envisage that chitin and chitosan-based nanomaterials with their potential applications would augment nanobiotechnology and biomedical industries.

     

Prevention of Death of Axotomized Hypoglossal Neurones and Promotion of Regeneration by Chitin Grafting

1. Chitin is known to promote skin wound healing. In this study, chitin, prepared from Zuwai crab shell, was used as a bridge between the proximal and distal stumps of cut hypoglossal nerves in shrews. We compared the effects of chitin on the regeneration of transected right hypoglossal nerve axons, with those of porcine dermis, bovine dermal aterocollagen, and autologous nerve bundles.2. To assess the survival of neurones, the size of neuronal cell body, and number of motoneurones were determined in the absence of any bridged material and in the presence of porcine dermis, bovine dermal aterocollagen, chitin, or autologous nerve bundles as a bridge.3. Our results revealed a significantly better outcome in chitin and autologous nerve bridged groups; the size of neuronal cell body and number of hypoglossal neurones were higher than in the other groups. Chitin also enhanced the regeneration of neurones; the number of horseradish peroxide positive neurones indicative of repaired axonal processes was significantly higher in chitin and autologous nerve-bridged groups than in other groups.4. Our results demonstrated that the use of chitin sheet or autograft successfully prevented the death of severed neurones and promoted the regeneration of the lesioned nerve. Although the mechanisms underlying the effects of chitin are still unknown, chitin seems to be a potentially useful biocompatible material for nerve repair and regeneration.     

Alternate method of calculating the free-energy change accompanying the growth of saccharomyces cerevisiae (Hansen) on three substrates.

A method is described for determining the free energy of formation of the cells of Saccharomyces cerevisiae (Hansen) that are formed as the result of anaerobic growth on glucose, and aerobic growth on glucose and ethanol. The method is based on the direct relationship that exists between the enthalpy changes and the free-energy changes that accompany the oxidation of 1 g cellular material formed during these growth reactions and the degree of reduction of the same material. When the results of these calculations are used together with the free energies of formation of the reactants and of other products of a given growth reaction, it becomes possible to calculate the free-energy change accompanying this reaction. These free-energy changes are in excellent agreement with those calculated by another method based on the hypothesis that the free-energy change accompanying the conversion of the substrate plus other reactants into cellular material plus other products is equal to zero.     

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo,Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.     

Lime treatment of shrimp head waste for the generation of highly digestible animal feed

In addition to approximately 20% ash, shrimp processing by-products contain 64% protein and chitin, both of which can be used to generate several valuable products. Chitin and chitosan production is currently based on several crustacean wastes, and at the present time the protein fraction is not being used. This paper describes the thermo-chemical treatment of shrimp processing wastes with lime to generate a protein-rich material with a well-balanced amino acid content that can be used as a monogastric animal feed supplement. The residual solid, rich in calcium carbonate and chitin, can still be used to generate chitin and chitosan through well-established processes.