Isolation and characterization of chitin from bumblebee (Bombus terrestris)

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.     

Extraction of chitin and chitosan from housefly,Musca domestica,pupa shells

Chitins and chitosans are some of the most abundant natural polysaccharide materials, and are used to increase innate immune response and disease resistance in humans and animals. In this work, chitin and chitosan from housefly, Musca domestica, pupa shells were obtained by treatment with HCl and NaOH. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve decalcification and deproteinization, respectively. For chitosan extraction, 50% NaOH solution was used to achieve deacetylation. The yields of chitin and chitosan from pupa shells of M. domestica were 8.02% and 5.87%, respectively. The deacetylations of chitosan (from chitin C1 and C2) were 89.76% and 92.39%, respectively, after the first alkali treatment with 50% NaOH (w/w) solution at 105 °C for 3 h and 5 h, respectively. The viscosities of the chitosans (from chitin C1 and C2) were 33.6 and 19.2 cP, respectively.     

α-Chitin dissolution,N-deacetylation and valorization in deep eutectic solvents

Chitin displays a highly rigid structure due to the vast intra- and intermolecular hydrogen bonding, thus hindering its dissolution and deacetylation using most solvents. Deep eutectic solvents (DESs) are special and environmentally friendly solvents composed of a hydrogen bond acceptor and a hydrogen bond donor. This allows them to dissolve chitin by disturbing its natural hydrogen bonding while establishing new bonds, hence turning the polymer more susceptible to solvents. Therefore, four distinct DESs (choline chloride-lactic acid ([Ch]Cl:LA), choline chloride:oxalic acid ([Ch]Cl:OA), choline chloride:urea ([Ch]Cl:U) and betaine-glycerol (Bet:G)) were applied in chitin dissolution, being the most performant ones further applied in its homogenous N-deacetylation with NaOH. In this work, a milder and more biocompatible approach was carried out by using 30 wt% NaOH at 80°C, instead of the typical ≥40 wt% NaOH at temperatures ≥100°C. Herein, the reaction process took up to 18 hours, being the results analyzed through ATR-FTIR. Chitin was converted into chitosan with a 70-80% degree of deacetylation (DDA) in a short period while using homogenous conditions. These promising results provide the first proof of concept of the ability of Bet:G and [Ch]Cl:LA-based DESs to be used as a greener approach for the chitin homogeneous N-deacetylation.     

Optimization of chitin extraction from shrimp shells

The aim of this paper is to define optimal conditions for the extraction of chitin from shrimp shells. The kinetics of both demineralization and deproteinization with, in the latter case, the role of temperature are studied. The characterization of the residual calcium and protein contents, the molecular weights, and degrees of acetylation (DA) allows us to propose the optimal conditions as follows. The demineralization is completely achieved within 15 min at ambient temperature in an excess of HCl 0.25 M (with a solid-to-liquid ratio of about 1/40 (w/v)). The deproteinization is conveniently obtained in NaOH 1 M within 24 h at a temperature close to 70 degrees C with no incidence on the molecular weight or the DA. In these conditions, the residual content of calcium in chitin is below 0.01%, and the DA is almost 95%.     

A hemagglutinating substance in chitin.

Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance having a strong capacity to agglutinate red blood cells was eluted from the chitin during a weak acid desorption step. The chitin agglutinin is a complex structure containing protein and poly-GlcNAc. Chitin samples from four biochemical suppliers were tested; all contained the elutable agglutinin. Acid (0.05 N HCl or 0.1 N acetic acid) appears to hydrolyze the material from the solid chitin. NaOH at 0.5 N does not remove the agglutinin. Since agglutination is the assay used to monitor lectin purification, care must be taken to avoid the native agglutinin if chitin is used as an affinity matrix.     

Chitin gels

Chitin dissolved in N,N-dimethylacetamide, N-methyl-2-pyrrolidone and their mixed solvents in the presence of 5% LiCl was treated with acetic anhydride-pyridine, and the mixture solution was heated at 100 degrees C for 6 h to give a partially O-acetylated chitin gel. Chitin dissolved in these solvents in the presence of 5% LiCl was mixed with pyridine, and the mixture solution was heated at 60 degrees C for 5 h to give a chitin gel. Both the gels were rigid and transparent, and their properties and the rate of the hydrolysis of the chitin xerogel by hen-egg white lysozyme were essentially similar to those of N-acetylchitosan gel prepared by chemical N-acetylation of chitosan.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.     

Production of chitin and chitosan from the exoskeleton of adult two‐spotted field crickets (Gryllus bimaculatus)

Chitin and chitosan were extracted from all specimens of Type I and II two‐spotted field crickets (Gryllus bimaculatus) following chemical treatment with an acid and alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve demineralization and deproteinization, respectively. For chitosan extraction, 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis, respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis, respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and 7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %, 90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi Poise), respectively. The molecular weight of chitosan from adult exoskeletons of G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G. bimaculatus could be used as a source of chitin and chitosan for use as functional additives in industrial animal feeds.     

Characterization of chitin and its hydrolysis to GlcNAc and GlcN

Proton NMR spectra of chitin dissolved in concentrated and deuterated hydrochloric acid (DCl) were found to be a simple and powerful method for identifying chitin from samples of biological origin. During the first hour after dissolving chitin in concentrated DCl (25 degrees C), insignificant de-N-acetylation occurred, meaning that the fraction of acetylated units (FA) of chitin could be determined. FA of demineralized shrimp shell samples treated with 1 M NaOH at 95 degrees C for 1-24 h were determined and were found to decrease linearly with time from 0.96 to 0.91 during the treatment with NaOH. Extrapolation to zero time suggested that chitin from shrimp shells has a FA of 0.96, that is, contains a small but significant fraction of de-N-acetylated units. Proton NMR spectra of chitin ( FA = 0.96) dissolved in concentrated DCl were obtained as a function of time until the samples were almost quantitatively hydrolyzed to the monomer glucosamine (GlcN). The initial phase of the reaction involves mainly depolymerization of the chitin chains, resulting in that almost 90% (molar fraction) of the chitin is converted to the monomer N-acetyl-glucosamine (GlcNAc).Thus, effective conversion of chitin to GlcNAc in concentrated acid is reported for the first time. GlcNAc is then further de-N-acetylated to GlcN. A new theoretical model was developed to simulate the experimental data of the kinetics of hydrolysis of chitin in concentrated acid. The model uses three different rate constants; two for the hydrolysis of the glycosidic linkages following an N-acetylated or a de-N-acetylated sugar unit and one for the de-N-acetylation reaction. The three rate constants were estimated by fitting model data to experimental results. The rate of hydrolysis of a glycosidic linkage following an N-acetylated unit was found to be 54 times higher as compared to the rate of de-N-acetylation and 115 times higher than the rate of hydrolysis of a glycosidic linkage following a de-N-acetylated unit. Two chitin samples with different F A values (0.96 and 0.70) were incubated in concentrated DCl until the samples were converted to the maximum yield of GlcNAc and the oligomer composition analyzed, showing that the maximum yield of GlcNAc was much higher when prepared from the chitin with the highest F A value.     

Technical note: Examining the use of ethylenediaminetetraacetic acid for humic extraction of ancient bone

We examined the efficacy of ethylenediaminetetraacetic acid (EDTA) for removing humic contaminants from collagen extracted from ancient bone. Humic contaminants must be removed to obtain reliable stable isotope values from ancient bone collagen, given that humic acids have consistently lower δ¹³C values than collagen. The purpose of our research was to examine if EDTA treatment could effectively remove humic contaminants from bone collagen and thus serve as an alternative to the commonly implemented sodium hydroxide (NaOH) treatment, which may be associated with large collagen losses in poorly preserved samples. We compared the isotopic and elemental composition of ancient samples treated with EDTA alone, samples demineralized in hydrochloric acid (HCl) and rinsed in EDTA, samples treated with HCl alone, and samples demineralized in HCl and rinsed with NaOH. The samples used in the analyses were selected because they presented evidence of substantial humic contamination. We found that NaOH was the most effective agent for reducing humic contaminants as evidenced by the samples treated with this agent having higher δ¹³C values and lower C:N ratios relative to other treatments. The results from samples treated with EDTA suggest that this chemical cannot effectively remove humic contaminants given that these samples had significantly higher C:N ratios and lower δ¹³C and δ¹⁵N values relative to the HCl/NaOH treatment. Our results demonstrate that when performing stable isotope analysis of ancient bone collagen suspected to be contaminated with humic acids, NaOH is the most effective chemical agent to remove humic contaminants, while EDTA cannot perform this task.     

Structural characterization of chitin and chitosan obtained by biological and chemical methods

Chitin production was biologically achieved by lactic acid fermentation (LAF) of shrimp waste (Litopenaeus vannameii) in a packed bed column reactor with maximal percentages of demineralization (D(MIN)) and deproteinization (D(PROT)) after 96 h of 92 and 94%, respectively. This procedure also afforded high free astaxanthin recovery with up to 2400 μg per gram of silage. Chitin product was also obtained from the shrimp waste by a chemical method using acid and alkali for comparison. The biologically obtained chitin (BIO-C) showed higher M(w) (1200 kDa) and crystallinity index (I(CR)) (86%) than the chemically extracted chitin (CH-C). A multistep freeze-pump-thaw (FPT) methodology was applied to obtain medium M(w) chitosan (400 kDa) with degree of acetylation (DA) ca. 10% from BIO-C, which was higher than that from CH-C. Additionally, I(CR) values showed the preservation of crystalline chitin structure in BIO-C derivatives at low DA (40-25%). Moreover, the FPT deacetylation of the attained BIO-C produced chitosans with bloc copolymer structure inherited from a coarse chitin crystalline morphology. Therefore, our LAF method combined with FPT proved to be an affective biological method to avoid excessive depolymerization and loss of crystallinity during chitosan production, which offers new perspective applications for this material.     

Deacetylation of Chitin under Homogeneous Conditions

Chitin isolated enzymatically from Antarctic krill shells was dissolved in aqueous NaOH by freezing and thawing to create homogeneous conditions. Deacetylation was performed at room temperature or under heating. The degree of deacetylation, molecular weight, and dynamic viscosity of solutions were estimated in chitosan samples. Deacetylation of chitin under homogeneous conditions was optimized. Chitosans with molecular weights of 180–220 and 250–300 kDa were obtained from the chitins of Antarctic krill and northern shrimp, respectively.     

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.     

Flexible chitin films: structural studies

Chitin gels were transformed into thin, flexible chitin films with minimal dimensional shrinkage and maximum flexibility and thickness in the range of 25-80 microm by a cold-press process. Solvent residue was removed by heating the films at 50 degrees C for 12 h, followed by rinsing in 95% ethanol. The crystallinity and mechanical properties of the flexible chitin films were found to be a function of the amount of shrinkage from the gel to the final film that was obtained. For 28-microm thick films with 30% shrinkage, transparency of up to 90% was found. X-ray diffractometry (XRD) showed that the number of diffraction peaks appearing at 2theta;=23 degrees and 2theta;=27 degrees became increasingly sharper with shrinkage. Topographical information obtained from scanning electron microscopy (SEM) and atomic force microscopy (AFM) attributed the structural morphology of the films to the formation of sub-microscopic micelles. Scanning transmission electron microscopy (STEM) showed that shrinkage resulted in coarser microstructure, affecting tensile properties, where the ductility and toughness were proportional to the amount of shrinkage. These flexible chitin films have potential as wound dressing materials.     

Cell wall composition and protoplast regeneration in Candida albicans

The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.     

A new bifunctional reagent for the intramolecular crosslinking of insulin (author's transl)]

Reaction of bis-[2-(succinimidooxycarbonyloxy)ethyl]sulfone [SO2(Eoc-ONSu)2] with insulin in 1N NaHCO3/dimethylformamide forms NalphaA1,NepsilonA1,NepsilonB29-2,2'-sulfonylbis(ethoxycarbonyl)insulin [SO2(Eoc)2-insulin] in 20 - 35% yield. The product can be purified by partition chromatography. After cleavage of the disulfide bridges, reoxidation in very dilute solution reconstitutes about 60% of the original insulin activity. Cleavage of the crosslinking moiety can be achieved with 0.5N NaOH at 0 degrees C in only a few seconds, rendering a biologically fully active insulin.     

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

Stability of chitin synthetase in cell-free preparations of a wild-type strain and a ''slime'' variant of Neurospora crassa

Chitin synthetase activity in cell-free preparations from a wild-type strain and a 'slime' variant of Neurospora crassa was monitored over many days in samples stored at 0 degrees C. Total activity in whole-cell-free extracts and low-speed supernatants from both organisms was very unstable, losing more than 90% of the initial activity on storage at 0 degrees C for 96 h. Chitin synthetase detection was not masked by chitinase activity present in the preparations. Gel-filtration chromatography of these preparations increased the stability of the activity from the 'slime' variant, whereas removal of particulate structures by high-speed centrifugation stabilized the chitin synthetase activity in the supernatant, particularly in the wild type. These results suggest that factor(s) involved in the regulation of chitin synthetase may be differentially located or altered in 'slime' cells.     

Solution properties of chitin in alkali

The solution properties of alpha-chitin dissolved in 2.77 M NaOH are discussed. Chitin samples in the weight-average molecular weight range 0.1 x 10(6) g/mol to 1.2 x 10(6) g/mol were prepared by heterogeneous acid hydrolysis of chitin. Dilute solution properties were measured by viscometry and light scattering. From dynamic light scattering data, relative similar size distributions of the chitin samples were obtained, except for the most degraded sample, which contained aggregates. Second virial coefficients in the range 1 to 2 x 10(-3) mL.mol.g(-2) indicated that 2.77 M NaOH is a good solvent to chitin. The Mark-Houwink-Sakurada equation and the relationship between the z-average radius of gyration (Rg) and the weight-average molecular weight (Mw) were determined to be [eta] = 0.10Mw0.68 (mL.g(-1)) and Rg = 0.17Mw0.46 (nm), respectively, suggesting a random-coil structure for the chitin molecules in alkali conditions. These random-coil structures have Kuhn lengths in the range 23-26 nm.