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.     

Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia

Chitin samples in a alpha-form structure were isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia by treatment with 1 N HCl and 1 N NaOH. Chitosan was prepared by treating them in 40% NaOH containing NaBH(4). Chitin and chitosan were analyzed by X-ray, [13C]CP/MAS NMR, [13C]FT-NMR, and scanning electron microscopy (SEM) methods. Insect chitin degraded more readily than shrimp chitin when treated with 6 N HCl and the enzyme-chitinase. After treatment with 2 N HCl at 100 degrees C, the insect chitin crystallinity increased. N-deacetylation of insect chitin was easier than that of crustaceous chitin, and about 94% of the N-acetyl groups were removed in one treatment with 40% NaOH for 4 h at 110 degrees C. After treatment with 2 N HCl, 55% of the N-acetyl groups of silkworm chitin were removed under the same conditions. Beetle chitin showed a higher affinity for chitinase than shrimp chitin.     

Characterization of Some Fungal Chitosans

Chitosan-like materials were extracted from five different fungal cells with NaOH and acetic acid, with the yields varying from 1.2 to 10.4% of the dry fungal cell weight. The degree of N-acetylation of the extracts measured by the colloidal titration method varied considerably depending on the individual species. By IR measurements and the Elson-Morgan method, four kinds of the extracts were characterized as chitosan while another one was not.

The degree of N-acetylation and the Cu²⁺ adsorption capacity of the fungal chitosans were measured and compared with those of authentic samples with various degrees of N-acetylation, which were prepared by chemical treatment of authentic chitin and chitosan derived from Crustacea. The Cu²⁺ adsorption capacity of the fungal chitosans was higher than that of the authentic chitosan samples with similar degrees of N-acetylation and independent of the molecular weight of the chitosans from the various sources.     

Extraction of chitin and chitosan from larval exuvium and whole body of edible mealworm,Tenebrio molitor

The purpose of this study was to investigate the production of chitin and chitosan from both the exuvium and whole body of mealworm (Tenebrio molitor) larvae. Chitin from the exuvium and whole body of T. molitor larvae was chemically extracted with acid and alkali solutions to achieve demineralization (DM) and deproteinization (DP), respectively. The average DM (%) and DP (%) on a dry weight (DW) basis was 32.56 and 73.16% from larval exuvium, and 41.68 and 91.53% from whole body, respectively. To obtain chitosan, chitin particles from the exuvium and whole body of T. molitor larva were heated at various temperatures in different concentrations of NaOH. Average chitin yields were 18.01% and 4.92% of DW from the exuvium and whole body, respectively. The relative average yield of chitosan from whole body was 3.65% of DW. On average, over 90% of chitosan derived from whole body was deacetylated. The viscosity of chitosan from whole body was ranged from 48.0 cP to 54.0 cP. The chitin content of dry and wet byproducts from whole body were 17.32% and 16.94% respectively, compared to dry weight. The chitosan contents of byproducts on a DW basis were 14.48% in dry and 13.07% in wet byproduct. These results indicate that the exuvium and whole body of T. molitor larva may serve as a source of chitin and chitosan for use in domestic animal feed.     

TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

A chitinase with high activity toward partially<Emphasis Type="Italic"> N</Emphasis>-acetylated chitosan from a new,moderately thermophilic,chitin-degrading bacterium,<Emphasis Type="Italic"> Ralstonia</Emphasis> sp. A-471

A moderately thermophilic bacterium, strain A-471, capable of degrading chitin was isolated from a composting system of chitin-containing waste. Analysis of the 16S rDNA sequence revealed that the bacterium belongs to the genus Ralstonia. A thermostable chitinase A (Ra-ChiA) was purified from culture fluid of the bacterium grown in colloidal chitin medium. Purification of the enzyme was achieved mainly by exploiting its binding to the colloidal chitin. The molecular mass of the enzyme was estimated to be 70 kDa and the isoelectric point approximately 4.7. N-terminal amino acid sequencing revealed a sequence of ADPYLKVAYYP, which had high homology (66% identity) with that of chitinase A1 from Bacillus circulans WL-12. The pH and temperature optima were determined to be 5.0 and 70°C, respectively. The enzyme was classified as a retaining glycosyl hydrolase and was most active against partially N-acetylated chitosans. Its activities towards the partially N-acetylated chitosans, i.e. chitosan 7B, chitosan 8B, and chitosan 9B, were about 11-fold, 9-fold, and 5-fold higher than towards colloidal chitin, respectively. Ra-ChiA cleaved (GlcNAc)₆ almost exclusively into (GlcNAc)_2. Activation of Ra-ChiA was observed by the addition of 1 mM Cu²⁺, Mn²⁺, Ca²⁺_, or Mg²⁺. Degradation of the partially N-acetylated chitosan produced oligosaccharides with a degree of polymerization ranging from 1–8; these are products that offer potential application for functional oligosaccharide production.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

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.     

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.     

Polysaccharides from mycelium of the fungus <Emphasis Type="Italic">Cunninghamella japonica</Emphasis>

Preliminary data on the polysaccharide composition of mycelium of the fungus Cunninghamella japonica (synonymous with C. echinulata) grown by the method of submerged cultivation were obtained. Mild acidic hydrolysis of mycelium resulted in the formation of glucose, mannose, and galactose; while the treatment with acid under drastic conditions afforded glucosamine as a product of hydrolysis of chitin and chitosan, their total content was about 35%. Several polysaccharide fractions were isolated from mycelium by successive extraction with hot water, 2% aqueous NaOH, and 10% AcOH; their monosaccharide composition was characterized. The yield of chitosan extracted with AcOH was insignificant. Additional purification of the fraction obtained after extraction with alkali afforded polysaccharide which was a linear (1 → 3)-α-D-glucopyranan according to the data of NMR spectroscopy and the chemical methods of structural analysis. The presence of this polysaccharide, as well as a low content of chitosan and polyuronides, distinguishes the studied strain C. japonica from most of the known Mucorales.     

Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane

Different molecular weight, 90% deacetylated chitosans were obtained by ultrasonic degradation on 90% deacetylated chitosan at 80 °C for various times.

Ninety percent deacetylated chitosan was prepared from alkali treatment of chitin that was obtained from red shrimp waste. Number average-, viscosity average- molecular weights were measured by gel permeation chromatography and the viscometric method, respectively. Degree of deacetylation was measured by the titration method. Enthalpy, maximum melting temperature, tensile strength and elongation of the membranes, flow rate of permeates and water are properties measured to elucidate the effect of molecular weight of chitosan on the above thermal, mechanical, and permeation properties, respectively of the prepared membranes. Results show tensile strength, tensile elongation, and enthalpy of the membrane prepared from high molecular weight chitosans were higher than those from low molecular weight. However, the permeability show membranes prepared from high molecular weight chitosans are lower than that from those of low molecular weight.     

Antimicrobial Characteristics of Chitosans against Food Spoilage Microorganisms in Liquid Media and Mayonnaise

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

Antioxidant properties of chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and its antioxidant properties studied. Chitosan exhibited showed antioxidant activities of 58.3–70.2% at 1 mg/mL and showed reducing powers of 0.32–0.44 at 10 mg/mL. At 10 mg/mL, the scavenging ability of chitosan C60 on 1,1-diphenyl-2-picrylhydrazyl radicals was 28.4% whereas those of other chitosans were 46.4–52.3%. At 0.1 mg/mL, scavenging abilities on hydroxyl radicals were 62.3–77.6% whereas at 1 mg/mL, chelating abilities on ferrous ions were 82.9–96.5%. All EC₅₀ values of antioxidant activity were below 1.5 mg/mL. With regard to antioxidant properties assayed, the effectiveness of chitosans C60, C90 and C120 correlated with their N-deacetylation times. Overall, crab chitosan was good in antioxidant activity, scavenging ability on hydroxyl radicals and chelating abilities on ferrous ions and may be used as a source of antioxidants, as a possible food supplement or ingredient in the pharmaceutical industry.     

Purification and characterization of a chitosanase from Streptomyces N174

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.Correspondence to: R. Brzezinski     

Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise.

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25 degrees C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

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%.     

Antitumor activity of chitosan from mayfly with comparison to commercially available low,medium and high molecular weight chitosans

Insects’ cuticles have a potential to be evaluated as a chitin source. Especially adults of aquatic insects like mayflies (order Ephemeroptera) swarm in enormous numbers in artificially lit areas while mating in spring and then die by leaving huge amounts of dead insects’ bodies. Here in this study, mayfly corpses were harvested and used for production of low MW chitosan. Dried mayfly bodies had 10.21% chitin content; mayfly chitin was converted into chitosan with efficiency rate of 78.43% (deacetylation degree, 84.3%; MW, 3.69 kDa). Cytotoxicity and anti-proliferative activity of mayfly and commercially available shrimp chitosans (low, medium, and high MW) were determined on L929 fibroblast and three different cancer types including HeLa, A549, and WiDr. Apoptosis and necrosis stimulating potential of mayfly and commercial chitosans were also evaluated on A549 and WiDr cells using acridine orange and propidium iodide dual staining to observe morphological changes in nuclei and thus to reveal the predominant cell death mechanism. The effects of chitosans have varied depending on cell types, concentration, and chitosan derivatives. Mayfly and low MW chitosans had a cytotoxic effect at a concentration of 500 μg mL^?1 on non-cancer cells. At concentrations below this value (250 μg mL^?1), mayfly and commercial chitosans except high MW one exhibited strong inhibitory activity on cancer cells especially A549 and WiDr cells. Mayfly chitosan induced early and late apoptosis in A549 cells, but late apoptosis and necrosis in WiDr cells. This study suggests that dead bodies of mayflies can be used for production of low MW chitosan with anti-proliferative activity.     

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.     

The genusCandida berkhout

The aim of the study was to verify the accuracy of the taxonomic classification of rough variants of the speciesCandida guilliermondii on the basis of comprehensive study of phenetic manifestation and to determine differences in cell wall structure with special reference to polysaccharides (1) According to their phenotype, the test strainsCandida guilliermondii (Cast.) Langeron et Guerra andCandida guilliermondii var.membranaefaciens Lodder et Kreger-Van Rij belong to the speciesCandida guilliermondii, whileCandida guilliermondii var.nitratophila Diddens et Lodder is phenotypically closer toCandida pelliculosa. (2) Observation of native and hydrolysed cell walls in the electron microscope showed no differences between the test strains. (3) The results of X-ray phase analysis of cell walls differentiatedCandida guilliermondii var.nitratophila from the other two, however. (4) Electron microscopy photomicrographs and diffractograms of cell walls indicated that, after 2% HCl extraction at 100 C, the cell walls contain chitin, which is isolated by further extraction with 30% HCl. After 3% NaOH hydrolysis the chitin diffractogram is not clear.