Improved method for production of chitin and chitosan from shrimp shells.

In this study, pretreatment procedures have been investigated preceding the standard production of chitin and chitosan. These steps can be used in industrial processes to preserve raw shrimp shells as long as the amount of material is not enough for one production batch. After these treatments, shrimp shells are clean and are facile for further demineralization, deproteinization and deacetylation processes. The prepared chitin and chitosan show a high purity with very low ash (less than 0.3%) and protein residues (less than 0.5%), along with their high molecular weight and high crystallinity. This modified approach has potential for application in large-scale production due to its ease of operation and reduction of environmental concerns.

Preparation of water soluble hydrochloric chitosan from low molecular weight chitosan in the solid state.

In this study, low molecular weight chitosan salt (LMWC-HCl) highly soluble in water was prepared from low molecular weight chitosan (LMWC) in the solid state exposed to hydrogen chloride gas as a reagent. The effects of chitosan particle size, exposure conditions, reaction temperature and reaction time were investigated on the solubility and the molecular weight of obtained products. The formation of the chloride salt was observed after 3 h in a range of temperatures from 4 to 50 °C. The solubility of prepared LMWC-HCl was over 98% for all samples, much higher than that of the original LMWC. The average molecular weight of the LMWC-HCl was about 20-90 kDa with a quite narrow distribution and lower compared to the LMWC. LMWC-HCl and LMWC showed the same high antibacterial activity against Bacillus cereus and Vibrio parahaemolyticus. This facile and efficient process for solubilization of LMWC has potential for industrial application of chitosan.

Decomposition of myceliar matrix and extraction of chitosan from Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076.

Free chitosan, 2 g/100g mycelia from Gongronella butleri and 6.5 g/100g mycelia from Absidia coerulea were isolated by 1M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h. Both myceliar matrixes did not break down under these conditions. However, myceliar matrix could be decomposed by treatment with 11 M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h and then extracted the total chitosan, 8-9 g/100g mycelia from both fungi. According to these results, G. butleri has higher amount of complexed chitosan and A. coerulea has higher amount in free chitosan.

Ionotropic cross-linked chitosan microspheres for controlled release of ampicillin.

The solubility of non cross-linked chitosan in weak acid solutions restricts its utility in microspheres for drug delivery. The primary aim of this study was to produce pentasodium tripolyphosphate cross-linked chitosan microspheres with higher acid resistance for controlled release of ampicillin. The microspheres were prepared by two different microencapsulation procedures (by emulsification and by spray-drying) and characterized by their particle size, surface morphology, stability, drug entrapment efficiency and drug release. The size of the microspheres was <10 microm with a narrow size distribution. The entrapment of ampicillin in the microspheres was more than 80%. Stability of uncross-linked and cross-linked microspheres was affected by the pH of simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5). The inclusion of the enzymes pepsin and pancreatin did not affect the stability of the microspheres. The inclusion of lysozyme in phosphate buffer saline resulted in increased solubilization. The release of the drug was affected by cross-linking of microspheres with tripolyphosphate (TPP). The cross-linked microspheres were more stable in simulated gastric fluid and showed slower but sustained release of ampicillin. The antimicrobial activity of the released ampicillin was confirmed by Staphylococcus aureus bioassay.

Evaluation of an improved acid hydrolysis-HPLC assay for the acetyl content in chitin and chitosan.

Functional properties of the amino polysaccharides, chitin and chitosan, vary significantly with their acetyl content. The acid hydrolysis-HPLC method offers good accuracy and precision to assay the acetyl content regardless of the solubility of the sample. In this research, the hydrolysis parameters were changed, and the analytical method was counterchecked with other methods. Complete hydrolysis was achieved by mixing chitosan with 1.4 mM oxalic acid and 12 M sulfuric acid followed by treatment at 110 degrees C for 40 min. A sealed glass ampule was used instead of a vacuum hydrolysis tube. The acetic acid released during acid hydrolysis was measured quantitatively by HPLC. A high correlation (r(2) = 0.98) was obtained between the modified HPLC assay and the solid-state (13)C CP/MAS NMR method for the samples of various crustacean sources with a wide range of acetyl contents. The modified HPLC method was also highly correlated (r(2) = 0.99) with the first derivative UV method for soluble chitosan.

Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles.

The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column.

Functional characteristics of shrimp chitosan and its membranes as affected by the degree of deacetylation.

The functional properties of three shrimp chitosan preparations with different degrees of deacetylation (75%, 87% and 96% DD) but with a constant molecular weight (about 810 kDa) were investigated. Chitosan with 75% DD had a 1.5 times higher water absorption, probably due to its 20% lower level of crystallinity. Membranes cast from this chitosan also exhibited 1.5 times more water absorption and 2 times higher permeability. However, chitosan with 87% and 96% DD had 1.5-2 times higher absorption of fat and the orange II dye. This is attributed to the higher content of positively charged amine groups in the polymer. Cast into membrane, chitosan of higher degree of deacetylation showed a higher tensile strength and a higher elongation at break, probably due to the higher level of crystallinity.

Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae.

Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.

Chitosan-alginate multilayer beads for controlled release of ampicillin.

The aim of this study is to develop multilayer beads with improved properties for controlled delivery of the antibiotic ampicillin. Ionotropic gelation was applied to prepare single and multilayer beads using various combinations of chitosan and Ca(2+) as cationic components and alginate and polyphosphate as anions. Beads prepared with higher concentrations of chitosan entrapped more ampicillin. During incubation in simulated gastric fluid, the beads swelled and started to float but did not show any sign of erosion. Single layer chitosan-alginate beads released 70% of the drug within 4 h. Multilayer beads released only 20-30% in the same period of time. During subsequent incubation in simulated intestinal fluid, both single and multilayer beads continued to release drug. At least part of this release is due to disintegration of the beads. The rate of release both in gastric and intestinal fluid and the kinetics of disintegration in intestinal fluid can be controlled by changing the chitosan concentration in the coagulation fluid. The release of the drug can also be controlled by the degree of cross-linking using polyphosphate. Cross-linked multilayer beads were prepared that released only 40% of the entrapped drug during 24 h. It is concluded that chitosan-alginate multilayer beads, cross-linked with polyphosphate offer an opportunity for controlled gastrointestinal passage of compounds with low molecular weight like ampicillin.

Chitosan membrane as a wound-healing dressing: characterization and clinical application.

Chitosan prepared from natural biopolymer chitin and cast into membranes has been tested as wound dressing at the skin-graft donor site in patients. Bactigras, a commonly used impregnated tulle gras bandage, served as a control. Chitosan membrane, prepared with a 75% degree of deacetylation and a thickness of 10 microm, was used in nonmesh or mesh form. The progress in wound healing was compared by clinical and histological examination. Itching and pain sensitivity of the wound dressed area was scored with the use of a visual analogue scale. Mesh chitosan membrane in contrast to the nonmesh membrane allowed blood to ooze into the surrounding gauze. After 10 days, the chitosan-dressed area had been healed more promptly as compared with the Bactigras dressed area. Moreover, the chitosan mesh membrane showed a positive effect on the re-epithelialization and the regeneration of the granular layer. The data confirm that chitosan mesh membrane is a potential substitute for human wound dressing.

Selection of a practical assay for the determination of the entire range of acetyl content in chitin and chitosan: UV spectrophotometry with phosphoric acid as solvent.

The rapidly expanding use of chitomaterials in biomedical applications demands accurate and precise analytical methods to determine physico-chemical characteristics, especially the acetyl content of the sample. The analytical methods available for the determination of the acetyl content of the biomaterials are quite different in efficiency, accuracy and precision. Out of 22 analytical methods reviewed, XRD, DSC, FTIR (KBr pellet), solid state (13)C CP/MAS NMR, and acid hydrolysis-HPLC and the spectrophotometry assay using phosphoric acid as solvent (PUV) were selected in this study. The validity and applicability of these methods were investigated with a wide range of chitin and chitosan samples varying acetyl content, preparation methods, and sources. The XRD, DSC, and FTIR (KBr pellet) methods showed poor accuracy with the samples of diverse preparations and sources. The PUV method was modified and accuracy of the method was examined against absolute methods: solid state (13)C CP/MAS NMR and acid hydrolysis-HPLC methods. The correlations between these three methods were >0.9. Therefore, the PUV method was selected as the most generally acceptable method based on its accuracy, reliability, simplicity, and instrument availability.

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe).

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.

Characterization of decrystallized chitosan and its application in biosorption of textile dyes.

Decrystallized chitosan was produced from shrimp shells with a low degree of crystallinity (10%) and a high anionic dye binding capacity. Raw, mixed dye wastewater from a textile factory was efficiently decolorized using decrystallized chitosan that was more efficient than using normal chitosan and activated carbon. Decolorization reached 90% within 10 min and could be carried out from pH 4.5 to 8.1. Decrystallized chitosan can be regenerated by 2 M H2SO4 and was reusable more than 10 times. It is, therefore, an attractive candidate for the removal of dyes from textile wastewater.

Quantification and characterization of insoluble chitinous materials in viscous chitosan solutions.

Insoluble chitinous materials in highly viscous chitosan solutions can be quantified using the viscosity-lowering action of transglucosidase (EC 2.4.1.24). In chitosan, commonly produced by high temperature deacetylation (90 degrees C), between 70-90% of insoluble chitinous materials were recovered by this enzymatic method whereas only 25% recovery was obtained by the nitrous acid method. The insoluble material recovered after enzyme treatment had a higher degree of deacetylation and a lower degree of crystallization than that after nitrous acid treatment. The results are explained by difference in penetration by enzyme and nitrous acid into the insoluble particle.

Transdermal delivery controlled by a chitosan membrane.

The release of a drug from a transdermal delivery system with a rate controlling chitosan membrane was analyzed in vitro and in vivo. Lidocaine hydrochloride, a local anesthetic, was used as the model drug. The in vitro permeability of various chitosan membranes for the drug was investigated using a Franz diffusion cell. Drug release was slower through chitosan membranes with a higher degree of deacetylation (% DD) and with a larger thickness. A transdermal chitosan patch was developed using a chitosan membrane for rate control and a chitosan hydrogel as a drug reservoir. The most prolonged release in vitro was obtained with a 95% DD chitosan rate controlling membrane. The transport mechanism was found to be non-Fickian. The functionality of this transdermal patch was studied on the forearm of human volunteers by assessing the anesthetic effect. Patches with 70% and 95% DD membranes delayed the anesthetic effect, increasing the delay with increasing % DD. It was concluded that a combination of chitosan membrane and chitosan hydrogel is a good transparent system for controlled drug delivery and that the release kinetics in vitro at least for lidocaine have a predictive value for its anesthetic effect in vivo. The demonstration of a direct relationship between in vitro drug membrane permeability and its physiological effect might be considered as quite unique.

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization.

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.

Chitosan-alginate multilayer beads for gastric passage and controlled intestinal release of protein.

Chitosan-alginate beads loaded with a model protein, bovine serum albumin (BSA) were investigated to explore the temporary protection of protein against acidic and enzymatic degradation during gastric passage. Optimum conditions were established for preparation of homogenous, spherical, and smooth chitosan-alginate beads loaded with BSA. Multilayer beads were prepared by additional treatment with either chitosan or alginate or both. The presence of chitosan in the coagulation bath during bead preparation resulted in increased entrapment of BSA. During incubation in simulated gastric fluid (SGF pH 1.2), the beads showed swelling and started to float but did not show any sign of erosion. Inclusion of pepsin in the gastric fluid did not show a further effect on the properties of the beads. Release studies were done in simulated gastric fluid (SGF pH 1.2) and subsequently in simulated intestinal fluid (SIF pH 7.5) to mimic the physiological gastrointestinal conditions. After transfer to intestinal fluid, the beads were found to erode, burst, and release the protein. Microscopic and macroscopic observations confirmed that the release of protein was brought about by the burst of beads. Chitosan-reinforced calcium-alginate beads showed delay in the release of BSA. The multilayer beads disintegrated very slowly. The enzymes pepsin and pancreatin did not change the characteristics of BSA-loaded chitosan-alginate beads. Single layer chitosan-alginate beads released 80-90% of the model protein within 12h while multilayer beads released only 40-50% in the same period of time. The release from chitosan-alginate beads and multilayer beads in SIF was further delayed without prior incubation in SGF. It is concluded that alginate beads reinforced with chitosan offer an excellent perspective for controlled gastrointestinal passage of protein drugs.