Biological Properties of Chitosan Films with Different Degree of Deacetylation

Chitin and chitosan films were prepared by solution casting method. Chitosan specimens used in this study were deacetylated by 50.4%, 69.2%, 85.5% and 96.3%. Their water content, protein adhesion ability, cytocompatibility, cell adhesion ability, in vitro and vivo degradability and biocompatibility were evaluated. Results indicated that with the degree of deacetylation （DD） between 50% and 70%, the chitosan showed higher water content. The higher the DD, the stronger protein adhesion ability the chitosan had. All the films have good cytocompatibility and the films with higher DD have better cell adhesion ability. Chitin films degraded more rapidly than others, which disappeared in 2 to 4 weeks after they were implanted in subcutaneous tissue and musculature. Their inflammatory reaction became weaker as the films degraded. As the DD got higher, the films degraded slower. The films of DD 85.5% and DD 90.3% even didn＇t disappeared in 12 weeks after they were implanted. Their inflammatory reaction was mild at the beginning of degradation, and became severe in 4 to 8 weeks, then weaken at last. This basic result can be very helpful for tissue engineering.

Extraction and Characterization of Litopenaeus vannamei’s Shell as Potential Sources of Chitosan Biopolymers

Chitin is the second most abundant polysaccharide,produced mainly as an industrial waste stream during crustacean processing.Chitin can be derived into chitosan through the deacetylation process.Conversion of shrimp waste into chitosan via the deacetylation process could be considered a practical approach for shell waste remediation.In this study,chitosan’s physicochemical characteristics extracted from two types of Pacific white leg shrimp,L.vannamei’s shell(i.e.,rough and smooth),were compared with commercial chitosan.The yield,moisture,ash,solubility,water and fat binding capacity were measured.The degree of deacetylation(DDA)was calculated using FTIR,and their chemical Structure was confirmed using XRD and SEM-EDS.Both extracted chitosan showed no significant difference in yield,moisture,ash,solubility and water binding capacity but showed a significant difference with commercial chitosan.Moreover,the fat binding capacity of commercial chitosan showed the lowest percentage(408.34±0.83%)as compared to extracted chitosan(smooth shell 549.59±12.48%;rough shell 500.55±12.10%).The DDA indicated that extracted chitosan from the smooth and rough shell was considered good chitosan as compared to commercial chitosan with 84.08±1.27%,80.78±0.79%and 74.99±1.48%,respectively.Additionally,the presence of hydroxyl and amino groups from FTIR and a good crystallinity index was recorded using XRD of extracted chitosan.Based on observed characteristics,shrimp shell waste from L.vannamei can achieve chitosan standard quality as a biopolymer and highly potential to be applied in various industrial applications.

Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

Eco-friendly preparation of chitooligosaccharides with different degrees of deacetylation from shrimp shell waste and their effects on the germination of wheat seeds

Production of chitosan and its derivatives by traditional methods involves the excessive use of a reaction solution comprisedof sodium hydroxide and hydrochloric acid. Waste water resulting from this process has lim让ed the application of chitosanas a fertilizer as the process causes serious environmental pollution. Specifically, the resulting waste water contains highlevels of dissolved nitrogen and minerals from shrimp shells. In this study, an eco-friendly method was established to produce chitooligosaccharides (COS) with different degrees of deacetylation (DDAs) from shrimp shell waste. At a solid-tosolventratio of 1:6, the degree of demineralization was above 90% with the treatment of 30 g-L_1 H3PO4, and the degree ofdeproteinization was above 80% when treated with 30 g-L_1 KOH at 70 °C. Chitosans with different DDAs were obtainedby microwave-assisted KOH metathesis and the COS with Mw approximately 1500 Da were then prepared by oxidativedegradation. In summary, 33.73 kg H3PO4,12.77 kg, and 241.31 kg KOH were supplied during the processes of demineralization,deproteinization, and deacetylation of 100 kg shrimp shell waste, respectively. The process water was totally recycled,demonstrating that the shrimp shell could be wholly transformed into fertilize The entire process created a product withthe fractions of N:P2O5:K2O:COS = 7.94:24.44:10.72:1 &27. The test on the germination promotion of wheat seeds revealedthat the COS with 72」2% DDA significantly promoted germination. This work demonstrated the use of an eco-friendlypreparation method of COS with a specific degree of deacetylation that can be applied as a fertilizer.

Chitosan Obtained from Cell Wall of Aspergillus Niger Mycelium

Chitin from cell walls of Aspergillus Niger mycelium was prepared. A new method for the preparation of high deacetylation degree chitosan was studied in a dilute sodium hydroxide solution at a high pressure. The experimental results indicate that the deacetylation degree of the chitosan can reach 80% under the condition of a 5.00 mol/L sodium hydroxide solution at 0.1 MPa of pressure for 1 h. This method shows the advantages of the applications in the industry production and environment protection.

Bone remodeling effect of a chitosan and calcium phosphate-based composite

Chitosan is a biocompatible polymer that has been widely studied for tissue engineering purposes.The aim of this research was to assess bone regenerative properties of an injectable chitosan and calcium phosphate-based composite and identify optimal degree of deacetylation(%DDA)of the chitosan polymer.Drill holes were generated on the left side of a mandible in Sprague-Dawley rats,and the hole was either left empty or filled with the implant.The animals were sacrificed at several time points after surgery(7–22 days)and bone was investigated using micro-CT and histology.No significant new bone formation was observed in the implants themselves at any time points.However,substantial new bone formation was observed in the rat mandible further away from the drill hole.Morphological changes indicating bone formation were found in specimens explanted on Day 7 in animals that received implant.Similar bone formation pattern was seen in control animals with an empty drill hole at later time points but not to the same extent.A second experiment was performed to examine if the%DDA of the chitosan polymer influenced the bone remodeling response.The results suggest that chitosan polymers with%DDA between 50 and 70%enhance the natural bone remodeling mechanism.