Effect of ultrasonic treatment on the biochemphysical properties of chitosan

Four chitosans with different molecular weights and degrees of deacetylation degree and 28 chitosans derived from these initial chitosans by ultrasonic degradation have been characterized by gel permeation chromatography (GPC), FT-IR spectroscopy, X-ray diffraction and titrimetric analyses. Antimicrobial activities were investigated against E. coli and S. aureus using an inhibitory rate technique. The results showed that ultrasonic treatment decreased the molecular weight of chitosan, and that chitosan with higher molecular weight and higher DD was more easily degraded. The polydispersity decreased with ultrasonic treatment time, which was in linear relationship with the decrease of molecular weight. Ultrasonic degradation changed the DD of initial chitosan with a lower DD (<90%), but not the DD of the initials chitosan with a higher DD (>90%). The increased crystallinity of ultrasonically treated chitosan indicated that ultrasonic treatment changed the physical structure of chitosan, mainly due to the decrease of molecular weight. Ultrasonic treatment enhanced the antimicrobial activity of chitosan, mainly due to the decrease of molecular weight.     

Effect of chitosan type on protein and water recovery efficiency from surimi wash water treated with chitosan-alginate complexes

Previous research has shown that soluble protein recovery by chitosan (Chi) complexes with polyanions such as alginate (Alg) is more effective than using chitosan alone. In this study, Chi-Alg complexes were used to recover soluble proteins from surimi wash water (SWW) slightly acidified to pH 6. Six Chi samples differing in molecular weight (MW) and degree of deacetylation (DD) were used at 20, 40 and 100mg/L SWW Chi-Alg complexes prepared with a Chi:Alg mixing ratio previously optimized (MR=0.2). FTIR analysis of the solids recovered revealed the three characteristic amide bands observed in the same region for untreated SWW confirming protein adsorption by Chi-Alg. The superior effectiveness of Chi complexes was confirmed but differences among chitosan types could not be correlated to MW and DD. Experimental Chi samples with 94%, 93%, 75% and 93% DD and 22, 47, 225 and 3404 x 10(3)Da, respectively, showed 73-76% protein adsorption while a commercial chitosan sample with 84% DD and 3832 x 10(3)Da had 74-83% protein adsorption. An experimental chitosan, SY-1000 with 94% DD and 1.5 x 10(6)Da, showed the highest protein adsorption (79-86%) and turbidity reduction (85-92%) when used at 20mg/L SWW.     

The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation

In the design of chitosan-based drug delivery systems and implantable scaffolds, the biodegradation rate of the chitosan matrix represents a promising strategy for drug delivery and the function of carriers. In this study, we have investigated the degradation of chitosan with different degrees of N-acetylation, with respect to weight loss, water absorption, swelling behavior, molecular weight loss of bulk materials, and reducing sugar content in the media. Chitosan matrices were prepared by compression molding. The results revealed that the initial degradation rate, equilibrium water absorption, and swelling degree increased with decreasing degree of deacetylation (DD) and a dramatic rise began as DD of the chitosan matrix decreased to 62.4%. Chitosan matrices with DD of 52.6%, 56.1%, and 62.4% had the weight half-life of 9.8, 27.3, and more than 56 days, respectively, and the weight half-life of average molecular weight 8.4, 8.8, and 20.0 days, respectively. For chitosan matrices with DD of 71.7%, 81.7%, and 93.5%, both types of half-life exceeded 84 days because of the much slower degradation rate. The dimension of chitosan matrices during degradation was determined by the process of swelling and degradation. These findings may help to design chitosan-based biomedical materials with predetermined degradation timed from several days to months and proper swelling behaviors.     

快速膨胀片层多孔壳聚糖止血海绵的制备及生物相容性研究

目的:探索快速膨胀片层多孔壳聚糖止血海绵的制备工艺,评价止血海绵的理化性能及生物相容性,并探讨原料脱乙酰度对止血海绵性能的影响。方法:考察止血海绵的理化性质,包括扫描电子显微镜(SEM)观察表观形貌,检测力学性能、吸水率、快速吸水膨胀时间和膨胀率,研究其体内外的生物相容性,包括体外细胞毒性实验、动物皮内刺激实验和皮下植入实验。结果:确定了止血海绵的制备工艺,采用该工艺制备的止血海绵均具有片层多孔结构,且具有较高的力学强度和快速膨胀的特点。证实高脱乙酰度原料(DD=95.14%)制备的止血海绵力学性能、吸水率、膨胀率均优于低脱乙酰度原料(DD=69.70%)制备的止血海绵。脱乙酰度69.70%和脱乙酰度95.14%的壳聚糖止血海绵,拉伸强度分别为10.1 N和15.4 N,吸水率分别为1904%和2131%,吸水膨胀时间分别为13.4 s和14.0 s,膨胀率分别为8.4倍和10.8倍。体外细胞毒性实验表明脱乙酰度为95.14%的壳聚糖止血海绵更有利于细胞的增殖,皮内刺激和皮下植入实验结果表明脱乙酰度为95.14%的壳聚糖海止血海绵表现出更小的组织炎性反应。结论:脱乙酰度为95.14%的壳聚糖止血海绵具有优良的力学性能、优异的吸水膨胀能力以及良好的生物相容性,在临床止血特别是腔隙止血方面具有广阔的应用前景。     

Controlling chitosan’s molecular weight via multistage deacetylation

Chitosan samples with different molecular weights (Mw) and degree of deacetylation (DD) were prepared by controlling operating conditions throughout the multistage alkali treatment. The temperature of the reaction, time duration and number of reaction steps were considered effective parameters. A database was developed for chitosan preparation in order to achieve high degrees of deacetylation and control the molecular weight of chitosan without changing other molecular structures. The number of treatments and the duration of each step of deacetylation significantly affected molecular weight so that two samples were obtained with a DD of 99% and two different molecular weights ranging from 4.66×10⁵ to 2.93×10⁵ Based on these results, the highest molecular weight obtained using the multistage treatment without decreasing DD was 5.32×10⁵, with a DD of 96.67%. Also, the morphological studies indicate that the molecular weight of chitosan has a significant effect on the pore size of the prepared scaffolds. However, this effect is critical. In other words, the pore size will increase by increasing molecular weight of chitosan from low upto medium molecular weight and when it reached to high molecular weight the pore size is decreased.     

Successful removal of p-quinone with chitosan in an aqueous phase in relation to degree of deacetylation

Phenol oxidant is successfully removed by using chitosan particles in the aqueous phase. Removal of p-quinone by chitosan from crab shells was investigated kinetically from molecular weight (MW) of chitosan, deacetylation degree (DD) and reaction temperature. The rate constant assuming first-ordered reaction on removal of p-quinone in aqueous phase primarily depended on the MW of chitosan, not on the DD. Quantities of chitosan exceeding 5 x 10(5) MW are able to obtain a sufficiently high rate constant (10(-3) s(-1)). At higher temperatures, higher rate constants were obtained in the entire experimental MW and DD. The activation energy obtained was 43.8 kJ x mol(-1).     

Determination of the Mark-Houwink equation for chitosans with different degrees of deacetylation.

The values of k and alpha in the Mark-Houwink equation have been determined for chitosans with different degrees of deacetylation (DD) (69, 84, 91 and 100% respectively), in 0.2 M CH3COOH/0.1 M CH3COONa aqueous solution at 30 degrees C by the light scattering method. It was shown that the values of alpha decreased from 1.12 to 0.81 and the values of k increased from 0.104 x 10(-3) to 16.80 x 10(-3) ml/g, when the DD varied from 69 to 100%. This is due to a reduction of rigidity of the molecular chain and an increase of the electrostatic repulsion force of the ionic groups along the polyelectrolyte chain in chitosan solution, when the DD of chitosan increases gradually.     

Cytotoxic activities of water-soluble chitosan derivatives with different degree of deacetylation

Chitosans with different degree of deacetylation (DD) (90% and 50% deacetylated chitosan) were prepared by N-deacetylation followed by grafted onto chitosan to form water-soluble aminoethyl-chitosan (AE-chitosan), and dimetylaminoethyl-chitosan (DMAE-chitosan), diethylaminoethyl-chitosan (DEAE-chitosan). In the present study, cytotoxic activities of the chitosan derivatives were evaluated using three tumor cell lines and two normal cell lines, and structure-activity relationship was suggested. The cytotoxic activity was dependent on their DD and substituted group.     

胍乙酸壳聚糖的合成及其对黄瓜的保鲜研究

以自制的不同脱乙酰度的壳聚糖和1-氯胍乙酸为原料合成了胍乙酸壳聚糖,研究了胍乙酸壳聚糖对黄瓜的保鲜效果。结果表明,由脱乙酰度为96%的壳聚糖制得的胍乙酸壳聚糖平均分子量为5287。随着脱乙酰度的增加,黄瓜失重率的增加逐渐减缓,随着贮存时间延长总叶绿素含量先升高然后缓慢下降,而维生素C含量则一直缓慢下降;脱乙酰度为96%的壳聚糖制得的胍乙酸壳聚糖贮存35 d后,黄瓜的质量损失为0.7%;贮存20 d后,总叶绿素含量仍然可达1.34 mg/g;贮存时间20 d后,维生素C含量可达0.18 mg/g。     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.     

Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction

A new method to determine the degree of deacetylation (DD) of alpha-chitin and chitosan in the range of 17-94% DD using X-ray powder diffraction (XRD) is proposed. The results were calibrated using (1)H NMR spectroscopy for chitosan and FTIR for chitin, in comparison with the potentiometric titration method. The results showed a good linear correlation between the CrI020 from XRD and the calibrated DD value. This method is found to be simple, rapid and nondestructive to the sample.     

Effects of concentration, degree of deacetylation and molecular weight on emulsifying properties of chitosan

Chitosan has excellent emulsifying properties. Emulsifying activity and stability of chitosan were determined by integrated light scattering technique and turbidimetric method. The effects of concentration, degree of deacetylation and molecular weight on emulsifying properties of chitosan were systematically studied in the paper. Emulsifying activity of chitosan initially increased, arrived at the peak at 0.75% and then declined, while emulsifying stability continuously increased with a rise of chitosan concentration from 0.25% to 1.25%. Emulsifying activity and stability of chitosan initially decreased and reached the minimum, then increased with the rise of degree of deacetylation. Chitosan with DD 60.5% and 86.1% showed superior emulsifying activity and stability. Chitosan with low Mw exhibited better emulsifying activity than those with high Mw. Chitosan with Mw 410 kDa and 600 kDa showed superior emulsifying activity in the test range. Emulsifying stability of chitosan increased with a rise of Mw.     

Physical properties of fungal chitosan

Fungi are promising alternative sources of chitosan. This study evaluated the physical properties of fungal chitosan from Absidia coerulea (AF 93105), Mucor rouxii (Ag 92033), and Rhizopus oryzae (Ag 92033). FT-IR and X-ray diffraction of the extracted products showed typical chitosan peak distributions which confirmed the extracted products to be chitosan. All of their glucosamine contents and degrees of deacetylation (DD) were over 80%, not showing obvious differences respectively. However, differences had been observed in their molecular weight (M_w), ranging from 6.6  to 560 kDa. The results of this study demonstrated that different fungi could produce different M_w chitosan with high DD and high purity.     

A new linear potentiometric titration method for the determination of deacetylation degree of chitosan

The degree of deacetylation (DD) is one of the most important properties of chitosan. Therefore, a simple, rapid and reliable method for the determination of DD of chitosan is essential. In this report, two new potentiometric titration functions are derived for the determination of DD of chitosan. The effects of the precipitation and the errors induced in pH measurement are discussed in detail. To make this method more simple and reliable, two universal pH regions for the accurate plotting of different DD chitosan samples are proposed for the new potentiometric titration functions. The DD values of three chitosan samples obtained with this new method show good agreement with those yielded from elemental analysis and ¹H-NMR.     

Chitosan as an enabling excipient for drug delivery systems. I. Molecular modifications

Chitosan was physicochemically modified for its potential use as a matrix for an implantable antibiotic delivery system that could sustain bactericidal concentrations in the vicinity of an implant or prosthesis. Deacetylation and depolymerization of chitosan were implemented in order to increase the number or accessibility of the reactive amino groups on the polymer backbone for better polymer-drug interaction. The deacetylation process involved reaction of particulate chitosan/depolymerized chitosan with alkali. The rate of deacetylation of chitosan was directly proportional to the reaction temperature up to 80 degrees C; beyond 80 degrees C, rapid degradation of the polymer occurred. The depolymerization of chitosan involved acid digestion of the polymer followed by application of mechanical agitation. This depolymerized product, although water insoluble, possessed a molecular weight that was one to two orders of magnitude lower than that of commercially available chitosans. These products not only exhibited improved reactivity, but also showed increased crystallinity when compared with the parent chitosan. The reactivity was found to be inversely proportional to chitosan's molecular weight. The depolymerization and deacetylation treatments afforded formation of chitosan having a greater number of amino groups available for interactions with the anionic actives.     

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.     

Chitosan as a Biomaterial: Influence of Degree of Deacetylation on Its Physiochemical,Material and Biological Properties

Chitosan is a biomaterial with a range of current and potential biomedical applications. Manipulation of chitosan degree of deacetylation (DDA) to achieve specific properties appears feasible, but studies investigating its influence on properties are often contradictory. With a view to the potential of chitosan in the regeneration of nerve tissue, the influence of DDA on the growth and health of olfactory ensheathing cells (OECs) was investigated. There was a linear increase in OEC proliferation as the DDA increased from 72 to 85%. This correlated with linear increases in average surface roughness (0.62 to 0.78 μm) and crystallinity (4.3 to 10.1%) of the chitosan films. Mitochondrial activity and membrane integrity of OECs was significantly different for OECs cultivated on chitosan with DDAs below 75%, while those on films with DDAs up to 85% were similar to cells in asynchronous growth. Apoptotic indices and cell cycle analysis also suggested that chitosan films with DDAs below 75% were cytocompatible but induced cellular stress, while OECs grown on films fabricated from chitosan with DDAs above 75% showed no significant differences compared to those in asynchronous growth. Tensile strength and elongation to break varied with DDA from 32.3 to 45.3 MPa and 3.6 to 7.1% respectively. DDA had no significant influence on abiotic and biotic degradation profiles of the chitosan films which showed approximately 8 and 20% weight loss respectively. Finally, perceived patterns in property changes are subject to change based on potential variations in DDA analysis. NMR examination of the chitosan samples here revealed significant differences depending upon which peaks were selected for integration; 6 to 13% in DDA values within individual samples. Furthermore, differences between DDA values determined here and those reported by the commercial suppliers were significant and this may also be a source of concern when selecting commercial chitosans for biomaterial research.     

Preparation of low-molecular-weight chitosan using phosphoric acid

Two types of low degree of polymerisation (DP) chitosan were prepared by homogeneous hydrolysis of chitosan in 85% phosphoric acid at room temperature for 1–6 weeks. The hydrolysates were collected by addition of excess ethanol, and were fractionated by solubility in water. The changes in yields of water-insoluble (higher DP) and water-soluble (lower DP) fractions were determined as a function of hydrolysis time. The hydrolysis proceeded with further deacetylation of chitosan, resulting in degree of deacetylation of more than 90%. The water-insoluble fraction prepared after the hydrolysis for 4 weeks (43% yield) had a weight-average DP ( ) of 16·8, and showed the ‘tendon’ type X-ray diffraction pattern. The water-soluble fraction (12·5% yield) had a of 7·3, and showed the ‘annealed’ type pattern.     

Heterogeneous molecular aggregation and fractal structure in chitosan/acetic acid systems

The influence of deacetylation degree on heterogeneous molecular aggregation has been investigated for chitosan solution in 2 wt % acetic acid aqueous solution using rheological and small-angle x-ray scattering (SAXS) methods. Three samples of chitosan, which were designated CS62, CS79, and CS96, were used, and the deacetylation degrees of these samples were 0.62, 0.79 and 0.96, respectively. Rheological properties show that the systems of CS62 and CS96 are homogeneous, and the system of CS79 has a certain heterogeneous structure with a long-time relaxation mechanism. According to the SAXS measurement, the heterogeneous system has a fractal structure and the fractal dimension is about 1.3.     

Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms

Low-molecular-weight chitosans with a viscosity-average molecular weight (Mv) of 5 to 27 kDa and equal degree of deacetylation (DD, 85%) were highly active against Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, and Bifidobacterium bifidum 791, causing death of 80 to 100% of cells. An exception to this tendency was Escherichia coli, for which the rate of cell death, induced by the 5-kDa chitosan, was 38%. The antibacterial effect was manifested as early as 10 min after incubation of 12-kDa chitosan with B. subtilis or E. coli cells. Candida krusei was almost insensitive to the above crab chitosans. However, Candida krusei was highly sensitive to chitosans with Mv 5, 6, 12, 15.7, and 27 kDa: the minimum inhibitory concentration (MIC) varied from 0.06 to 0.005%. Chitosans with M, 5, 12, and 15.7 kDa exerted an antibacterial effect on Staphylococcus aureus. Chitosans with Mv 5, 15.7, and 27 kDa had no effect on Bifidobacterium bifidum ATCC 14893. The antibacterial effect of the 4-kDa chitosan on E. coli and B. bifidum 791 increased with DD in the range 55-85%.