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.     

Effects of molecular weight and deacetylation degree of chitin/chitosan on wound healing

We studied the effects of chitin/chitosan on wound healing with reference to chemical properties using a linear incisional wound model in rats. Wound break strength of the chitosan group (D-glucosamine (GlcN), chito-oligosaccharide (COS), chitosan) was higher than the chitin group (N-acetyl-D-glucosamine (GlcNAc), chiti-oligosaccharide (NACOS), chitin). Collagenase activity was also higher in the chitosan group than the chitin group. There was no significant change between the concentration of the sample and the break strength and collagenase activity in all samples. In histological findings, collagen fibers run perpendicular against the incisional line in the oligosaccharide group (NACOS, COS), and many activated fibroblasts were observed around the wound in the chitosan group. As for the deacetylation degree, the higher the deacetylation degree becomes, the more the stronger the break strength becomes. Also, activated fibroblasts appeared more in the higher deacetylation degree.     

Effect of deacetylation time on the preparation, properties and swelling behavior of chitosan films

The objectives of the study were to propose a two-stage microfluidization combined with an ultrafiltration (UF) treatment for chitosan mass production and the manipulation of molecular weight and its distribution. The proposed methods are based on the degradation rate and rate constant of various process variables studied. Results obtained were that the rate constants were faster during the earlier reaction period, were higher for those operating at a higher pressure, were better for using concurrent UF treatment to remove small degraded fragments, and the degradation rate constants were faster for 30 °C solutions than that for 50 or 0 °C. A two-stage microfluidization process is proposed. The first stage constitutes of the highest possible concentration solution with concurrent UF treatment at 50 °C, and recycled 5 times. The second stage consists of the highest possible concentration of solutions with concurrent UF treatment at 30 °C, and recycled 5 times.     

Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree

Antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a Bactometer. The purpose of this investigation was to produce a practical, high-performance membrane for separation engineering. The antibacterial activity of powdered chitosan membrane was evaluated by the minimal inhibitory concentration (MIC). The MIC for Escherichia coli was almost 200 (mg-chitosan/ml-bacterial suspension), and for Staphylococcus aureus it was 40 (mg-chitosan/ml-bacterial suspension). Growth of the gram-positive sample (S. aureus) was more strongly inhibited by chitosan than the gram-negative sample (E. coli). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and depended on the shape and the specific surface area of the powdered chitosan membrane. The influence of the deacetylation degree (DD) of the chitosan on inhibiting the growth of S. aureus was investigated by two methods: incubation using a mannitol salt agar medium, and a conductimetric assay. By both methods, chitosan with a higher DD successfully inhibited growth of S. aureus. Our findings regarding the dominant role of the DD of chitosan will be useful for designing long-life, hygienic, membrane-based processes.     

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.     

Prolyl endopeptidase inhibitory activity of chitosan sulfates with different degree of deacetylation

Three kinds of partially deacetylated chitosan, 90% deacetylated chitosan, 75% deacetylated chitosan and 50% deacetylated chitosan, were prepared from crab chitin by N-deacetylation with 40% (w/w) sodium hydroxide solution for different durations. In order to improve biological activity and solubility, their sulfated derivatives were prepared, and prolyl endopeptidase (PEP) inhibitory activities were investigated. Fifty percent-deacetylated chitosan sulfate (50-CS) exhibited the highest inhibitory activity, and inhibition rate was a dose-dependant. In addition, Dixon plots suggested that 50-CS was act as competitive inhibitor, and the inhibition constant (K_i) was 2.6 mg/ml.     

Relationships between the molecular structure and moisture-absorption and moisture-retention abilities of carboxymethyl chitosan. II. Effect of degree of deacetylation and carboxymethylation

Carboxymethyl chitosans (CM-chitosan) of various degrees of deacetylation (DD 28-95%) and substitution (DS 0.15-1.21) were successfully prepared from N-acetylchitosans in NaOH of varying concentrations. Infrared spectroscopy (IR), elemental analysis, potentiometric titration, 13C NMR, X-ray diffraction and gel-permeation chromatographic (GPC) techniques were used to characterize their molecular structures. The moisture-absorption (R(a)) and -retention (R(h)) abilities of CM-chitosan are closely related to the DD and DS values. Under conditions of high relative humidity, the maximum R(a) and R(h) were obtained at DD values of about 50%, and when the DD value deviated from 50%, R(a) and R(h) decreased. Under dry conditions, when the DD value was 50%, the R(h) was the lowest. With the DS value increasing, R(a) and R(h) increased. However, further increase of the DS value above 1.0 reduced the increasing tendency of R(a) and R(h), and even some decreases in R(a) and R(h) were observed. Intermolecular hydrogen bonds play a very important role in moisture-absorption and retention ability of CM-chitosan.     

Effects of degree of deacetylation on enzyme immobilization in hydrophobically modified chitosan

Chitosan is a deacetylated form of the polysaccharide chitin. Over the last decade, researchers have employed reductive amination to hydrophobically modify chitosan to induce a micellar structure. These micellar polymers have been used for a variety of purposes including drug delivery and enzyme immobilization and stabilization. However, commercial sources of chitosan vary in their degree of deacetylation and there remains a paucity of information regarding how this can impact the modified polymer’s functionality for enzyme immobilization. This paper, therefore, evaluates the effect that the degree of deacetylation has on the hydrophobic modification of medium molecular weight chitosan via reductive amination with long chain aldehydes and the resulting changes in enzyme activity after the immobilization of glucose oxidase in the micellar polymeric structure. The chitosan was deacetylated to differing degrees via autoclaving in 40–45% NaOH solutions and characterized using NMR, viscosity measurements, and differential scan calorimetry. Results suggest that a high degree of deacetylation provides optimal enzyme immobilization properties (i.e. high activity), but that the deacetylation method begins to significantly decrease the polymer molecular weight after a 20 min autoclave treatment, which negatively affects immobilized enzyme activity.     

Enzymatic method for determination of the degree of deacetylation of chitosan.

A new method was developed for the accurate determination of the degree of deacetylation of chitosan. The method involves the complete hydrolysis of chitosan to glucosamine and N-acetylglucosamine by a cooperative action of chitosanolytic enzymes exo-beta-D-glucosaminidase, beta-N-acetylhexosaminidase, and chitosanase, and subsequent determination of the monosaccharides by specific colorimetric assays or HPLC. The conditions required for the complete hydrolysis of chitosan were examined and the degree of deacetylation of several chitosan samples was determined.     

Reactive oxygen species scavenging activity of aminoderivatized chitosan with different degree of deacetylation

Chitosans with different degree of deacetylation were prepared from crab shell chitin in the presence of alkali. Aminoderivatized chitosan derivatives were prepared in addition of amino functional groups at a hydroxyl site in the chitosan backbone. Six kinds of aminoderivatized chitosan such as aminoethyl-chitosan (AEC90), dimethylaminoethyl-chitosan (DMAEC90), and diethylaminoethyl-chitosan (DEAEC90), which were prepared from 90% deacetylated chitosan, and AEC50, DMAEC50 and DEAEC50, which were prepared from 50% deacetylated chitosan, were prepared and their reactive oxygen species (ROS) scavenging activities were investigated against hydroxyl radical, superoxide anion radical and hydrogen peroxide. The electron spin resonance (ESR) spectrum revealed that AEC90 showed the highest scavenging effects against hydroxyl and superoxide anion radical, the effects were 91.67% and 65.34% at 0.25 and 5 mg/mL, respectively. For hydrogen peroxide scavenging effect, DEAEC90 exhibited the strongest activity. These results suggest that the scavenging effect depends on their degree of deacetylation and substituted group.     

An investigation on the short-term biodegradability of chitosan with various molecular weights and degrees of deacetylation

Research efforts have been devoted to demonstrating that the pH-sensitive characteristics of poly NIPAAm/chitosan nanoparticles can be applied to targeting tumors. A copolymer of (NIPAAm) and chitosan (4:1, m/m) was synthesized, and its drug release characteristics investigated. The results revealed that drug-loaded nanoparticles which encapsulation and loading efficiencies were 85.7% and 9.6%, respectively, exhibited pH-sensitive responses to tumor pH. The cumulative release rate was significantly enhanced below pH 6.8 and decreased rapidly above pH 6.9 at 36.5 ± 0.5 °C. MTT assay and fluorescence microscopic study showed that drug release was drastically promoted in tumor surroundings while exerting less effect in normal conditions. For mice treated with nanoparticles, the decrease in body weight was limited, and significant tumor regression was observed with complete regression in more than 50% of the mice. The life span of tumor-bearing mice was significantly increased when they were treated with nanoparticles. Thereby, the super pH-sensitive poly NIPAAm/chitosan nanoparticles may provide outstanding advantages for anti-cancer drug delivery.     

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.     

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with M_w of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm³ of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: M_w 141,000 > M_w 199,000 > M_w 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest M_w chitosan (141,000) promoted the growth of human skin cells more than those of the others.     

Novel starch/chitosan blending membrane: Antibacterial,permeable and mechanical properties

Antibacterial membranes were prepared from a mixture of hydrolyzed starch and chitosan. Glycerin was incorporated in the membranes to as plasticizer agent. The effects of component ratio on the mechanical and permeable properties of the prepared membranes were investigated. The elongation-at-break and water vapor transmission rate of starch/chitosan blending membranes were largely improved compared with each single component due to the interaction formed between the hydroxyl groups of starch and the amino ones of chitosan, which was confirmed by FT-IR characterizations. With the help of optical microscope, the influence of component ratio on the morphologies of starch/chitosan membranes was systematically investigated. It comes to a conclusion that extreme low or high starch content will cause an asymmetric membrane surface. To prove the antibacterial activity of obtained membranes, Escherichia coli (E. coli) was chosen as the target bacteria via optical density method. The resulted starch/chitosan membranes exhibited an outstanding antibacterial activity against E. coli.     

Limitations of pH-potentiometric titration for the determination of the degree of deacetylation of chitosan

The degree of deacetylation (DDA) of chitosan determines the biopolymer's physico-chemical properties and technological applications. pH-Potentiometric titration seems to offer a simple and convenient means of determining DDA. However, to obtain accurate pH-potentiometric DDA values, several factors have to be taken into consideration. We found that the moisture content of the air-dry chitosan samples can be as high as 15%, and a reasonable fraction of this humidity cannot be removed by ordinary drying. Corrections have to be made for the ash content, as in some samples it can be as high as 1% by weight. The method of equivalence point determination was also found to cause systematic variations in the results and in some samples extra acid as high as 1 mol% of the free amino content was also identified. To compensate for the latter effect, the second equivalence point of the titration has to be determined separately and the analytical concentration of the acid be corrected for it. All the corrections listed here are necessary to obtain DDA values that are in reasonable agreement with those obtained from (1)H NMR and IR spectroscopic measurements. The need for these corrections severely limits the usefulness of pH-metry for determining accurate DDA values and thus potentiometry is hardly able to compete with other standard spectroscopic procedures, that is, (1)H NMR spectroscopy.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phaseolus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 μg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule—glucosamine and N-acetylglucosamine—exhibited no antiviral activity.     

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.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phasoleus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 microg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule-glucosamine and N-acetylglucosamine--exhibited no antiviral activity.     

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.