Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4–14% and a yield of 60–80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coli. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02–0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Galactosylated derivatives of low-molecular-weight chitosan: production and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-beta-D-glucopyranose and 2-amino-2-deoxy-beta-D-glucopyranose residues linked in different proportions via beta-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

Galactosylated derivatives of low-molecular-weight chitosan: Obtaining and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues linked in different proportions via β-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.     

Effect of chitosan derivatives on the reproduction of Coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosan N-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 in Escherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited bactericidal activity. The efficiency of inhibition of viral infection and the bactericidal activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or bactericidal activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability of E. coli may also be due to the bactericidal effect of chitosan.     

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.     

Nonnatural branched polysaccharides: synthesis and properties of chitin and chitosan having disaccharide maltose branches

Synthesis and properties of chitin and chitosan derivatives having beta-maltoside branches at C-6 have been studied. Chitosan was first transformed into an organosoluble acceptor having a reactive group only at C-6, 3-O-acetyl-2-N-phthaloyl-6-O-trimethylsilylchitosan. Glycosylation with an ortho ester from d-maltose was performed successfully at room temperature in dichloromethane in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst. The degree of substitution could be controlled by the reaction conditions and was up to 0.56. Full deprotection gave chitosan with maltoside branches, and the subsequent N-acetylation resulted in the formation of the corresponding chitin derivative. The introduced disaccharide unit improved hydrophilic properties considerably compared to monosaccharide units as confirmed by high solubility in water and moisture absorption and retention ability. The enzymatic degradability and antimicrobial activity were moderate probably because of the bulky nature of the branches.     

Study of the Protective Activity of Chitosan Hydrolyzate Against Septoria Leaf Blotch of Wheat and Brown Spot of Tobacco

Chitosan hydrolyzate containing low-molecular-weight chitosan (≤24 kDa) and its oligomers (≤1.2 kDa) has been obtained via chemical depolimerization of high-molecular-weight chitosan by nitric acid. The fractions of the obtained chitosan hydrolyzate have been characterized by high performance gel permeation chromatography and proton magnetic resonance. The test performed on detached leaves of wheat has shown that the hydrolyzate completely inhibits the development of Stagonospora nodorum, a casual agent of the septoria leaf blotch, at a concentration of 200 μg/mL. A similar test with detached tobacco leaves has shown that the hydrolyzate at a concentration of 100 μg/mL also inhibits the development of Alternaria longipes, which causes brown spot of tobacco, by 75%.     

Chitosan�CEDTA New Combination is a Promising Candidate for Treatment of Bacterial and Fungal Infections

Chitosan is an attractive preparation widely used as a pharmaceutical excipient. This study aimed to evaluate the antimicrobial activities of chitosan derivatives, EDTA, and the newly developed chitosan-EDTA combination against Gram-negative and Gram-positive bacteria as well as Candida albicans. Antimicrobial activity was studied. Both minimal Inhibitory Concentrations (MIC) and minimal biocidal concentrations (MBC) were determined. Chitosan acetic acid recorded lower MIC values against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans than those exhibited by EDTA. EDTA failed to have inhibitory activity against Enterococcus faecalis as well as MBC against any of the studied microorganisms. Chitosan acetic acid's MBC were recorded to all examined species. Checkerboard assay results indicated a synergistic antimicrobial activity of the new combination against Staphylococcus aureus and an additive effect against other microorganisms. Moreover, a short microbial exposure to chitosan-EDTA (20-30 min) caused complete eradication. Due to the continuous emergence of resistant strains, there is an urgent need to discover new antimicrobial agents. Our findings suggest the use of chitosan as an enhancing agent with antibacterial and antifungal properties in combination with EDTA in pharmaceutical preparations.     

Regioselective Sequential Modification of Chitosan via Azide-Alkyne Click Reaction: Synthesis,Characterization, and Antimicrobial Activity of Chitosan Derivatives and Nanoparticles

Recently, the attention of researchers has been drawn toward the synthesis of chitosan derivatives and their nanoparticles with enhanced antimicrobial activities. In this study, chitosan derivatives with different azides and alkyne groups were synthesized using click chemistry, and these were further transformed into nanoparticles by using the ionotropic gelation method. A series of chitosan derivatives was successfully synthesized by regioselective modification of chitosan via an azide-alkyne click reaction. The amino moieties of chitosan were protected during derivatization by pthaloylation and subsequently unblocked at the end to restore their functionality. Nanoparticles of synthesized derivatives were fabricated by ionic gelation to form complexes of polyanionic penta-sodium tripolyphosphate (TPP) and cationic chitosan derivatives. Particle size analysis showed that nanoparticle size ranged from 181.03 ± 12.73 nm to 236.50 ± 14.32 nm and had narrow polydispersity index and positive surface charge. The derivatives and corresponding nanoparticles were evaluated in vitro for antibacterial and antifungal activities against three gram-positive and gram-negative bacteria and three fungal strains, respectively. The minimum inhibitory concentration (MIC) of all derivatives ranged from 31.3 to 250 µg/mL for bacteria and 188 to1500 µg/mL for fungi and was lower than that of native chitosan. The nanoparticles with MIC ranging from 1.56 to 25 µg/mLfor bacteria and 94 to 750 µg/mL for fungi exhibited higher activity than the chitosan derivatives. Chitosan O-(1-methylbenzene) triazolyl carbamate and chitosan O-(1-methyl phenyl sulfide) triazolyl carbamate were the most active against the tested bacterial and fungal strains. The hemolytic assay on erythrocytes and cell viability test on two different cell lines (Chinese hamster lung fibroblast cells V79 and Human hepatic cell line WRL68) demonstrated the safety; suggesting that these derivatives could be used in future medical applications. Chitosan derivatives with triazole functionality, synthesized by Huisgen 1,3-dipolar cycloaddition, and their nanoparticles showed significant enhancement in antibacterial and antifungal activities in comparison to those associated with native, non-altered chitosan.     

Enhanced microbial safety of channel catfish (Ictalurus punctatus) fillet using recently invented medium molecular weight water-soluble chitosan coating

Chitosan with higher molecular weight exhibited higher antimicrobial efficacy against foodborne pathogens. However, the poor water solubility of higher or medium molecular weight chitosan limits its applications. To overcome the challenge, our research team searched for simple preparation procedure for fast-dissolving medium molecular weight chitosan in water. Throughout the process, we were able to obtain a higher concentration of medium molecular weight water-soluble (MMWWS) chitosan (400 kDa). The MMWWS chitosan showed physicochemical properties that are suitable for edible coating. Antibacterial activities of 400-kDa chitosan coating prepared in acetic acid (1% v/v) or aspartic acid (1% or 3% w/v) were examined. The surface of catfish cubes was inoculated with six foodborne pathogens and then coated with chitosan solutions. The survival of each pathogen was evaluated during shelf life storage. Compared with the control, 3% w/v chitosan coating in aspartic acid solution exhibited the most effective antibacterial activities among other coating treatments, completely inhibiting Vibrio parahaemolyticus on the surface of catfish. The study suggested that chitosan dissolved in aspartic acid has the potential for use as an alternative antimicrobial coating for catfish fillet.     

Production of polysaccharides by callus cultures of common duckweed

Callus lines of common duckweed produced acid arabinogalactan and pectin in an amount varying from 1 to 3% of dry weight. The arabinogalactan specimens from the cell lines studied displayed a similar monosaccharide composition. The duckweed callus lines whose arabinogalactans contained apiose residues (1-2%) were found. All pectin specimens had a similar qualitative monosaccharide composition but differed in the quantitative content of monosaccharide residues. The lines with high contents of galactose, arabinose, and apiose in pectin specimens were obtained. The total content of neutral monosaccharide residues in pectins varied from 26 to 50%.     

Effect of chitosan derivatives on the reproduction of coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosanN-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 inEscherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited antibacterial activity. The efficiency of inhibition of viral infection and the antibacterial activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or antibacterial activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability ofE. coli may also be due to the antibacterial effect of chitosan.     

Formation and characterization of three dimensional human hepatocyte cell line spheroids on chitosan matrix for in vitro tissue engineering applications

Chitosan was used as a matrix to induce three-dimensional spheroids of HepG2 cells. Chitosan films were prepared and used for culturing Hep G2 cells. Attachment kinetics of the cells was studied on the chitosan films. The optimum seeding density of the Hep G2 cells, required for three-dimensional spheroid formation was determined and was found to be 5 × 10⁴/ml. The growth kinetics of Hep G2 cells was studied using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay, and morphology of the cells was studied through optical photographs taken at various days of culture. The liver cell functions of the spheroids were determined by measuring albumin and urea secretions. The results obtained from these studies have shown that the culture of Hep G2 cells on chitosan matrix taking appropriate seeding density resulted in the formation of three-dimensional spheroids and exhibited higher amount of albumin and urea synthesis compared to monolayer culture. These miniature “liver tissue like” models can be used for in vitro tissue engineering applications like preliminary evaluation of the toxicity of drugs and chemicals.     

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.     

Antifungal efficacy of chitosan and its thiourea derivatives upon the growth of some sugar-beet pathogens

Chitosan (CS) was modified by reaction with benzoyl thiocyanate to give a thiourea derivative (TUCS). The antifungal behavior of chitosan and its thiourea derivative was investigated in vitro on the mycelial growth, sporulation and germination of conidia or sclerotia of the following sugar-beet: Beta vulgaris pathogens isolated in Egypt, Rhizoctonia solani Kühn (AG(2-2)) Sclerotium rolfsii Sacc. and Fusarium solani (Mart.) Sacc. All the prepared thiourea derivatives had a significant inhibiting effect on the different stages of development on the germination of conidia or sclerotia of all the investigated fungi in the polymer concentration range of 5-1000 microg ml(-1). In the absence of chitosan and its derivative, R. solani exhibited the fastest growth of the fungi studied. However, growth tolerance of the modified chitosan was highest for F. solani and lowest for R. solani. The most sensitive to the modified chitosan stress with regard to their germination and number produced were the sclerotia of S. rolfsii. It has been found that the TUCS is a much better fungicidal agent (about 60 times more) than the pure CS against most of the fungal strains tested. The molecular weight and the degree of deacetylation were found to have an important effect on the growth activities of the pathogens.     

The use of single walled carbon nanotubes dispersed in a chitosan matrix for preparation of a galactose biosensor

Chitosan was chosen as a natural polymer for dispersion of single walled carbon nanotubes (SWNT) based on its ability to efficiently solubilize SWNTs to form a stable dispersion. Moreover, chitosan films deposited on a surface of a glassy carbon (GC) electrode are mechanically stable. Further stabilisation of the chitosan film containing SWNT (CHIT-SWNT) was done by chemical crosslinking with glutaraldehyde and free aldehyde groups produced a substrate used for covalent immobilisation of galactose oxidase (GalOD). Different galactose biosensor configurations were tested with optimisation of composition of inner and outer membrane; and enzyme immobilisation procedure, as well. Detection of oxygen uptake by GalOD on CHIT-SWNT layer at -400 mV is robust and, when flow injection analysis (FIA) was applied for assays, a low detection limit (25 microM) and very high assay throughput rate (150 h-1) was achieved. This new galactose biosensor offers highly reliable detection of galactose with R.S.D. well below 2% and it has been successfully applied to assaying galactose in a blood sample with recovery index between 101.2 and 102.7%.     

Effect of chitosan derivatives on the development of phage infection in cultured Bacillus thuringiensis

The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infection of Bacillus thuringiensis by phage 1-97A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 mg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate and N-succinate-6-O-sulfate did not inactivate phage, did not influence bacterial growth, and did not inhibit the process of viral infection.     

One-pot synthesis in aqueous system for water-soluble chitosan-graft-poly(ethylene glycol) methyl ether

Chitosan is functionalized with poly(ethylene glycol) methyl ether (mPEG) at the amino and hydroxyl groups via a single step reaction in a homogeneous aqueous system. A chitosan aqueous solution obtained from the mixture of chitosan and hydroxybenzotriazole (HOBt) in water is a key factor in providing mild conditions to conjugate mPEG by using a carbodiimide conjugating agent. The reaction at ambient temperature for 24 h gives chitosan-g-mPEG with water solubility with mPEG content as high as 42%. This work demonstrates that a water-soluble chitosan-HOBt complex is an effective system for the preparation of chitosan derivatives via the aqueous system without the use of acids or organic solvents.     

Synthesis of organosoluble chitosan derivatives with polyphenolic side chains

A one-pot synthesis was used to produce chitosan derivatives with polyphenolic side chains via a regioselective phenolic coupling reaction. Under Mannich reaction conditions, treatment of chitosan with formaldehyde and methyl 2,4-dihydroxybenzoate gave N-(2,6-dihydroxy-3-methoxycarbonylphenyl)methylated chitosan in good yield (87%). Formation of a CC bond occurred regioselectively at the C(3) position of methyl 2,4-dihydroxybenzoate. Chitosan derivatives having various phenolic compounds as a side chain were easily synthesized in a similar manner. The chitosan derivatives showed good biodegradability and improved their solubility in methanol (9.8mgmL(-1)) and 2-methoxyethanol (> 10mgmL(-1)). The UV protection provided by the derivatives with phenolic benzophenone side chain was evaluated using UV spectra of polyethylene terephthalate and poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) films coated with the derivatives and the derivatives absorbed effectively in the UV-A region (<60%). Self-aggregation of the chitosan derivatives with the phenolic side chain was observed by using a fluorescent probe in aqueous solution.     

Studies on the antibacterial activities and mechanisms of chitosan obtained from cuticles of housefly larvae

Chitosan was obtained from cuticles of the housefly (Musca domestica) larvae. Antibacterial activities of different Mw chitosans were examined against six bacteria. Antibacterial mechanisms of chitosan were investigated by measuring permeability of bacterial cell membranes and observing integrity of bacterial cells. Results show that the antibacterial activity of chitosan decreased with increase in Mw. Chitosan showed higher antibacterial activity at low pH. Ca2+ and Mg2+ could markedly reduce the antibacterial activity of chitosan. The minimum inhibitory concentrations of chitosans ranged from 0.03% - 0.25% and varied with the type of bacteria and Mw of chitosan. Chitosan could cause leakage of cell contents of the bacteria and disrupt the cell wall.