Divergicin M35-Chitosan Film: Development and Characterization

Chitosan films loaded with bacteriocin were examined by FTIR spectroscopy, tested for color, puncture strength, water vapor permeability, and as antimicrobials of Listeria innocua HPB13. Divergicin M35, a bacteriocin produced by Carnobacterium divergens, was incorporated into films made with chitosan of molecular mass 2 kDa, 20 kDa, or 100 kDa and de-acetylated either 87% or 95%. Only 100 kDa chitosan yielded films that could be peeled and handled easily. The higher degree of de-acetylation increased the total color factor (ΔE) of bacteriocin-loaded films, their permeability, and puncture strength. Incorporation of divergicin M35 into the films increased amide I peak intensity but otherwise did not induce significant structural change. The FTIR spectra of divergicin M35 shed from the films did not differ from those of the original free bacteriocin, except in overall peak intensity. The release of active divergicin M35 from the film was faster into the buffer than into tryptic soy broth and peaked at 10–12 h in both cases. Chitosan 95% de-acetylated and loaded with divergicin M35 was the most active, producing a six-log drop in Listeria innocua HPB13 viable count within 24 h. These results suggest that the biocompatible and biodegradable films developed here have the potential for application as antimicrobials of Listeria spp. in foods, especially ready-to-eat, minimally processed products.

     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

Antimicrobial Peptide JH-3 Effectively Kills Salmonella enterica Serovar Typhimurium Strain CVCC541 and Reduces Its Pathogenicity in Mice

Salmonella is an important zoonotic pathogen and is a major cause of gastrointestinal diseases worldwide. The current serious problem of antibiotic abuse has prompted the search for new substitutes for antibiotics. JH-3 is a small antimicrobial peptide with broad-spectrum bactericidal activity. In this study, we showed that JH-3 has good bactericidal activity towards the clinical isolate Salmonella enterica serovar Typhimurium strain CVCC541. The minimum inhibitory concentration (MIC) of JH-3 against this bacterium was determined to be 100 μg/mL, which could decrease the number of CVCC541 cells by 1000-fold in vitro within 5 h. The transmission electron microscopy (TEM) results showed that JH-3 can damage the cell wall and membrane of CVCC541, leading to the leakage of cell contents and subsequent cell death. To measure the bactericidal activity of CVCC541-infected mice were treated intraperitoneally 40 or 10 mg/kg JH-3 at 2 h or 3 days postinfection. Our results showed that treatment with 40 mg/kg JH-3 at 2 h postinfection had the best therapeutic effect and could significantly protect mice from a lethal dose of CVCC541. Furthermore, the clinical symptoms, bacterial burden in blood and organs, and intestinal pathological changes were all decreased and were close to normal. This study examined the therapeutic effect of the antimicrobial peptide JH-3 against S. enterica CVCC541 infection for the first time and determined the therapeutic effect of different JH-3 doses and treatment times, laying the foundation for studies of new antimicrobial agents.

     

The contribution of acidulant to the antibacterial activity of acid soluble α- and β-chitosan solutions and their films

This study evaluated individual contributions of dissolving acids (acetic acid, lactic acid, and hydrochloric acid) or acid solubilized chitosan to the antibacterial activity against Listeria innocua and Escherichia coli as solutions and dried films. Solutions containing chitosan showed significantly (P?<?0.05) different inhibitory activity (measured as percentage of inhibition (PI), in percent) against L. innocua and E. coli, compared to equivalent acid solutions. This increase was calculated as additional inhibition (AI, in percent), which could be as high as 65 % in solutions containing 300–320 kDa chitosan depending on the acid type, bacterial species, and the chitosan form (α or β). Solutions containing 4–5 kDa chitosan had lower AI and showed much greater variability among the different chitosan forms, acid types, and bacterial species. Higher molecular weight (Mw) chitosan also showed significantly higher levels of adsorption to bacterial cells than that of lower Mw samples, suggesting that the observed increase in inhibition was the result of surface phenomena. The contribution of acids to the antibacterial activity of chitosan films was assessed by comparing non-rinsed and rinsed films (rinsed in the appropriate broth to remove residual acids and active fragments formed on the dried film). Rinsing β-chitosan films has reduced PI by as much as 28 % compared with non-rinsed films, indicating that part of the antibacterial activity of chitosan films is due to the presence of soluble acid compounds and/or other active fragments. Overall, both acidulant and chitosan were found to contribute to the antibacterial activity of acid solubilized α- and β-chitosan, with the exact antibacterial activity of chitosan varying based on the solution and film properties, suggesting a complex interaction.     

Root cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> subsp. <Emphasis Type="Italic">angustifolium</Emphasis> elicited with chitosan and production of xanthone-rich extracts with antifungal activity

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l⁻¹ was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g⁻¹ dry weight [DW]) and release (2.64 ± 0.13 mg g⁻¹ DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 μg ml⁻¹ against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.     

Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi

An investigation was undertaken to understand the mechanism(s) by which chitosan exerts its antifungal effects against the wood-degrading fungi Sphaeropsis sapinea and Trichoderma harzianum. Exposure to increasing concentrations of chitosan caused an increase in the amount of hydrogen peroxide accumulation in cultures of S. sapinea, which was accompanied by a decrease in superoxide formation. The same effect was not observed in T. harzianum. Potassium ion leakage was an early event for both test fungi, leakage being more pronounced for S. sapinea than T. harzianum for the first 5 min, particularly at higher concentrations of chitosan treatment. Fluorescence microscopy provided evidence that the effect of chitosan on fungal hyphae was mediated through alterations in the plasma membrane properties. Chitosan also severely affected fungal morphology. Increasing concentrations of chitosan induced excessive branching, vacuolation, and a reduction in hyphal diameter. Transmission electron microscopy, which showed more severe ultrastructural changes in S. sapinea hyphae from chitosan treatment as compared to T. harzianum, provided valuable complementary information. The data suggest that the plasma membrane may be the primary target of chitosan action, and that the two fungi differ in the extent to which they are affected.     

Production of chitin and chitosan from the exoskeleton of adult two‐spotted field crickets (Gryllus bimaculatus)

Chitin and chitosan were extracted from all specimens of Type I and II two‐spotted field crickets (Gryllus bimaculatus) following chemical treatment with an acid and alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve demineralization and deproteinization, respectively. For chitosan extraction, 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis, respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis, respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and 7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %, 90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi Poise), respectively. The molecular weight of chitosan from adult exoskeletons of G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G. bimaculatus could be used as a source of chitin and chitosan for use as functional additives in industrial animal feeds.     

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.     

Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution

The kinetics and mechanisms of depolymerization of aqueous chitosan and alginate solutions at elevated temperatures have been investigated. Chitosan salts of different degree of acetylation (F_A), type of counterions (-glutamate, -chloride) and degree of purity were studied. One commercially available highly purified sodium alginate sample with high content of guluronic acid (G) was also studied. Furthermore, the influence of oxygen, H⁺ and OH⁻ ions on the initial depolymerization rates was investigated. Depolymerization kinetics was followed by measuring the time courses of the apparent viscosity and the intrinsic viscosity. The initial rate constants for depolymerization were determined from the intrinsic viscosity data converted to a quantity proportional to the fraction of bonds broken. The activation energies of the chitosan chloride and chitosan glutamate solutions with pH close to 5 and the same degree of acetylation, F_A = 0.14, were determined from the initial rate constants to be 76 ± 13 kJ/mol and 80 ± 11 kJ/mol, respectively.The results reported herein suggest that the stability of aqueous chitosan and alginate solutions at pH values 5–8 will be influenced by oxidative–reductive depolymerization (ORD) as the primary mechanism as long as transition metal ions are presented in the samples. Acid – and alkaline depolymerization will be the primary mechanisms for highly purified samples.     

Preparation of N-vanillyl chitosan and 4-hydroxybenzyl chitosan and their physico-mechanical, optical, barrier, and antimicrobial properties

Chitosan derivatives such as N-vanillyl chitosan and 4-hydroxybenzyl chitosan were prepared by reacting chitosan with 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxybenzaldehyde. Amino groups on chitosan reacts with these aldehydes to form a Schiff base intermediate, which is later on converted into N-alkyl chitosans by reduction with sodium cyanoborohydride. The chemical reaction was monitored by ¹H NMR spectroscopy and the absence of aldehydic proton at 9.83 ppm in NMR spectra was observed for both the modified chitosan derivatives confirming the reaction. Modified chitosan films were later prepared by solution casting method and their physico-mechanical, barrier, optical and thermal properties were studied. The results clearly indicated significant change in tensile strength, water vapour transmission rate, and haze properties of modified chitosans. Modified chitosan films were also studied for their antimicrobial activity against Aspergillus flavus. The results showed a marked reduction of aflatoxins produced by the fungus in the presence of the N-vanillyl chitosan and 4-hydroxybenzyl chitosan film discs to 98.9% and non-detectable levels, respectively.     

Effect of chitosan for the control of potato diseases caused by Fusarium species

The antifungal activity of chitosan against Fusarium spp. was investigated based on in vitro and in vivo assays, and its possible modes of action were also explored. Chitosan applied at 4.0 g/L of acetic acid-distilled water solution significantly decreased the mycelial growth of Fusarium oxysporum, Fusarium sambucinum and Fusarium graminearum by 88.4%, 89.0% and 89.8%, respectively. Tuber treatment by chitosan (4.0 g/L) of acetic acid-distilled water solution, prior to inoculation, reduced dry rot severity induced by F. oxysporum and F. sambucinum by 60.0% and 48.2%, respectively. When tested as plant treatment, potato plants inoculated with Fusarium species, exhibited 33.5%–45.3% less wilting severity as compared to the control. This abiotic treatment improved the phenolic compounds activities and defence-related enzymes such as peroxidase and polyphenoloxidase in potato tubers inoculated with Fusarium spp. Results clearly demonstrated that chitosan could be explored as an alternative agent to chemical fungicides for the control of tuber dry rot and Fusarium wilt through induction of the plant defence system.     

Development and Evaluation of Buccal Films Based on Chitosan for the Potential Treatment of Oral Candidiasis

In this work, chitosan films were prepared by a casting/solvent evaporation methodology using pectin or hydroxypropylmethyl cellulose to form polymeric matrices. Miconazole nitrate, as a model drug, was loaded into such formulations. These polymeric films were characterized in terms of mechanical properties, adhesiveness, and swelling as well as drug release. Besides, the morphology of raw materials and films was investigated by scanning electron microscopy; interactions between polymers were analyzed by infrared spectroscopy and drug crystallinity studied by differential scanning calorimetry and X-ray diffraction. In addition, antifungal activity against cultures of the five most important fungal opportunistic pathogens belonging to Candida genus was investigated. Chitosan:hydroxypropylmethyl cellulose films were found to be the most appropriate formulations in terms of folding endurance, mechanical properties, and adhesiveness. Also, an improvement in the dissolution rate of miconazole nitrate from the films up to 90% compared to the non-loaded drug was observed. The in vitro antifungal activity showed a significant activity of the model drug when it is loaded into chitosan films. These findings suggest that chitosan-based films are a promising approach to deliver miconazole nitrate for the treatment of candidiasis.     

Synergistic antimicrobial properties of active molecular chitosan with EDTA‐divalent metal ion compounds

The aim of this study was to elucidate the synergistic antimicrobial activity of chitosan hydrolysates derived from hydrolysis of high molecular weight chitosan (HMWC) with various kinds of divalent metal ions‐EDTA compounds against Penicillium italicum. Amongst hydrolysates, active molecular chitosan (AMC) ranging of molecular weight approximately from 0.8 to 3.0 kDa has been newly researched and demonstrated that it has great antimicrobial activity. Copper, manganese, magnesium, cobalt and zinc ions were used to manufacture chelates with EDTA using a microwave apparatus. Inhibitory effect of AMC or chelates on growth of P. italicum was investigated for up to 72 hr. As the results, AMC showed the highest growth inhibitory activity in the presence of Zn‐EDTA compound compared to the other ion chelates. According to these results, we demonstrated that although Zn‐EDTA‐chelating ligand alone does seem to have a little antimicrobial activity against P. italicum, however, it has synergistic effect against the fungal species when it combined with AMC. Taken together, we suggest that AMC‐EDTA‐Zinc combination (AEM‐Zn) can be a great candidate as natural pesticide for defending postharvest decay of citrus fruits by the fungi.     

Preparation of single or double-network chitosan/poly(vinyl alcohol) gel films through selectively cross-linking method

A selectively cross-linking method, which is based on the “di–diol” interaction between poly(vinyl alcohol) and borate and the strong electrostatic interaction between chitosan and tripolyphosphate, was developed. Chitosan/poly(vinyl alcohol) films cross-linked separately with borate, tripolyphosphate and borate/tripolyphosphate were then prepared in terms of this method. Water vapor permeation, mechanical strength, surface morphology and molecular interactions of the films were studied by water permeation test, texture test, atomic force microscopy and ATR-FTIR spectroscopy. With the introduction of cross-linking structure, there is a large improvement in elastic modulus from 271 ± 14.2 to 551 ± 14.7 MPa and a large decrease in water vapor permeability from (5.41 ± 0.21) × 10⁻⁷ g/m h Pa to (3.12 ± 0.24) × 10⁻⁷ g/m h Pa of chitosan/poly(vinyl alcohol) films. The surface morphology of the cross-linked films exhibits a nanoparticle aggregation structure. The size and aggregation behavior of these nanoparticles are strongly related to the type of cross-linker. Furthermore, ATR-FTIR results indicate that strong interaction between polymer matrix and cross-linker exists in our system. This work provides a simple and efficient way to prepare chitosan/poly(vinyl alcohol) films with controllable network structure.     

Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores

Aims: Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill‐kinetics of these solutions is limited. The aim of the study was to identify the kill‐rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte). Methods and Results: Standard suspensions of methicillin‐resistant Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus spores and Clostridium difficile spores were treated with anolyte in a quantitative suspension assay. For vegetative cells, all concentrations of anolyte tested reduced the viable population to below the detection limit within 10 s. At a concentration of 99%, anolyte produced a log₁₀ reduction factor of greater than five in viable B. atrophaeus endospores within 90 s and reduced numbers of C. difficile endospores to below the experimental detection limit within 20 s at concentrations of 5% or greater. Conclusions: Anolyte was highly effective in killing test‐bacteria and spores. The bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against C. difficile spores as low as 5%. Significance and Impact of Study: The results of this study demonstrate that ECAS are effective at lower concentrations and act more rapidly than previously reported. Potent bactericidal and sporicidal activity coupled with point‐of‐use generation, low production‐costs and environmental compatibility suggest that acidic ECAS has the potential to be a useful addition to the current armoury of disinfectants.     

Antioxidant properties of chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and its antioxidant properties studied. Chitosan exhibited showed antioxidant activities of 58.3–70.2% at 1 mg/mL and showed reducing powers of 0.32–0.44 at 10 mg/mL. At 10 mg/mL, the scavenging ability of chitosan C60 on 1,1-diphenyl-2-picrylhydrazyl radicals was 28.4% whereas those of other chitosans were 46.4–52.3%. At 0.1 mg/mL, scavenging abilities on hydroxyl radicals were 62.3–77.6% whereas at 1 mg/mL, chelating abilities on ferrous ions were 82.9–96.5%. All EC₅₀ values of antioxidant activity were below 1.5 mg/mL. With regard to antioxidant properties assayed, the effectiveness of chitosans C60, C90 and C120 correlated with their N-deacetylation times. Overall, crab chitosan was good in antioxidant activity, scavenging ability on hydroxyl radicals and chelating abilities on ferrous ions and may be used as a source of antioxidants, as a possible food supplement or ingredient in the pharmaceutical industry.     

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.     

Chitosan–caffeic acid–genipin films presenting enhanced antioxidant activity and stability in acidic media

The use of chitosan films has been limited due to their high degradability in aqueous acidic media. In order to produce chitosan films with high antioxidant activity and insoluble in acid solutions caffeic acid was grafted to chitosan by a radical mechanism using ammonium cerium (IV) nitrate (60 mM). Genipin was used as cross-linker. This methodology originated films with 80% higher antioxidant activity than the pristine film. Also, these films only lost 11% of their mass upon seven days immersion into an aqueous solution at pH 3.5 under stirring. The films surface wettability (contact angle 105°), mechanical properties (68 MPa of tensile strength and 4% of elongation at break), and thermal stability for temperatures lower than 300 °C were not significantly influenced by the covalent linkage of caffeic acid and genipin to chitosan. Due to their characteristics, mainly higher antioxidant activity and lower solubility, these are promising materials to be used as active films.     

Chitosan enhances leaf membrane stability and antioxidant enzyme activities in apple seedlings under drought stress

Chitosan is a cationic marine polysaccharide with unique bioactive properties that make it an effective scavenger of reactive oxygen species. Chitosan application has been suggested as an aid for reducing oxidative injury caused by drought stress in crop plants. In order to confirm the antioxidant effects of exogenous chitosan, cell membrane stability and antioxidant enzyme activities were analyzed in leaves of apple seedlings placed under a period of drought stress. Pretreatment of apple seedling leaves with chitosan solution (20, 50, 100, 150 and 200 mg l⁻¹) prior to drought stress significantly decreased electrolyte leakage and the production of malondialdehyde in the leaves, while increasing antioxidant enzyme activities (superoxide dismutase, catalase), following imposition of drought stress conditions. An optimum response was obtained at a chitosan concentration of 100 mg l⁻¹. When apple seedlings were pretreated with 100 mg l⁻¹ of chitosan, cell membrane stability and antioxidant enzyme activities were enhanced for 21 days of drought treatment. Following restoration of moisture and a repeated drought stress, similar results were obtained on day 35. It is proposed that chitosan may act as an exogenous antioxidant that enhances resistance to oxidative stress during drought.     

The influence of the cation of quaternized chitosans on antioxidant activity

Various quaternized chitosans (QCSs) were synthesized according to previous method. Their reducing power and antioxidant potency against hydroxyl radicals (OH) and hydrogen peroxide (H₂O₂) were explored by the established systems in vitro. The QCSs exhibited markedly antioxidant activity, especially TCEDMCS, whose IC₅₀ on hydroxyl radicals was 0.235 mg/mL. They showed 65–80% scavenging effect on hydrogen peroxide at a dose of 0.5 mg/mL. Generally, the antioxidant activity decreased in the order TCEDMCS > TBEDMCS > EDMCS > PDMCS > IBDMCS > Chitosan. Furthermore, the order of their OH and H₂O₂ scavenging activity was consistent with the electronegativity of different substituted groups in the QCSs. The QCSs showed much stronger antioxidant activity than that of chitosan may be due to the positive charge density of the nitrogen atoms in QCSs strengthened by the substituted groups.