Mucoadhesive property and biocompatibility of methylated N-aryl chitosan derivatives

The methylated N-aryl chitosan derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMCh) and methylated N-(4-pyridylmethyl) chitosan chloride (MPyMeCh), were synthesized by two steps, the reductive amination and the methylation. The physicochemical properties of chitosan derivatives were determined by ATR-FTIR, NMR, X-ray diffraction (XRD) and thermogravimetric (TG) techniques. The XRD analysis showed that the crystallinity and thermal stability of methylated chitosan derivatives were lower than those of chitosan. The effects of degree of quaternization (DQ), polymer structure and positive charge location on mucoadhesive property and cytotoxicity were investigated by using a mucin particle method and MTT assay compared to N,N,N-trimethylammonium chitosan chloride (TMChC). It was found that the mucoadhesive property and cytotoxicity increased with increasing DQ. At the DQ of 65%, the mucoadhesive property of the MDMCMCh was twofold lower than that of the TMChC. However, this phenomenon did not affect the mucoadhesive property when the DQ was higher than 65%. Surprisingly, the MPyMeCh showed the lowest toxicity even with the high DQ. These could be due to the resonance effect of the positive charge in the pyridine ring and the molecular weight after methylation. Finally, our result revealed that the mucoadhesive property was dependent on the DQ and polymer structure whereas the cytotoxicity was dependent on the combination of the polymer structure, positive charge location and molecular weight after methylation.     

Flocculation of algae using chitosan

Flocculation of three freshwater algae, Spirulina,Oscillatoria and Chlorella, and onebrackish alga, Synechocystis, using chitosan was studiedinthe pH range 4 to 9, and chlorophyll-a concentrations inthe range 80 to 800 mg m^–3, which produces aturbidity of 10 to 100 nephelometric turbidity units (NTU) in water. Chitosanreduced the algal content effectively by flocculation and settling. Theflocculation efficiency is very sensitive to pH, and reached a maximum at pH7.0for the freshwater species, but lower for the marine species. The optimalchitosan concentration that is required to effect maximum flocculation dependedon the concentration of alga. Flocculation and settling were faster whenconcentrations of chitosan higher than optimal are used. The settled algalcellsare intact and live, but will not be redispersed by mechanical agitation. Thede-algated water may be reused to produce fresh cultures of algae.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

不同浓度壳聚糖对炭黑曲霉和硫色镰刀菌生长和发育的影响

【背景】壳聚糖是广泛存在于甲壳动物的一种多糖，具有广谱的抗真菌活性，但壳聚糖是否影响炭黑曲霉(Aspergillus carbonarius)和硫色镰刀菌(Fusarium sulphureum)生长和发育尚未见报道。【目的】明确不同浓度壳聚糖对A. carbonarius和F. sulphureum生长和发育的影响。【方法】通过在PDA培养基中添加不同浓度壳聚糖，测定两种真菌的菌落直径、生物量和菌丝干重，观察产孢量、孢子萌发和芽管长度，比较抑菌的差异。【结果】壳聚糖处理可显著改变两种真菌的菌落形态，处理浓度越高菌落皱缩和变形越明显；壳聚糖还可以有效抑制两种真菌的菌落生长、菌丝干重和菌丝生物量，抑制效果呈明显的浓度依赖，对F. sulphureum的抑制效果更好。壳聚糖可抑制两种真菌的产孢量、孢子萌发和芽管伸长，处理浓度越高抑制效果越好，对F. sulphureum的抑制效果更为明显。壳聚糖对A. carbonarius和F. sulphureum的EC₅₀值分别为0.12 mg/mL和0.075 mg/mL。【结论】壳聚糖可有效抑制A. carbonarius和F. sulphureum的生长发育，抑制效果呈浓度依赖，F. sulphureum对壳聚糖更为敏感。     

茶多酚与壳聚糖复配溶液对香椿芽保鲜效果的研究

为研究茶多酚与壳聚糖复配溶液对香椿芽的保鲜效果,该研究以多年生香椿树嫩芽为材料,采用不同浓度茶多酚(TP)与壳聚糖复配溶液处理香椿芽,(4±1)℃条件下贮藏,测定贮藏期间香椿芽感官变化和生理生化指标以及腐烂率的变化。结果表明:一定浓度的茶多酚与壳聚糖复配溶液可维持香椿芽的感官品质、降低生理衰老程度并减少腐烂率。其中,0.3%茶多酚-0.5%壳聚糖复配液对香椿芽保鲜效果最显著。0.3%茶多酚-0.5%壳聚糖复配液处理的香椿芽在贮藏12 d时失重率仅为1.52%、维生素C保持率为63.08%、叶绿素损失率为20.67%,同时对降低香椿芽的腐烂率和脱叶率有明显效果,贮藏21 d香椿芽的完好率接近84%、脱叶率为8%。1%茶多酚会加剧香椿芽的腐败脱叶,掩盖了香椿芽原有的香味,并导致褐变现象。因此,0.3%茶多酚-0.5%壳聚糖复配液在香椿芽保鲜方面具有较好的应用价值。     

Biosorption of nonylphenol on dead biomass of Rhizopus arrhizus encapsulated in chitosan beads

The nonylphenol (NP) biosorption and desorption potential for fungal biomass used under batch conditions was investigated using kinetics and isotherm models. Fungal biomass of Rhizopus arrhizus TISTR 3610 exhibited preferential uptake of NP, an endocrine disrupting chemicals. Sporangiospores, asexual spores, were immobilised in chitosan beads. The biosorption data of NP on the moist heat inactivated R. arrhizus–chitosan beads were analyzed using four popular adsorption isotherms and, by using non-linear least-regression with the solver add-in in Microsoft Excel, correlated in order with the Fritz–Schluender > Redlich–Peterson > Freundlich > Langmuir isotherms. The pseudo first-order kinetics was found to have the best fit with the experimental data. The diffusivity of NP in the R. arrhizus–chitosan beads was calculated using the shrinking core model, and the diffusivity values were in the ranges of 2.3736 × 10⁻⁴–1.8950 × 10⁻⁴ cm² s⁻¹. Desorption to recover the adsorbed NP from the beads was performed in methanol and was best described using a pseudo second-order kinetic model.     

Binding and biological properties of lipopolysaccharide Proteus vulgaris O25 (48/57)–chitosan complexes

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.     

Methylated N-aryl chitosan derivative/DNA complex nanoparticles for gene delivery: Synthesis and structure–activity relationships

In this study, three kinds of methylated chitosan containing different aromatic moieties were synthesized by two steps, reductive amination and methylation, respectively. The chemical structures of all methylated derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMChC), methylated N-(4-N,N-dimethylaminobenzyl) chitosan chloride (MDMBzChC), and methylated N-(4-pyridinylmethyl) chitosan chloride (MPyMeChC) were characterized by ATR–FTIR and ¹H NMR spectroscopy. The complexes between the chitosan derivatives and plasmid DNA at different N/P ratios were characterized by gel electrophoresis, dynamic light scattering, and atomic force microscopic techniques. The smallest particle sizes of these complexes were obtained at N/P ratio of 5 and ranged from 95 to 124 nm while the zeta-potentials were in the range of 18–27 mV. Transfection efficiencies of these complexes were investigated by expression of the plasmid DNA encoding green fluorescence protein (pEGFP-C2) on human hepatoma cells (Huh 7 cells) compared to N,N,N-trimethyl chitosan chloride (TMChC). The rank of transfection efficiency was MPyMeChC > MDMBzChC > TMChC > MDMCMChC, respectively. The cytotoxicity of these complexes was also studied by MTT assay where the MPyMeChC complex exhibited less toxicity than other derivatives even at high N/P ratios. Therefore, MPyMeChC demonstrated potential as its safe and efficient gene carrier.     

Colorimetric assay of chitosan in presence of proteins and polyelectrolytes by using o-phthalaldehyde

A novel approach of colorimetric quantification of chitosan based on the derivatization reaction of its primary amino groups with o-phthalaldehyde and a thiol – N-acetyl-l-cysteine has been developed. The reaction of equal volumes of sample solution and the reagent solution was allowed to proceed for 1 h, and then the absorbance values were measured at 340 nm against the reference solution. The procedure conditions have been optimized for chitosan assay in the presence of polyanionic electrolyte dextran sulphate (pH 8.9, the reagent solution: 4.0 mM o-phthalaldehyde, 2.6 mM N-acetyl-l-cysteine, 0.25 M NaCl). The method has proven to be convenient and reliable for quantitative determination of either the concentrations of chitosans of various molecular weights or their degree of deacetylation. The different reactivity of chitosans and proteins can be used in order to determine chitosan in presence of the protein. This approach ensured accurate assay within the chitosan concentrations ranging from 0.01 to 0.15 mg/ml and could be applied for quantitative analysis of chitosan in protein-loaded microparticles.     

嵌合鞭毛蛋白和壳聚糖/三聚磷酸(CS/TPP)纳米凝胶封装对鼻接种幽门螺杆菌黏附素诱导的免疫应答的佐剂作用

[目的] 幽门螺杆菌黏附素A（HpaA）是幽门螺杆菌疫苗的一种有希望的抗原。探索嵌合鞭毛蛋白（cFLN）和壳聚糖/三聚磷酸（CS/TPP）纳米凝胶包封对鼻递送抗原HpaA诱导的免疫应答增强的佐剂作用。[方法] 通过将鼠伤寒沙门氏菌鞭毛蛋白FliC的D0、D1结构域与幽门螺杆菌鞭毛FlaA的D2、D3结构域相结合,构建嵌合鞭毛蛋白cFLN。作为分子内佐剂,嵌合鞭毛蛋白cFLN与黏附素（HpaA）连接构建复合抗原cFLN-HpaA。cFLN、HpaA、cFLN-HpaA在重组大肠杆菌中表达,并通过Ni-NTA预装色谱柱进行纯化。使用离子凝胶法分别将cFLN、HpaA、cFLN-HpaA包封到壳聚糖/三聚磷酸（CS/TPP）纳米凝胶中。[结果] 成功表达和纯化了cFLN、HpaA和cFLN-HpaA,并用离子凝胶法将这3种抗原包封在CS/TPP/纳米凝胶中,制备了包封cFLN的CS/TPP纳米凝胶（cFLN-HpaA NG）、包封HpaA的CS/TPP纳米凝胶（HpaA NG）、包封cFLN-HpaA的CS/TPP/纳米凝胶（cFLN-HpaA NG）。经鼻免疫小鼠后,与HpaA相比,cFLN-HpaA分别导致血清中IgG、IgG1和IgA含量增加2.09倍、1.4倍和2.62倍。与cFLN、HpaA和cFLN-HpaA相比,cFLN NG、HpaA NG和cFLN-HpaA NG分别使血清中IgA、IgG、IgG1和IgG2a的含量增加了1.28至1.71倍。[结论] cFLN和CS/TPP NG可以显著增强鼻腔输送的HpaA诱导的体液免疫。通过检测鼻腔免疫后胃黏膜中IFN-γ、IL-4、IL-17和SIgA的含量,与HpaA相比,cFLN-HpaA分别诱导IFN-γ、IL-4和SIgA的含量增加1.38、1.16和1.58倍。与cFLN-HpaA相比,cFLN-HpaA NG分别使小鼠胃黏膜中IFN-γ、IL-4、IL-17和SIgA的含量增加1.81、1.71、2.16和2.1倍。表明cFLN和CS/TPP NG可以显著增强Th1和Th17型免疫应答。小鼠体内免疫原性研究表明,分子内佐剂cFLN和纳米凝胶包封不仅可以有效增强鼻腔输送HpaA诱导的体液免疫应答,而且还可以增强小鼠胃黏膜中的黏膜免疫应答——Th1和Th17型免疫应答。因此,这些结果表明,cFLN-HpaA NG可能是有希望的经鼻输送的用于预防幽门螺杆菌感染的疫苗系统。     

Polyelectrolyte complexes and layer-by-layer capsules from chitosan/chitosan sulfate

Polyelectrolyte complex formation of chitosans of varying average molecular weight and degree of acetylation with chitosan sulfate or poly(styrene sulfonate) was studied by static light scattering in dilute solution at various ionic strengths. Unlike the molecular weight, the degree of acetylation was found to have a significant effect on the resultant structural densities of the complexes. The same system was applied to the preparation of micrometer-sized hollow shells by means of a layer-by-layer technique (in total eight layers). Their behavior toward fluorescent probes such as fluorescein and rhodamin 6G or fluorescein isothiocyanate labeled dextrans at various ionic strengths and pH (observed by confocal laser light scanning microscopy) could be understood through a discussion of electrostatic forces between the highly charged shells and the probes to be dominant. At an ionic strength of 0.1 M and above, charge effects are largely suppressed (screening effect) and a size-dependent "cutoff" for the permeation of the macromolecular fluorophore was observed.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

Glucosamine-mediated detoxification of p-benzoquinone and its removal by chitosan

p-Benzoquinone non-enzymatically reacted with d-glucosamine at physiological pH and moderate temperature. The reaction of p-benzoquinone with glucosamine was signaled by changes in the UV and visible spectra. The reactivity proceeded fastest at pH values above 7, with a sharp drop from pH 6.5 to 7.0, and the reaction was negligible in acidic conditions. The order of reactivity of amino sugars was d-mannosamine > d-glucosamine > d-galactosamine. From the reaction mixture, four conversion products were isolated and none was toxic to Escherichia coli even at 500–700 g ml^–1, while p-benzoquinone was cytotoxic to E. coli at 20 g ml^–1. Chitosan could react with p-benzoquinone efficiently and remove this toxicant in aqueous solution.     

Biological control of Pythium aphanidermatum in cucumber with a combined application of Lysobacter enzymogenes strain 3.1T8 and chitosan

Pythium aphanidermatum (Edson) Fitzp., causing root and crown rot in cucumber, was successfully managed by Lysobacter enzymogenes strain 3.1T8. Greenhouse experiments were performed with cucumber plants grown in rockwool blocks up to 5 weeks with a recirculated nutrient solution. Application of L. enzymogenes 3.1T8 in combination with chitosan (the deacetylated derivative of chitin) reduced the number of diseased plants by 50–100% in four independent experiments relative to the Pythium control. Application of chitosan or the bacterial inoculant alone was not effective. Washed bacterial cells plus chitosan inhibited Pythium-induced disease, but the supernatant without bacterial cells combined with chitosan was not effective. The most effective and convenient type of commercially available chitosan was selected. Chitosan disappeared from the hydroponic system within 24 h after application, which we attribute to enzyme expression of L. enzymogenes 3.1T8 induced by the exposure to chitosan. Plate counts of the nutrient solution on a general bacterial medium showed the dominance of the inoculated strain, and an increased bacterial population growing on chitin and chitosan as single carbon source. The population density of L. enzymogenes 3.1T8 on the cucumber roots was investigated with a strain specific real-time TaqMan PCR. Highest chitosan concentrations applied (0.1 and 0.03 g/plant) resulted in the highest numbers of L. enzymogenes 3.1T8 present on roots; i.e. 10⁸–10⁹ cells/g root. Substantially higher numbers of bacterial cells were observed by scanning electron microscopy after application of chitosan; no morphological or other qualitative differences were found. The results indicate that addition of chitosan enhanced the biocontrol efficacy of L. enzymogenes 3.1T8; either chitosan serves as C- and N-source for the antagonist, induces antagonistic gene expression, or both.     

The N-permethylation of chitosan and the preparation of N-trimethyl chitosan iodide

Chitosan was N-permethylated by reaction with formaldehyde and sodium borohydride under controlled conditions (pH 4·0, 15°C, reaction times 12 and 8 h, respectively). The N-permethylated chitosan was reacted with methyl iodide at 35°C and N-trimethyl chitosan iodide with a quaternary nitrogen degree of 60% was obtained. This material may have uses as an antibiotic and an ion exchange material.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

Dyeing and antimicrobial characteristics of chitosan treated wool fabrics with henna dye

Chitosan, a naturally available biopolymer which is now increasingly being used as a functional finish on textile substrates to impart antimicrobial characteristics and increase dye uptake of fabrics was applied on wool fabrics. Henna a natural dye with proven bactericidal properties was applied on wool fabrics along with chitosan to impart antimicrobial characteristics. The effect of chitosan application on the dyeing properties of wool fabrics was studied by measuring the K/S values of the treated substrates at various concentrations of chitosan and the dye. The antimicrobial properties of chitosan and natural dyes both when applied independently and collectively on fabrics were assessed. The results proved that the chitosan treated wool fabrics showed increase dye uptake of fabrics. The treated fabrics were found to be antimicrobial and the chitosan treatment enhances the antimicrobial characteristics of the dyes. Fastness properties of the applied finish to washing, rubbing and perspiration have also been discussed.     

Novel chitosan membranes as support for lipases immobilization: Characterization aspects

Membranes of chitosan (QS), chitosan treated with glutaraldehyde (QGA) and chitosan crown ether (QCE) were utilized as carriers for immobilization of Candida antarctica and Candida rugosa lipases. Membrane supports were characterized by several techniques (Raman spectroscopy, elemental analysis by CHN determination and Energy Dispersive X-ray (EDX), water sorption isotherms, and surface area from nitrogen sorption data). To verify the presence of enzymes, some of these techniques were also used for lipase on chitosan biocatalytic systems. Measurements of protein load from Biuret assays and catalytic activity in esterification in nonaqueous media were also made for the immobilized enzymes. Sorption isotherms at 20, 30, 40 and 50 °C for QS, QGA and QCE supports were fitted to the Guggenheim, Anderson and Böer model. GAB monolayer moisture parameter, Xm, varied between 0.029 and 0.051 for QS, 0.039 and 0.058 for QGA and 0.039–0.075 g of water g⁻¹ s.s. for QCE membranes. Elemental analysis and Raman spectra measurements of the lipase, supports and immobilized lipase systems gave evidence of the presence of enzymes on supports. Chitosan supports with internal surface area (m2 g⁻¹) among 3.31 and 1.26 were obtained. Regardless of these low values, acceptable protein load (0.61 to 3.21%) and esterification initial rates were achieved (0.88–2.75 mmol min⁻¹ g of protein⁻¹).     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.