Degree Of N-acetylation Research Articles

Environmental Sustainability and Physicochemical Property Screening of Chitin and Chitin-Glucan from 22 Fungal Species.

Thanks to its biobased character with embedded biogenic carbon, chitin can aid in the transition to a sustainable circular economy by replacing fossil carbon from the geosphere. However, meeting current demands for material availability and environmental sustainability requires alternative methods limiting conventional chemical and energy-consuming chitin extraction from crustaceans. To assist future chitinous bioproduct development, this work analyzes the physicochemical properties and potential environmental sustainability of fungal chitin-glucan complexes. A conventional isolation procedure using sodium hydroxide, a weak acid, and short reaction times are applied to the fruiting body of 22 fungal species. Besides, the valorization of underutilized waste streams including Agaricus bisporus and Agaricus brunnescens stipes is investigated. The carbohydrate analysis renders chitin fractions in the range of 9.5-63.5 wt %, while yields vary from 4.2 to 29.9%, and the N-acetylation degree in found in between 53.0 and 98.7%. The sustainability of the process is analyzed using life cycle assessment (LCA), providing impact quantification for global warming potential, terrestrial acidification, freshwater eutrophication, and water use. With 87.5-589.3 kg·CO2-equiv per kilo, potentially lower global warming potential values in comparison to crustacean chitin are achieved. The crystallinity degree ranged from 28 to 78%, while the apparent chitin crystalline size (L020) is between 2.3 and 5.4 nm. Ten of the species yield α-chitin coexisting with semicrystalline glucans. Zwitterionic properties are observed in aqueous solutions, shifting from cationic to anionic at pH 4.5. With its renewable carbon content, fungal chitin is an environmentally sustainable alternative for high-value applications due to its balance of minimal treatment, low carbon footprint, material renewability, ease of isolation, thermal stability, zwitterionic behavior, biodegradability, and noncytotoxicity.

One-pot chitin pulping using recyclable superbase-based protic ionic liquid

The application of ionic liquids and deep eutectic solvents offers a promising opportunity for a more environmentally friendly and straightforward chitin purification process from crustacean shells. Nonetheless, the insufficient recyclability of these ionic solvents poses a challenge to the long-term sustainability of such extraction methods. Thus, there is a strong imperative to focus on employing easily recyclable ionic liquids for chitin isolation, enhancing the overall sustainability of the process.In this investigation, a direct chitin purification procedure that utilized pulping liquors consisting of the superbase-based protic ionic liquid 1,5-diazabicyclo[4.3.0]non-5-enium acetate and its precursor, acetic acid, was developed. It was demonstrated that these pulping liquors were capable of simultaneously deproteinate and demineralize shrimp shells to generate chitins with higher purity, degree of N-acetylation and crystallinity than commercially obtained chitin. More significantly, the pulping liquors can be recycled to their pure form in high quantity by simple distillation under reduced pressure, allowing the reuse of these mixtures, which give chitin of nearly identical purity.

Water-soluble bio-sourced resist interactions with fluorinated etching plasmas during the photolithography process

Lithography is one of the key steps in micro/nanofabrication, which involves the use of oil-based resists, organic solvents, and toxic chemicals. Nowadays, environmental issues and regulation have raised the need for developing greener materials and processes. Therefore, efforts have been devoted to developing greener resists, in particular, resists based on water-soluble bio-sourced polymers. Among these biopolymers, polysaccharides have gained a strong interest. However, their interaction with silica etching plasmas, in particular, fluorinated plasmas, remains scarcely studied and contradictory results are found in the literature. The present contribution reports on the study of the interaction of two chitosans exhibiting different degrees of N-acetylation with SF6/Ar and CHF3 etching plasmas. The surface modifications and in-depth modifications were studied with x-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, infrared spectroscopy, water contact angle, and size-exclusion chromatography. The effect of neutrals, ions, and vacuum ultraviolet (VUV) was considered. Our results suggest that the chitosan selectivity is greatly influenced by the deposition of a fluorocarbon film and that VUV seems to be involved in scissions of the polymer chains. No significant difference between the two chitosans was observed.

Green-Chemical Strategies for Production of Tailor-Made Chitooligosaccharides with Enhanced Biological Activities.

Chitooligosaccharides (COSs) are b-1,4-linked homo-oligosaccharides of N-acetylglucosamine (GlcNAc) or glucosamine (GlcN), and also include hetero-oligosaccharides composed of GlcNAc and GlcN. These sugars are of practical importance because of their various biological activities, such as antimicrobial, anti-inflammatory, antioxidant and antitumor activities, as well as triggering the innate immunity in plants. The reported data on bioactivities of COSs used to contain some uncertainties or contradictions, because the experiments were conducted with poorly characterized COS mixtures. Recently, COSs have been satisfactorily characterized with respect to their structures, especially the degree of polymerization (DP) and degree of N-acetylation (DA); thus, the structure-bioactivity relationship of COSs has become more unambiguous. To date, various green-chemical strategies involving enzymatic synthesis of COSs with designed sequences and desired biological activities have been developed. The enzymatic strategies could involve transglycosylation or glycosynthase reactions using reducing end-activated sugars as the donor substrates and chitinase/chitosanase and their mutants as the biocatalysts. Site-specific chitin deacetylases were also proposed to be applicable for this purpose. Furthermore, to improve the yields of the COS products, metabolic engineering techniques could be applied. The above-mentioned approaches will provide the opportunity to produce tailor-made COSs, leading to the enhanced utilization of chitin biomass.

Comparative Evaluation of the In Vitro Cytotoxicity of a Series of Chitosans and Chitooligosaccharides Water-Soluble at Physiological pH.

Chitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL).

Preparation of highly stable and ultrasmooth chemically grafted thin films of chitosan.

Chitosan-coated surfaces are of great interest for biomedical applications (antibacterial coatings, implants, would healing, single-cell microfluidics…). However, one major limitation of chitosan-based systems is the high solubility of the polymer under acidic aqueous conditions. Herein, we describe a simple procedure to prepare extremely smooth and stable chitosan coatings. In detail, chitosan films with a low degree of N-acetylation and of thicknesses varying from 40 nm to 10 μm were grafted onto epoxy-functionalized silicon wafers via an optimized water-temperature treatment (WTT). The formation of a grafted chitosan network insoluble in acidic aqueous media (pH 3.5) was evidenced and the films were stable for at least 2 days at pH 3.5. The film morphology and the swelling behavior were characterized by atomic force microscopy (AFM) and neutron reflectivity, which showed that the film roughness was extremely low. The physical cross-linking of the films was demonstrated using infrared spectroscopy, dynamic mechanical analysis (DMA) and wide-angle X-ray scattering (WAXS). Finally, we show that the swelling behavior of such films was largely influenced by the environmental conditions, such as the pH or ionic strength of the solution.

Impact of extended acid hydrolysis on polymeric, structural and thermal properties of microcrystalline chitin

In this study, microcrystalline chitin (MCCh) was prepared from commercial shrimp shell α-chitin by acid hydrolysis at ambient temperature for various treatment durations (30, 60, 90, 120, and 150 min), and their structural, polymeric and thermal properties were studied. Results showed that MCCh was distinct from native chitin structure, exhibiting loosened fibrillar structure with large pores and void diameter, increasing with treatment duration from <0.5 μm for native chitin to 1.653 μm for MCCh-150. The recovery rate obtained was in the range of 57.50 – 71.65%. The degree of N-acetylation reduced slightly from 89.86% for native chitin to 88.07% for MCCh-150 with increasing treatment duration. Crystallinity indexes increased from 85 to 92% after 30 min of acid treatment, then decreased slightly to 90% after 150 min. DSC analysis indicated MCCh were thermally stable. These results suggest that extended acid hydrolysis treatment can greatly improve MCCh polymeric and thermal properties. Furthermore, the method described is simple, easy, energy-efficient, and requires less acid compared to earlier studies, making it more practical and scalable for the production of MCCh.

Impact of HILIC Amino-Based Column Equilibration Conditions on the Analysis of Chitooligosaccharides

Obtaining well-defined Chitooligosaccharides (COS) structures is very important as their physicochemical and biological properties depends strongly on their degree of polymerization (DP) and degree of N-acetylation (DA). Hydrophilic Interaction Liquid Chromatography (HILIC), that is commonly used for COS analyses, suffers from several drawbacks, related to their complex structure, leading to broadening/splitting of the chromatogram signals. We investigated, herein, on the role of the equilibration step of an amino-based HILIC column on the separation of COS DP ≤ 5. We demonstrated that COS could be separated according to their DP after equilibrating the column by a NaHCO3 buffer solution (100 mM, pH = 10) and mild elution chromatographic parameters (neutral mobile phase, r.t.); or according to their DA after equilibrating the column by an NH4Ac buffer solution (50 mM, pH = 4.5). Also, the nature of the counterion of the column stationary phase was found to affect both retention times and signal profiles of analyzed COS.

A Simple Approach to Produce Tailor-Made Chitosans with Specific Degrees of Acetylation and Molecular Weights.

Chitin is a structural polysaccharide that is found in crustaceans, insects, fungi and some yeasts. Chitin deacetylation produces chitosan, a well-studied biopolymer with reported chemical and biological properties for diverse potential applications for drug delivery, metal ion absorption, scaffolds and tissue engineering. Most known properties of chitosan have been determined from samples obtained from a variety of sources and in different conditions, this is, from chitosans with a wide range of degrees of N-acetylation (DA) and molecular weight (MW). However, as for any copolymer, the physicochemical and mechanical characteristics of chitosan highly depend on their monomer composition (DA) and chain size (MW). This work presents a simple methodology to produce chitosans with specific and predictive DA and MW. Reaction with acetic anhydride proved to be an efficient method to control the acetylation of chitosan, DAs between 10.6% and 50.6% were reproducibly obtained. In addition to this, MWs of chitosan chains were reduced in a controlled manner in two ways, by ultrasound and by acidic hydrolysis at different temperatures, samples with MWs between 130 kDa and 1300 kDa were obtained. DAs were determined by 1H-NMR and MWs by gel permeation chromatography.

Characterization of chitin-glucan complex from Tremella fuciformis fermentation residue and evaluation of its antibacterial performance

Submerged fermentation of fungi is an efficient way to obtain extracellular polysaccharides, however, in this process, excess discarded biomass is produced. In this study, Tremella fuciformis mycelia were reused as the raw material to isolate a novel fungal chitin-glucan complex (CGC-TFM) using alkaline extraction. Characteristic analysis revealed that the CGC-TFM consisted of glucosamine/acetylglucosamine and glucose (GlcN:Glc = 26:74 mol%), indicating a reference to the β polymorphism of chitin-glucan complex, with the molecular weight and crystallinity index of 256 ± 3.0 kDa and 54.25 ± 1.04%, respectively. Fourier transform infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy analyses confirmed that the chitin portion of the CGC-TFM exhibited a typical β configuration and N-acetylation degree of 70.52 ± 2.09%. Furthermore, the CGC-TFM exhibited good thermal stability and effective Escherichia coli inhibition ability, indicating that it could be applied as a potential food packaging material.

Effects of acetyl grafting on the structural and functional properties of whey protein microgels

Whey protein microgels (WPMs) are self-assembled sub-micron particles, with diverse applications in food science such as emulsion and foam stabilization. This study evaluated the influence of acetylation at different acetic anhydride-to-protein molar ratios (AA-to-protein MRs) on structural characteristics, colloidal stability, and technological functionality of WPMs. N-acetylation degree progressively increased with increasing the AA-to-protein MR and reached 96% when AA-to-protein MR was 3270. O-acylation increased significantly only after N-acylation extensively (96%) occurred. Acetylation process increased the randomness of proteins in WPMs (assessed by circular dichroism and Fourier transform infrared spectroscopies) as well as conferred a less compact structure (assessed by intrinsic fluorescence spectroscopy). The isoelectric point (pI) of WPMs shifted from roughly 4.5 to 3.0–3.9 depending on the acetylation extent; the higher the acetylation degree, the lower was the pI. Acetylation at any extent did not improve the colloidal stability of WPMs at their corresponding pI. However, the emulsification activity of WPMs at pH values between 5.0 and 7.0 increased with increasing the acetylation extent. The highest foaming and oil absorption capacities of WPMs at pH 7.0 was obtained at a N-acetylation extent of 40%.

Characterization of chitin extracted from enzymatically deproteinized Acetes shell residue with varying degree of hydrolysis

Acetes shrimp is an unexploited tiny shrimp mainly landed as bycatch which is a good source for the recovery of protein and chitin. In the present study, the residual shell obtained after the hydrolysis of Acetes was used for the extraction of chitin by combining enzymatic and chemical treatments. Enzymatic hydrolysis with Alcalase was performed at different rates. Results showed that the protein removal efficiency increases with the increase in DH and the maximum deproteinzation was achieved at 30 % DH (93.68 %). The FTIR spectra showed two sharp bands for chemically prepared chitin and 30 % DH chitin at 1627–1629 and 1664−1665 cm−1 indicating that its alpha amorphous structure. The degree of N-acetylation was found to be higher in enzymatically prepared chitin in all different hydrolytic treatment rather than chemically prepared. The surface morphologies of chitin revealed the porous and nanofibrous structures for 30 % DH chitin and chemically prepared chitin.

Structural changes in α-chitin through nanofibrillation by high-pressure homogenization in water

Chitin nanofiber was prepared from purified crab shell chitin by repeated high-pressure homogenization in water. The chitin nanofiber/water dispersion thus prepared was viscous and translucent, and maintained a stable dispersion at room temperature for several months. AFM images showed that the chitin nanofibers had heterogeneous network structures with widths ranging from several nanometers to several tens of nanometers. Some kinks and twisted structures were also observed in the AFM images. X-ray diffraction patterns showed that both the crystallinity index and crystal width of the original alpha-chitin decreased with nanofibrillation. Solid-state 13C-NMR spectra showed that the chemical shifts of all carbons were unchanged before and after nanofibrillation, and that all C6–OH groups had the gauche-gauche conformation irrespective of the crystalline fibril surfaces and insides. The degree of N-acetylation increased from 0.83 to 0.98, which was probably due to C2–NH2 groups present in the original chitin being partially removed during high-pressure homogenization in water. The original chitin and chitin nanofiber were dissolved in 8% LiCl/DMAc, and the solutions after dilution to 1% LiCl/DMAc were subjected to size-exclusion chromatography combined with multi-angle laser-light scattering to determine their molar masses and molar mass distributions. The weight-average molar mass (Mw) value of the original chitin was 271,200 (degree of polymerization [DP] ~1340). The Mw value of the chitin nanofiber was 165,500 (DP 820), showing that the DP of the original chitin decreased by 40% through the nanofibrillation in water to form the chitin nanofiber. Chitin nanofibers were prepared from purified crab-shell chitin particles by repeated high-pressure homogenization in water. AFM images showed that the chitin nanofibers had heterogeneous network structures. X-ray diffraction patterns showed that both the crystallinity index and crystal width of the original α-chitin decreased by nanofibrillation. Solid-state 13C-NMR spectra showed that all C6–OH groups had the gauche–gauche conformation. The degree of N-acetylation increased from 0.83 to 0.98 by nanofibrillation, while the weight-average molar masses of the original chitin and chitin nanofibers were 271,200 and 165,500, respectively.

In situ synthesis of Fe3O4 nanoparticles coated by chito-oligosaccharides: physico-chemical characterizations and cytotoxicity evaluation for biomedical applications

Fe3O4 nanoparticles coated with chito-oligosaccharides (COS) were prepared in situ by a simple co-precipitation method through a mixing of iron ions (Fe3+ and Fe2+) and COS aqueous solutions followed by precipitation with ammonia. The impact of COS with different degree of polymerization (DP 10, 24 and 45) and degree of N-acetylation (DA) ∼ 24% and 50% (exhibiting high solubility) on the synthesis and physical properties of the coated magnetic nanoparticles was evaluated. Several advantages were found when the magnetic nanoparticles were prepared in the presence of the studied COS, such as: preparation of functionalized magnetic nanoparticles with narrower size distributions and, consequently, higher saturation magnetization (an increase of up to 22%); and an expressive increasing in the concentration of COS-coated magnetic nanoparticles (up to twice) in the cell viability test in comparison with pure Fe3O4 nanoparticles. Furthermore, among the analyzed samples, the magnetic nanoparticles coated by COS with DA ∼ 50% present a higher cytocompatibility. Our results allow envisioning various biomedical applications, valorizing the use of coated-magnetic nanoparticles for magnetic-field assisted drug delivery, enzyme or cell immobilization, or as a marker for specific cell tracking, among others.

Chilean crab (Aegla cholchol) as a new source of chitin and chitosan with antifungal properties against Candida spp

In the present work, the chemical composition of the Chilean freshwater crab Aegla cholchol exoskeleton was studied for the first time. α-Chitin was isolated from three main body parts (pincers, legs, carapace), and its content ranged from 9.0–10.4% (w/w). Moreover, chitosan was extracted by alkaline treatment at different temperature and time regimes. Aegla cholchol exoskeleton, chitin and chitosans were characterized by FTIR, TGA, and SEM. Chemical structure of chitin and chitosan was confirmed by NMR spectroscopy. Chitosan molecular weight determinations were carried out by GPC. The obtained chitosan samples had a degree of N-acetylation (DA) between 4 and 15% and molecular weight (Mw) in the range of 65–201 kDa. The antifungal activity of the chitosan samples and the chitooligomer were tested toward twenty isolated clinical strains of Candida yeast. Chitosan with lower DA (4%) and higher molecular weight showed the lowest minimum inhibitory concentration (MIC) values, which was evidenced by the highest antifungal activity toward Candida albicans, Candida tropicalis, Candida glabrata, and Candida parapsilosis. Results suggest that Aegla cholchol is an excellent natural source for production of bioactive materials with potential applications in the health system, to prevent infections associated with Candida strains.

Water-Soluble 2,5-Anhydro-d-mannofuranose Chain End Chitosan Oligomers of a Very Low Molecular Weight: Synthesis and Characterization.

Five chitosans with different degrees of N-acetylation (DAs), molar masses, and origins were depolymerized by nitrous acid treatment in acidic media, leading to water-soluble 2,5-anhydro-d-mannose chain end oligomers with DPn < 20. The kinetics of the reaction was studied, and the best work conditions were found to be 3 h reaction at 50 °C. It was shown that the DPn of oligomers only depends on the quantity of NaNO2 involved. Molar masses or DAs do not have an impact on the depolymerization process when targeting oligomers with less than 20 units. This depolymerization process also leads to free 2,5-anhydromannofuranose (AMF) that might react with the free amines of obtained oligomers to form imines. This reaction is pH-dependent and in acidic condition leads to the formation of 5-hydromethyl-2-furfural (HMF). At the end, the oligomers were purified by dialysis to get rid of most of the free AMF (<5%) and other residual salts and appeared to have no acute toxicity.

Preparation of α-chitin-based nanocomposite as an effective biocatalyst for microwave aided domino reaction

In this paper, chitin (Ch) was extracted by an optimized method from cuttlebone of the Persian Gulf cuttlefish (Sepiidae, Cephalopoda). The extracted chitin was characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and thermal gravimetric analysis (TGA) which showed that the extracted chitin was in alpha form. The degree of N-acetylation (DA) and degree of substitution (DS) of α-chitin were calculated using titration method and FTIR spectroscopy and found to be 80–82% and 19.57 respectively. The α-Chitin was used as biomolecules for the preparation of nanostructured Ch/ZnO via a hydrothermal method. The obtained nanocomposite was characterized using FT-IR, XRD, scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis. The antimicrobial aspect of Ch/ZnO nanocomposite was previously proposed. In this paper, attempt was made to add the catalytic feature to these traits. For this purpose, the nanostructured Ch/ZnO was used as reusable nanocatalyst in the green and efficient synthesis of Benzo[a]pyrano(2,3-c)phenazine derivatives thru a four components microwave aided domino reaction. Eco-friendly, easy work up and separation of the nanostructured catalyst are some of the highlighted features this protocol.

Influence of the process parameters on β-chitin and α-chitin extraction: probing about the grinding and particles size

Abstract Chitin occurs in biomass as two main polymorphs, named as β- and α-chitin, which differ on their arrangements on the solid state. The control of chitin physical-chemistry properties by adjustments in their extraction process is an important key to improve its applications and derivatization reactions. Thus, the influences of the extraction parameters process on the β and α-chitin characteristics were studied. Squid pens (Loligo sp.) and shrimp shells (Macrobrachium r.), were used as raw material for β-chitin and α-chitin extraction, respectively. The obtained chitin, regardless polymorphs, was subjected to grinding in a knives mill, sieved and classified in different fractions according to the particles size. The fractions were characterized according to the degree of N-acetylation (DA), viscosimetric molecular weight (Mv), crystallinity, morphology and thermal stability. According to the scanning electron microscopy β-chitin particles present a fiber-like characteristic while α-chitin exhibit a flake-like shape. The procedure of grinding affected the DA and Mv of α-chitin (63%

The Conformation of Chitosan Molecules in Aqueous Solutions

This paper presents a review focused on the study of the physicochemical properties of chitosans. The main attention is paid to the analysis of data on the conformation of the polymer molecules in aqueous solutions depending on the molecular weight and degree of N-acetylation of chitosan, as well as on external factors, such as the nature of the solvent, the pH, and the ionic strength of the solutions.

Chitin nanocrystals prepared by oxidation of α-chitin using the O2/laccase/TEMPO system

Laccase mediator oxidation was applied to chitin at pH 6.8 and 30 °C to prepare chitin nanocrystals with a catalytic amount of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO). When 40 mM TEMPO and a total of 500 U laccase were added to 1 g chitin, the yield of water-insoluble oxidized chitin was more than 95%, and the carboxylate content was 0.43 mmol/g. Adsorption of laccase molecules on chitin particles occurred in a buffer at pH 6.8, which may have been caused by electrostatic interactions between positively charged C2-ammonium groups of chitin and anionically charged groups of laccase. Rod-like chitin nanocrystals (ChNCs) were obtained with average lengths and widths of 480 ± 200 nm and 24 ± 17 nm, respectively, by sonication of the oxidized chitin/water suspensions. The O2/laccase/TEMPO oxidation caused no decrease in the degree of N-acetylation or the crystallinity of the original chitin based on FTIR and X-ray diffraction data.