Isolation and Structure Analysis of Chitin Obtained from Different Developmental Stages of the Mulberry Silkworm (Bombyx mori).

Chitin, a ubiquitous biopolymer, holds paramount scientific and economic significance. Historically, it has been primarily isolated from marine crustaceans. However, the surge in demand for chitin and the burgeoning interest in biopolymers have necessitated the exploration of alternative sources. Among these methods, the mulberry silkworm (Bombyx mori) has emerged as a particularly intriguing prospect. To isolate chitin from Bombyx mori, a chemical extraction methodology was employed. This process involved a series of meticulously orchestrated steps, including Folch extraction, demineralization, deproteinization, and decolorization. The resultant chitin was subjected to comprehensive analysis utilizing techniques such as attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), 13C nuclear magnetic resonance (NMR) spectroscopy, and wide-angle X-ray scattering (WAXS). The obtained results allow us to conclude that the Bombyx mori represents an attractive alternative source of α-chitin.

Unveiling the structural properties and induced resistance activity in rice of Chitin/Chitosan-Glucan Complex of Rhizoctonia solani AG1 IA inner cell wall.

Phytopathogen cell wall polysaccharides have important physiological functions. In this study, we isolated and characterized the alkali-insoluble residue on the inner layers of the Rhizoctonia solani AG1 IA cell wall (RsCW-AIR). Through chemical composition and structural analysis, RsCW-AIR was mainly identified as a complex of chitin/chitosan and glucan (ChCsGC), with glucose and glucosamine were present in a molar ratio of 2.7:1.0. The predominant glycosidic bond linkage of glucan in ChCsGC was ß-1,3-linked Glcp, both the α and ß-polymorphic forms of chitin were presented in it by IR, XRD, and solid-state NMR, and the ChCsGC exhibited a degree of deacetylation measuring 67.08 %. RsCW-AIR pretreatment effectively reduced the incidence of rice sheath blight, and its induced resistance activity in rice was evaluated, such as inducing a reactive oxygen species (ROS) burst, leading to the accumulation of salicylic acid (SA) and the up-regulation of SA-related gene expression. The recognition of RsCW-AIR in rice is partially dependent on CERK1.

Design of Inhibitors Targeting Chitin-Degrading Enzymes by Bioisostere Substitutions and Scaffold Hopping for Selective Control of Ostrinia furnacalis.

Chitin-degrading enzymes are critical components in regulating the molting process of the Asian corn borer and serve as potential targets for controlling this destructive pest of maize. Here, we used a scaffold-hopping strategy to design a series of efficient naphthylimide insecticides. Among them, compound 8c exhibited potent inhibition of chitinase from OfChi-h and OfChtI at low nanomolar concentrations (IC50 = 1.51 and 9.21 nM, respectively). Molecular docking simulations suggested that 8c binds to chitinase by mimicking the interaction of chitin oligosaccharide substrates with chitinase. At low ppm concentrations, compound 8c performed comparably to commercial insecticides in controlling the highly destructive plant pest, the Asian corn borer. Tests on a wide range of nontarget organisms indicate that compound 8c has very low toxicity. In addition, the effect of inhibitor treatment on the expression of genes associated with the Asian corn borer chitin-degrading enzymes was further investigated by quantitative real-time polymerase chain reaction. In conclusion, our study highlights the potential of 8c as a novel chitinase-targeting insecticide for effective control of the Asian corn borer, providing a promising solution in the quest for sustainable pest management.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

Activities of proteases in deep eutectic solvents and removal of protein from chitin by subtilisin A in betaine/glycerol.

This research intended to remove residual protein from chitin with proteases in deep eutectic solvents (DESs). The activities of some proteases in several DESs, including choline chloride/p-toluenesulfonic acid, betaine/glycerol (Bet/G), choline chloride/malic acid, choline chloride/lactic acid, and choline chloride/urea, which are capable of dissolving chitin, were tested, and only in Bet/G some proteases were found to be active, with subtilisin A, ficin, and bromelain showing higher activity than other proteases. However, the latter two proteases caused degradation of chitin molecules. Further investigation revealed that subtilisin A in Bet/G did not exhibit "pH memory", which is a universal characteristic displayed by enzymes dispersed in organic phases, and the catalytic characteristics of subtilisin A in Bet/G differed significantly from those in aqueous phase. The conditions for protein removal from chitin by subtilisin A in Bet/G were determined: Chitin dissolved in Bet/G with 0.5 % subtilisin A (442.0 U/mg, based on the mass of chitin) was hydrolyzed at 45 °C for 30 min. The residual protein content in chitin decreased from 5.75 % ± 0.10 % to 1.01 % ± 0.12 %, improving protein removal by 57.20 % compared with protein removal obtained by Bet/G alone. The crystallinity and deacetylation degrees of chitin remained unchanged after the treatment.

Dynamic microvilli sculpt bristles at nanometric scale.

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.

Exploring the purity of chitin from crustacean sources using deep eutectic solvents: A machine learning approach.

OBJECTIVE: Chitin a natural polymer is abundant in several sources such as shells of crustaceans, mollusks, insects, and fungi. Several possible attempts have been made to recover chitin because of its importance in biomedical applications in various forms such as hydrogel, nanoparticles, nanosheets, nanowires, etc. Among them, deep eutectic solvents have gained much consideration because of their eco-friendly and recyclable nature. However, several factors need to be addressed to obtain a pure form of chitin with a high yield. The development of an innovative system for the production of quality chitin is of prime importance and is still challenging. METHODS: The present study intended to develop a novel and robust approach to investigate chitin purity from various crustacean shell wastes using deep eutectic solvents. This investigation will assist in envisaging the important influencing parameters to obtain a pure form of chitin via a machine learning approach. Different machine learning algorithms have been proposed to model chitin purity by considering the enormous experimental dataset retrieved from previously conducted experiments. Several input variables have been selected to assess chitin purity as the output variable. RESULTS: The statistical criteria of the proposed model have been critically investigated and it was observed that the results indicate XGBoost has the maximum predictive accuracy of 0.95 compared with other selected models. The RMSE and MAE values were also minimal in the XGBoost model. In addition, it revealed better input variables to obtain pure chitin with minimal processing time. CONCLUSION: This study validates that machine learning paves the way for complex problems with substantial datasets and can be an inexpensive and time-saving model for analyzing chitin purity from crustacean shells.

Tunable Oxidized-Chitin Hydrogels with Customizable Mechanical Properties by Metal or Hydrogen Ion Exposure.

This study focuses on the optimization of chitin oxidation in C6 to carboxylic acid and its use to obtain a hydrogel with tunable resistance. After the optimization, water-soluble crystalline ß-chitin fibrils (ß-chitOx) with a degree of functionalization of 10% were obtained. Diverse reaction conditions were also tested for α-chitin, which showed a lower reactivity and a slower reaction kinetic. After that, a set of hydrogels was synthesized from ß-chitOx 1 wt.% at pH 9, inducing the gelation by sonication. These hydrogels were exposed to different environments, such as different amounts of Ca2+, Na+ or Mg2+ solutions, buffered environments such as pH 9, PBS, pH 5, and pH 1, and pure water. These hydrogels were characterized using rheology, XRPD, SEM, and FT-IR. The notable feature of these hydrogels is their ability to be strengthened through cation chelation, being metal cations or hydrogen ions, with a five- to tenfold increase in their storage modulus (G'). The ions were theorized to alter the hydrogen-bonding network of the polymer and intercalate in chitin's crystal structure along the a-axis. On the other hand, the hydrogel dissolved at pH 9 and pure water. These bio-based tunable hydrogels represent an intriguing material suitable for biomedical applications.

Valorization of Hermetia illucens breeding rejects by chitins and chitosans production. Influence of processes and life cycle on their physicochemical characteristics.

Breeding of the black soldier fly is carried out to produce proteins. It is accompanied by releases during the life cycle of this insect. This work is a study of the valorization of these rejects through the production of chitins and chitosans with controlled characteristics. An extraction process is developed with an order of treatments and reaction conditions that provide chitins with high contents. These contents increase as the stages of the life cycle progress and drop for the adult. However, the exuviae chitins present organic impurities which will be eliminated at the N-deacetylation reaction for pupe and after a purification treatment for chitosan from larval stages. All these chitins have an α structure although certain physicochemical characteristics of the larval exuviae chitins are close to those presented by γ chitin. The observed shifts are linked to the effect of impurities rather than to a difference in structure. N-deacetylation of chitins makes possible the valorization of all rejects by the production of pure chitosans with high yields which retain a porous structure for the exuviae and fibrous for the adult which allow complementary applications. These chitosans are highly to completely deacetylated and their molar masses can vary depending on the process and life stage.

Synthesis of poly(ionic liquid-OH) mediated deacetylated chitin and its hydrogels: A study on their applications in controlled release of paracetamol and urea.

Due to the biodegradable and biocompatible nature of chitin and chitosan, they are extensively used in the synthesis of hydrogels for various applications. In this work, deacetylation of chitin is carried out with alkaline poly(dimethyldiallylammonium-hydroxide) that gave a higher amount of water-soluble chitin (with 84 % of the degree of deacetylation = chitosan0.84) compared to deacetylation using NaOH. The water-soluble chitosan0.84 is used as intercalating chains for the preparation of acrylic acid and vinylimidazole-based hydrogels. The quaternization of imidazole groups is done with 1,ω-dibromoalkanes, which sets off the crosslinking in the above polymer network. A set of three chitosan0.84 intercalated hydrogels, namely Cs-C4-hydrogel, Cs-C5-hydrogel, and Cs-C10-hydrogel are prepared bearing butyl, pentyl, and decyl chains as respective crosslinkers. The swell ratios of these intercalated hydrogels are compared with those of non-intercalated hydrogels (C4-hydrogel, C5-hydrogel, and C10-hydrogel). Chitosan0.84 intercalated Cs-C10-hydrogel has excellent swelling properties (2330 % swelling ratio) among six synthesized hydrogels. SEM analysis reveals that decyl crosslinker-bearing hydrogels are highly porous. The multi-functionality of Cs-C10-hydrogel and C10-hydrogel is explored towards -the controlled release of paracetamol/urea, and methyleneblue dye absorption. These studies disclose that chitosan0.84 intercalated hydrogels are showing superior-swelling behavior, high paracetamol/urea loading capacities and better dye entrapment than their non-intercalated counterparts.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Cryomilling-assisted high purity ß-chitin extraction from Uroteuthis edulis pens.

We report on the extraction of ß-chitin from pens (or Gladius) of Uroteuthis edulis, a squid species prevalent in the Pacific coastal regions of East Asia. In particular, we employ cryogenic mechanical grinding (or cryomilling) as a pre-treatment process for the raw squid pens. We show that the cryomilling step enables an effective pulverization of the raw materials, which facilitates the removal of protein residues allowing the extraction of high-purity ß-chitin with a high acetylation degree (∼97 %) and crystallinity (∼82 %). We also demonstrate that the Uroteuthis edulis extract ß-chitin affords a free-standing film with excellent optical transmittance and mechanical properties.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii.

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul-1 h-1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol-1, 100.59 kJ mol-1, 102.64 kJ mol-1 and 5.95 J mol-1 K-1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2-4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2-4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

Alternative processes to produce chitin, chitosan, and their oligomers.

Sustainable recovery of chitin and its derivatives from shellfish waste will be achieved when the industrial production of these polymers is achieved with a high control of their molecular structure, low costs, and acceptable levels of pollution. Therefore, the conventional chemical method for obtaining these biopolymers needs to be replaced or optimized. The goal of the present review is to ascertain what alternative methods are viable for the industrial-scale production of chitin, chitosan, and their oligomers. Therefore, a detailed review of recent literature was undertaken, focusing on the advantages and disadvantages of each method. The analysis of the existing data allows suggesting that combining conventional, biological, and alternative methods is the most efficient strategy to achieve sustainable production, preventing negative impacts and allowing for the recovery of high added-value compounds from shellfish waste. In conclusion, a new process for obtaining chitinous materials is suggested, with the potential of reducing the consumption of reagents, energy, and water by at least 1/10, 1/4, and 1/3 part with respect to the conventional process, respectively.

Advances in the preparation, characterization, and biological functions of chitosan oligosaccharide derivatives: A review.

Chitosan oligosaccharide (COS), which represent the positively charged basic amino oligosaccharide in nature, is the deacetylated and degraded products of chitin. COS has become the focus of intensive scientific investigation, with a growing body of practical and clinical studies highlighting its remarkable health-enhancing benefits. These effects encompass a wide range of properties, including antibacterial, antioxidant, anti-inflammatory, and anti-tumor activities. With the rapid advancements in chemical modification technology for oligosaccharides, many COS derivatives have been synthesized and investigated. These newly developed derivatives possess more stable chemical structures, improved biological activities, and find applications across a broader spectrum of fields. Given the recent interest in the chemical modification of COS, this comprehensive review seeks to consolidate knowledge regarding the preparation methods for COS derivatives, alongside discussions on their structural characterization. Additionally, various biological activities of COS derivatives have been discussed in detail. Lastly, the potential applications of COS derivatives in biomedicine have been reviewed and presented.

A review on chitin dissolution as preparation for electrospinning application.

Electrospinning has been acknowledged as an efficient technique for the fabrication of continuous nanofibers from polymeric based materials such as polyvinyl alcohol (PVA), cellulose acetate (CA), chitin nanocrystals and others. These nanofibers exhibit chemical and mechanical stability, high porosity, functionality, high surface area and one-dimensional orientation which make it extremely beneficial in industrial application. In recent years, research on chitin - a biopolymer derived from crustacean and fungal cell wall - had gained interest due to its unique structural arrangement, excellent physical and chemical properties, in which make it biodegradable, non-toxic and biocompatible. Chitin has been widely utilized in various applications such as wound dressings, drug delivery, tissue engineering, membranes, food packaging and others. However, chitin is insoluble in most solvents due to its highly crystalline structure. An appropriate solvent system is required for dissolving chitin to maximize its application and produce a fine and smooth electrospun nanofiber. This review focuses on the preparation of chitin polymer solution through dissolution process using different types of solvent system for electrospinning process. The effect of processing parameters also discussed by highlighting some representative examples. Finally, the perspectives are presented regarding the current application of electrospun chitin nanofibers in selected fields.

Identification and characterization of chitinase producing marine microorganism: Unleashing the potential of chitooligosaccharides for bioethanol synthesis.

The dwindling supply of the petroleum product and its carbon footprint has initiated search for a sustainable fuel and alternate feed-stocks. One such underexplored feedstock is chitin, a waste derived from sea food processing. The limitation of insolubility and crystallinity inherent in chitin is addressed with the chitin hydrolysates. In the present study, a chitinases producing marine isolate was isolated from the sediments of Arabian Sea from a depth of 20 m. In order to increase the expression of the chitinases, sequential optimisation using one factor at a time and Taguchi experimental designs were employed which resulted in a yield of 13.46 U/mL which was 2.62 fold higher than the initial bioprocess condition values. In a two-step refinery protocol, Candida albicans was evolved towards chitooligosaccharides using chemically synthesized hydrolysates. In a fed -batch fermentation design the Candida yielded a 12.8 % conversion of these commercial chitin oligosaccharides into bioethanol in a run time of 48 h. This is the first report demonstrating the potential of Candida to utilise chitin oligosaccharides for the production of bioethanol.

Chitin nanocrystals/alginate complex for tuning stability, rheology and bioavailability of cholecalciferol in Pickering emulsions.

In this paper we investigate polyelectrolyte complexes of sodium alginate (Alg) and chitin nanocrystals (ChNC). Formation, stability and transport properties of sunflower oil-in-water emulsions stabilized by ChNC-Alg complex were studied using dynamic light scattering (DLS), laser Doppler electrophoresis, optical microscopy, potentiometric titration, rheology and simulated digestion. It has been established that during emulsions formation, the ChNC-Alg complex is rearranged at the interface and the formation of a two-layer coating of the droplet occurs. Stabilized O/W emulsions are stable during storage, in the pH range 2-9 and centrifugal acceleration up to 2000 RCF. Presence of Ca2+ and Na+ ions in the range up to 150 mM has virtually no effect on the droplet size. Inclusion of 5 wt% Alg in the ChNC-based emulsion stabilizer system leads to a drop in Gibbs adsorption >16 times compared to the ChNC-stabilized emulsion, increase in viscosity and rheopexy index of the systems. We found that chemical properties of colloidal phase surface and rheological properties of emulsions stabilized by ChNC-Alg are mostly dependent on the droplet size, not the type of oil as a result of a comparative study of sunflower oil/liquid paraffin oil. Emulsion drops of an optimized composition are stable in the upper parts of the model gastrointestinal tract system and transport vitamin D3 to the small intestine without significant losses. The bioavailability of vitamin D3 in emulsions stabilized with the ChNC-Alg complex is higher than for emulsions stabilized with ChNC alone.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production.

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 µmole min-1 and a Km of 4.6 mg mL-1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg-1 and 625 U mg-1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

Pretreatment of shrimp shells with an acidic deep eutectic solvent system for chitin extraction and its enhanced performance as a carrier for immobilized lipase.

Existing methods for chitin extraction usually produce substantial waste, which poses ecological hazards. Natural deep eutectic solvent (NADES) offers a promising one-step pretreatment alternative, replacing the resource-intensive demineralization (DM) and deproteinization (DP) process. Hence, in this study, the influence of various acidic NADES, on achieving a simplified one-step DM and DP in the chitin extraction process was investigated. The study yielded chitin with 87.73 % purity, and microstructural analysis showed that NADES pretreatment minimally affected chitin quality without deacetylation. In addition, chitin extracted using choline chloride-oxalic acid as a carrier displayed excellent performance in the immobilization of Geobacillus thermocatenulatus lipase 2 (GTL2) because of obvious Ca2+ activation effect. This process contributed to enhancement of immobilized enzyme activity. The immobilized GTL2 showed excellent hydrolytic capabilities, with its highest activity reaching 547.80 ± 20.62 U/mg, significantly better than the five commercial lipases that exhibited <40 % of the enzyme activity. Furthermore, the hydrolytic capacity of immobilized GTL2 was notably high for 4-nitrophenyl butyrate, measuring 935.47 ± 51.60 U/mg. This study provided a constructive approach for the one-step pretreatment of shrimp shells with organic acid-based NADES to isolate and purify chitin and its potential application as an immobilized carrier to enhance enzyme activity.