Insect Chitin Research Articles

Coassembly of a New Insect Cuticular Protein and Chitosan via Liquid-Liquid Phase Separation.

Insect cuticle is a fiber-reinforced composite material that consists of polysaccharide chitin fibers and a protein matrix. The molecular interactions between insect cuticle proteins and chitin that govern the assembly and evolution of cuticles are still not well understood. Herein, we report that Ostrinia furnacalis cuticular protein hypothetical-1 (OfCPH-1), a newly discovered and most abundant cuticular protein from Asian corn borer O. furnacalis, can form coacervates in the presence of chitosan. The OfCPH-1-chitosan coacervate microdroplets are initially liquid-like but become gel-like with increasing time or salt concentration. The liquid-to-gel transition is driven by hydrogen-bonding interactions, during which an induced β-sheet structure of OfCPH-1 is observed. Given the abundance of OfCPH-1 in the cuticle of O. furnacalis, this liquid-liquid phase separation process and its aging behavior could play critical roles in the formation of the cuticle.

Modulation of Atlantic salmon (Salmo salar) gut microbiota composition and predicted metabolic capacity by feeding diets with processed black soldier fly (Hermetia illucens) larvae meals and fractions

BackgroundBlack soldier fly (Hermetia illucens) is a promising insect species to use as a novel ingredient in fish feeds. Black soldier fly larvae consists of three major fractions, namely protein, lipid, and exoskeleton. These fractions contain bioactive compounds that can modulate the gut microbiota in fish such as antimicrobial peptides, lauric acid, and chitin. However, it is not certain how, or which fractions of black solider fly would affect gut microbiota in fish. In the present study, black soldier fly larvae were processed into three different meals (full-fat, defatted and de-chitinized) and two fractions (oil and exoskeleton), and included in diets for Atlantic salmon (Salmo salar). Atlantic salmon pre-smolts were fed with these diets in comparison with a commercial-like control diet for eight weeks to investigate the effects of insect meals and fractions on the composition and predicted metabolic capacity of gut microbiota. The gut microbiota was profiled by 16S rRNA gene sequencing, and the predicted metabolic capacities of gut microbiota were determined using genome-scale metabolic models.ResultsThe inclusion of insect meals and fractions decreased abundance of Proteobacteria and increased abundance of Firmicutes in salmon gut. The diets that contained insect chitin, i.e., insect meals or exoskeleton diets, increased abundance of chitinolytic bacteria including lactic acid bacteria and Actinomyces in salmon gut, with fish fed full-fat meal diet showing the highest abundances. The diets that contained insect lipids, i.e., insect meals and oil diets enriched Bacillaceae in fish gut. The fish fed diets containing full-fat insect meal had a unique gut microbiota composition dominated by beneficial lactic acid bacteria and Actinomyces, and showed a predicted increase in mucin degradation compared to the other diets.ConclusionsThe present results showed that the dietary inclusion of insect meals and fractions can differently modulate the composition and predicted metabolic capacity of gut microbiota in Atlantic salmon pre-smolts. The use of full-fat black soldier fly larvae meal in diets for salmon is more favorable for beneficial modulation of gut microbiota than larvae processed by separation of lipid or exoskeleton fractions.

Molecular cloning and functional characterization of two insect chitin deacetylases from the American white moth, Hyphantria cunea

Molecular cloning and functional characterization of two insect chitin deacetylases from the American white moth, Hyphantria cunea

Chitin Determination in Residual Streams Derived From Insect Production by LC-ECD and LC-MS/MS Methods

Chitin, a biopolymer present in fungi and arthropods, is a compound of interest for various applications, such as in the agricultural and medical fields. With the recently growing interest in the development of insect farming, the availability of chitin-containing residual streams, particularly the molting skins (exuviae), is expected to increase in the near future. For application purposes, accurate quantification of chitin in these insect sources is essential. Previous studies on chitin extraction and quantification often overlooked the purity of the extracted chitin, making the outcomes inconsistent and prone to overestimation. The present study aims to determine chitin content in the exuviae of three insect species mass-reared worldwide: black soldier fly (BSF), mealworm, and house cricket. Chitin was chemically extracted using acid and alkali treatments to remove minerals and proteins. The purity of extracted chitin was evaluated by hydrolyzing the chitin into glucosamine, followed by quantitative determination of the latter using two liquid chromatography methods: electrochemical detection (ECD) and tandem mass spectrometry (MS/MS). Both methods proved accurate and precise, without the need for labor-intensive derivatization steps. Pearson's correlation and Bland-Altman plots showed that the glucosamine determination results obtained by the two methods were comparable, and there is no consistent bias of one approach vs. the other. The chitin content in extracted residues ranged between 7.9 and 18.5%, with the highest amount found in BSF puparium. In summary, the study demonstrated that (1) the residual streams of the insect farming industry have a great potential for utilization as an alternative chitin source, and (2) both LC-ECD and LC-MS/MS are reliable for the quantitative determination of glucosamine in insect chitin.

Deciphering the ChitoCode: fungal chitins and chitosans as functional biopolymers

Chitins and chitosans are among the most widespread and versatile functional biopolymers, with interesting biological activities and superior material properties. While chitins are evolutionary ancient and present in many eukaryotes except for higher plants and mammals, the natural distribution of chitosans, i.e. extensively deacetylated derivatives of chitin, is more limited. Unequivocal evidence for its presence is only available for fungi where chitosans are produced from chitin by the action of chitin deacetylases. However, neither the structural details such as fraction and pattern of acetylation nor the physiological roles of natural chitosans are known at present. We hypothesise that the chitin deacetylases are generating chitins and chitosans with specific acetylation patterns and that these provide information for the interaction with specific chitin- and chitosan-binding proteins. These may be structural proteins involved in the assembly of the complex chitin- and chitosan-containing matrices such as fungal cell walls and insect cuticles, chitin- and chitosan-modifying and -degrading enzymes such as chitin deacetylases, chitinases, and chitosanases, but also chitin- and chitosan-recognising receptors of the innate immune systems of plants, animals, and humans. The acetylation pattern, thus, may constitute a kind of ‘ChitoCode’, and we are convinced that new in silico, in vitro, and in situ analytical tools as well as new synthetic methods of enzyme biotechnology and organic synthesis are currently offering an unprecedented opportunity to decipher this code. We anticipate a deeper understanding of the biology of chitin- and chitosan-containing matrices, including their synthesis, assembly, mineralisation, degradation, and perception. This in turn will improve chitin and chitosan biotechnology and the development of reliable chitin- and chitosan-based products and applications, e.g. in medicine and agriculture, food and feed sciences, as well as cosmetics and material sciences.

Discovery of Biphenyl-Sulfonamides as Novel β-N-Acetyl-d-Hexosaminidase Inhibitors via Structure-Based Virtual Screening.

Novel insecticidal targets are always in demand due to the development of resistance. OfHex1, a β-N-acetyl-d-hexosaminidase identified in Ostrinia furnacalis (Asian corn borer), is involved in insect chitin catabolism and has proven an ideal target for insecticide development. In this study, structure-based virtual screening, structure simplification, and biological evaluation are used to show that compounds with a biphenyl-sulfonamide skeleton have great potential as OfHex1 inhibitors. Specifically, compounds 10k, 10u, and 10v have Ki values of 4.30, 3.72, and 4.56 μM, respectively, and thus, they are more potent than some reported nonglycosyl-based inhibitors such as phlegmacin B1 (Ki = 26 μM), berberine (Ki = 12 μM), 2 (Ki = 11.2 μM), and 3 (Ki = 28.9 μM). Furthermore, inhibitory kinetic assessments reveal that the target compounds are competitive inhibitors with respect substrate, and based on toxicity predictions, most of them have potent drug properties. The obtained results indicate that the biphenyl-sulfonamide skeleton characterized by simple chemical structure, synthetic tractability, potent activity, and low toxicity has potential for further development in pest management targeting OfHex1.

In silico characterization of chitin deacetylase genes in the Diaphorina citri genome.

Chitin deacetylases (CDAs) are one of the least understood components of insect chitin metabolism. The partial deacetylation of chitin polymers appears to be important for the proper formation of higher order chitin structures, such as long fibers and bundles, which contribute to the integrity of the insect exoskeleton and other structures. Some CDAs may also be involved in bacterial defense. Here, we report the manual annotation of four CDA genes from the Asian citrus psyllid, Diaphorina citri, laying the groundwork for future study of these genes.

Insect Chitin-Based Nanomaterials for Innovative Cosmetics and Cosmeceuticals

Chitin and its derivatives are attracting great interest in cosmetic and cosmeceutical fields, thanks to their antioxidant and antimicrobial properties, as well as their biocompatibility and biodegradability. The classical source of chitin, crustacean waste, is no longer sustainable and fungi, a possible alternative, have not been exploited at an industrial scale yet. On the contrary, the breeding of bioconverting insects, especially of the Diptera Hermetia illucens, is becoming increasingly popular worldwide. Therefore, their exoskeletons, consisting of chitin as a major component, represent a waste stream of facilities that could be exploited for many applications. Insect chitin, indeed, suggests its application in the same fields as the crustacean biopolymer, because of its comparable commercial characteristics. This review reports several cosmetic and cosmeceutical applications based on chitin and its derivatives. In this context, chitin nanofibers and nanofibrils, produced from crustacean waste, have proved to be excellent cosmeceutical active compounds and carriers of active ingredients in personal care. Consequently, the insect-based chitin, its derivatives and their complexes with hyaluronic acid and lignin, as well as with other chitin-derived compounds, may be considered a new appropriate potential polymer to be used in cosmetic and cosmeceutical fields.

Comparison of Sustainable Lipid and Protein Removal Methods for the Isolation of Insect Chitin from Black Soldier Fly Exoskeleton

Black soldier fly (Hermetia illucens), an insect known for feeding on waste biomass and converting it into useful nutrients such as proteins, lipids, and chitin, has been reared on insect farms on a large scale. In this study, chitin was isolated from the exoskeleton of the black soldier fly using different extraction protocols, such as chemical solvents, enzyme-assisted fractionation, and supercritical carbon dioxide extraction. The levels of effectiveness of the recovery of the chitin fraction using different extraction methods were then evaluated by determining the physicochemical properties of the extracted insect chitin using Fourier transform infrared spectroscopy, elemental analysis, and X-ray diffraction. The results show that the use of eco-friendly acids and solvents such as lactic acid, acetic acid, and ethanol as well as bacterial proteases holds promise for the defatting, demineralization, and deproteinization of the exoskeleton to yield good quality chitin, albeit with impurities.

Molecular cloning and functional characterization of two insect chitin deacetylases from the American white moth, Hyphantriacunea

AbstractChitin deacetylases (CDAs) convert chitin into chitosan and play crucial roles in the insect biological processes, including growth and development. In this study, two CDA‐encoding genes, HcCDA1 and HcCDA4, were identified from Hyphantria cunea. Conserved Domain Database for HcCDA1 identified a chitin‐binding peritrophin‐A domain (ChBD), a low‐density lipoprotein receptor class A domain (LDLa), and a putative CDA‐like catalytic domain (CDA), whereas HcCDA4 lacked the LDLa domain. Phylogenetic comparison with other insect CDAs revealed that HcCDA1 belonged to the insect group I CDA and HcCDA4 belonged to group III. qRT‐PCR results showed that HcCDA1 and HcCDA4 were more highly expressed in newly moulted 5th‐instar larvae, and HcCDA1 was highly expressed in the integument, whereas HcCDA4 was abundantly transcribed in the integument and hindgut. Western blot revealed that the expression of HcCDA1 and HcCDA4 in translational levels were consistent with the transcription. Double‐stranded RNA injection of HcCDA1 and HcCDA4 into H. cunea larvae significantly reduced HcCDA transcript levels, resulted in larval–pupal moulting difficulty, and produced high larval mortality. These results demonstrate important contributions of HcCDA1 and HcCDA4 to insect moulting and development and provide potential new targets for designing effective double‐stranded RNA‐based pesticides to protect crops.

An overall look at insect chitin deacetylases: Promising molecular targets for developing green pesticides.

Chitin deacetylase (CDA) is a key enzyme involved in the modification of chitin and plays critical roles in molting and pupation, which catalyzes the removal of acetyl groups from N-acetyl-D-glucosamine residues in chitin to form chitosan and release acetic acid. Defects in the CDA genes or their expression may lead to stunted insect development and even death. Therefore, CDA can be used as a potential pest control target. However, there are no effective pesticides known to target CDA. Although there has been some exciting research progress on bacterial or fungal CDAs, insect CDA characteristics are less understood. This review summarizes the current understanding of insect CDAs, especially very recent advances in our understanding of crystal structures and the catalytic mechanism. Progress in developing small-molecule CDA inhibitors is also summarized. We hope the information included in this review will help facilitate new pesticide development through a novel action mode, such as targeting CDA.

Nutritional value of insects and ways to manipulate their composition

This article reports on the nutrients present in insects and factors affecting their variability. Data on protein content and amino acid profiles of a variety of insect species are discussed and their amino acid profiles compared to nutrient requirements of growing broiler chicks, catfish, trout, swine, and human adults and young children. Bothin vitroandin vivoprotein digestibility data for a variety of insect species is presented and factors affecting these data are discussed. Furthermore, the fat content and fatty acid profiles of a variety of insect species is reviewed, with special attention on omega-6 and omega-3 fatty acids. Information on carbohydrates, fibre and chitin in insects is shown along with potential effects on nutrient availability. This is followed by a discussion of essential minerals in insects with an emphasis on calcium and phosphorus. Data on insect vitamin content is shown along with a discussion of antinutritional factors such as phytate and thiaminase, which can adversely affect their nutritional value. Dietary effects on insect nutrient composition are reviewed with an emphasis on essential minerals, heavy metals, vitamin E, and carotenoids. Lastly, the effects of processing, including protein extraction and various cooking methods on insect composition are discussed. In summary, this article provides an overview of the nutrient content of insects, and how select nutrients can be altered.

Exploring Simplified Methods for Insect Chitin Extraction and Application as a Potential Alternative Bioethanol Resource.

Simple SummaryThe studies on chitin utilization as a source for bioethanol production are still very few. The present study explores some simple methods for insect chitin extraction and application in bioethanol production. Using insect chitin in bioethanol production, may help decreasing the dependence on energy crops as a carbon source for bioethanol. Fungal strains of Mucor circinelloides were reported previously to bio-convert chitin directly to ethanol in submerged fermentation systems. In our study, we explored the bioconversion of insect chitin to bioethanol using two different strains of Mucor circinelloides in submerged fermentation systems. An insect-isolated M. circinelloides strain was found to bio-convert the extracted chitin directly to ethanol in submerged fermentation system. The source of strain isolation and the pH of the production medium were showed to influence the chitin bioconversion directly to bioethanol. All fermentation processes can be conducted easily, using the whole growing microorganism instead of using purified enzymes. These results highlight the insect biomass as a potential new, cheap and renewable source for bioethanol production simply, using a potent insect-isolated M. circinelloides strain.Chitin, the second most plentiful biopolymer in nature, is a major component of insect cuticle. In searching for alternative resources for fossil fuels, some fungal strains of Mucor circinelloides from an insect-source were found to produce bioethanol directly using insect chitin as a substrate. Herein, simplified methods for insect chitin extraction and application as a substrate in submerged fermentation for bioethanol production were explored. Chitin of the American cockroach (Periplaneta americana (L.)) was isolated by refluxing the cockroaches dried exoskeletons with 4% NaOH. The purity of the extracted chitin was assessed to be high when the physicochemical properties of the extracted chitin matched these of commercially available crab and shrimp samples. The extracted chitin was employed as a substrate in submerged fermentation using two strains of M. circinelloides. One of these, strains M. circinelloides 6017 showed immense potential for bioethanol production directly. It could to bio-transform 15 g/L of colloidal chitin directly to 11.22 ± 0.312 g/L of bioethanol (74% of the initial chitin mass) after 6 days of incubation. These results confirm the possibility of using insect biomass as a potential alternative resource for bioethanol production in a simple manner thus contributing to the creation of an alternate energy source.

Identification of azadirachtin responsive genes in Spodoptera frugiperda larvae based on RNA-seq

The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a polyphagous pest with 353 plant species as its hosts, including maize, sorghum, cotton, and rice. Azadirachtin is one of the most effective botanical insecticides. The effect of azadirachtin against S. frugiperda remains to be determined. Here we report strong growth inhibition of azadirachtin on S. frugiperda larvae under either 1.0 or 5.0 μg/g azadirachtin. To explore the relevant mechanisms, the larvae fed with normal artificial diet and with 1.0 μg/g azadirachtin exposure for 3 days were collected as samples for RNA-Seq. RNA-Seq on S. frugiperda larvae under different treatments identified a total of 24,153 unigenes, including 3494 novel genes, were identified. Among them, 1282 genes were affected by 1.0 μg/g azadirachtin exposure, with 672 up-regulated and 610 down-regulated. The impacted genes include 61 coding for detoxification enzymes (31 P450s, 7 GSTs, 11 CarEs, 7 UGTs and 5 ABC transporters), 31 for cuticle proteins, and several proteins involved in insect chitin and hormone biosynthesis. Our results indicated that azadirachtin could regulate the growth of S. frugiperda by affecting insect chitin and hormone biosynthesis pathway. The enhanced expression of detoxification enzymes might be related to detoxifying azadirachtin. These findings provided a foundation for further delineating the molecular mechanism of growth regulation induced by azadirachtin in S. frugiperda larvae.

Development of ultra-high performance liquid chromatographic and fluorescent method for the analysis of insect chitin

A precise quantification of insect chitin is needed in order to avoid overestimation of crude protein due to chitin-bound nitrogen. An UPLC/FLR method was optimized and validated for the determination of glucosamine (GlcN) hydrolyzed from chitin in insect materials. The method was applied for quantifying the chitin content in mealworms (Tenebrio molitor) and crickets (Acheta domesticus). A baseline separation was obtained using an Acquity HSS T3 C18 column, with an external calibration curve of excellent linearity, and a low limit of detection and quantification of GlcN. Even though the recovery of GlcN from spiked cricket material was slightly lower compared to that using spectrophotometric method, the UPLC/FLR method proved a sensitive and specific method of quantification of insect chitin. Chitin contents in T. molitor and A. domesticus were 4.6 ± 0.1% and 4.5 ± 0.0% on dry matter basis, respectively. Less than 0.01% of chitin was present in insect protein-enriched fractions extracted with 0.1 N NaCl at pH 10.

Enhanced virulence of Beauveria bassiana against Diatraea saccharalis using a soluble recombinant enzyme with endo- and exochitinase activity

Beauveria bassiana chitinases are involved in degrading the chitin in insects’ exoskeletons and internal structures, and thus are important virulence factors as they participate in initial to final steps of infection. In this work, the B. bassiana (Bv062 isolate) open reading frame (ORF) encoding a chitinase identified as Chit37 (orthologous Bvchit1) was molecularly cloned and expressed in Escherichia coli, and the potential of the recombinant protein (rChit37) to enhance the insecticidal activity of Bv062 conidia against second instar Diatraea saccharalis larvae was studied. rChit37 was produced in both soluble and insoluble fractions of an E. coli culture. Both fractions expressing endo- (90 mU/µL) and exochitinase (170 mU/µL) enzymatic activity, with optimum conditions for enzyme activity of 45 °C and pH 5.0. His-tag affinity chromatography was used to purify the rChit37 from the soluble fraction. Purified rChit37 was then diluted to 200 and 300 µg/mL for use as Bv062 conidia additive (1x106con/mL) in a laboratory bioassay against D. saccharalis larvae. No significant differences were observed between the efficacy of Bv062 conidia applied alone or mixed with 200 µg/mL purified rChit37. However, 300 µg/mL rChit37 increased BV062 conidia insecticidal activity, achieving 96.7% efficacy (14 days post-infection) and 6.2 days median lethal time (LT50), compared to 60% efficacy and 8.9 days for conidia alone. rChit37 addition did not affect conidial viability in terms of germination (96.6% after 24 h) or vigour estimated as germ-tube elongation rate. This work provides proof of concept about soluble recombinant chitinase as an additive to enhance B. bassiana virulence against D. saccharalis.

Electron Paramagnetic Resonance Imaging of Melanin in Honey Bee

Honey bees play a crucial role in the nature by pollinating wild flowers. Over the past years, there has been an increasing concern regarding the honey bee colony decline. Pesticides or environmental effects targeting the biochemistry of insect chitin and cuticle coating may be in part responsible for honey bee pathologies. We here propose the use of electron paramagnetic resonance imaging (EPRI) as a tool to image the melanin-chitin complexes as part of the exoskeleton of the honey bee. EPRI at 9.65 GHz was applied on intact freeze-dried bees. The imaging data were collected on the melanin peak. High-resolution images revealed that this compound is extensively distributed in the periphery of the animal, data consistent with the localization in the cuticle of the bee. While EPR of melanin has been so far explored in the context of melanoma characterization, it may offer new opportunities in research on honey bees and other insects.

Physicochemical Structure Analysis of Chitin Extracted from Pupa Exuviae and Dead Imago of Wild Black Soldier Fly (Hermetia illucens)

Chitin is the second most widely available natural fiber with diverse applications. Insect chitin is gaining popularity over the last decade. Black soldier fly is a widely known insect with waste management potential. In this study, chitin was isolated from Black soldier fly (Hermetia illucens) pupae exuvia (BSFE) and imago (BSFI). The chitin content was found to be 9% and 23% for pupae exuviae and imago respectively. Both the chitins were α-chitin. The degree of acetylation (DA) confirms that BSFE chitin has higher purity than BSFI chitin. The BSFE chitin is more amorphous than BSFI chitin. The crystallinity index (CrI) for BSFE and BSFI chitin was 25.20% and 49.4%, respectively. Both the chitins had good thermal stability with a maximum degradation (DTGMax) of BSFI and BSFE chitin at 363 °C and 371 °C, respectively. The Brunauer–Emmett–Teller (BET) study showed that chitin from BSFI was mesoporous with well defined cylindrical pore channels while the chitin from BSFE was non-porous. The surface area of BSFE and BSFI chitin was 1.63 and 23.00 m2/g respectively. Both the chitin had a smooth microfibrillar structure with repeating units. Based on the physicochemical characteristics of the BSF-derived chitin it can find promising commercial applications in tissue engineering, textile industry and as an adsorbent in water and wastewater treatment.

High-Strength Nanostructured Films Based on Well-Preserved α-Chitin Nanofibrils Disintegrated from Insect Cuticles.

The α-chitin nanofibril is an alternative to nanocellulose as a building-block for strong films and other nanomaterials. The hypothesis of high film strength for films based on mildly treated insect cuticles was tested. Fibrils from the cuticle of Ruspolia differens (a long-horned bush cricket grasshopper locally known as senene) are disintegrated by a mild process, subsequently characterized by transmission electron microscopy, NMR, Fourier transform infrared spectroscopy, and XRD, and used to prepare strong and transparent films. A mild process (with 20% NaOH treatment for 2 weeks and at room temperature) was used to largely remove the strongly bound protein associated with chitin. The purpose was to reduce chitin degradation. The native structure of chitin was indeed well preserved and close to the native state, as is supported by data for degree of acetylation, molar mass, crystallinity, and crystallite dimensions. The diameter of the smallest chitin fibrils was as small as 3-7 nm (average 6 nm) with lengths larger than or around 1 μm. A stable and well-dispersed colloidal chitin fibril suspension in water was achieved. A nanostructured chitin film prepared by filtration showed high optical transmittance (∼90%) and very high tensile strength (220 MPa). The high tensile strength was attributed to the well-preserved chitin structure, high intrinsic fibril strength, and high colloidal stability of the fibril suspension. Strong, transparent insect chitin films offer interesting alternatives to nanocellulose films because of different resource origins, surface chemistries, and potential antimicrobial properties.

Sustainable ecofriendly phytoextract mediated one pot green recovery of chitosan

Chitin and chitosan are biopolymers that have diverse applications in medicine, agriculture, food, cosmetics, pharmaceuticals, wastewater treatment and textiles. With bio-origins, they easily blend with biological systems and show exemplified compatibility which is mandatory when it comes to biomedical research. Chitin and chitosan are ecofriendly, however the processes that are used to recover them aren’t ecofriendly. The focus of this work is to attempt an ecofriendly, sustainable phytomediated one pot recovery of chitosan from commercial chitin and from crab and shrimp shells and squid pen solid wastes. Graviola extracts have been employed, given the fact file that their active ingredients acetogenins actively interact with chitin in insects (resulting in its application as an insecticide). With that as the core idea, the graviola extracts were chosen for orchestrating chitin recovery and a possible chitin to chitosan transformation. The results confirm that graviola extracts did succeed in recovery of chitosan nanofibers from commercial chitin flakes and recovery of chitosan particles directly from solid marine wastes of crab, shrimp and squids. This is a first time report of a phyto-extract mediated novel chitosan synthesis method.