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.

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.

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.

The Preparation and Physiochemical Characterization of Tenebrio molitor Chitin Using Alcalase.

Chitin is mostly produced from crustaceans, but it is difficult to supply raw materials due to marine pollution, and the commonly used chemical chitin extraction method is not environmentally friendly. Therefore, this study aims to establish a chitin extraction process using enzymes and to develop edible insect-derived chitin as an eco-friendly new material. The response surface methodology (RSM) was used to determine the optimal conditions for enzymatic hydrolysis. The optimal conditions for enzymatic hydrolysis by RSM were determined to be the substrate concentration (7.5%), enzyme concentration (80 µL/g), and reaction time (24 h). The solubility and DDA of the mealworm chitosan were 45% and 37%, respectively, and those of the commercial chitosan were 61% and 57%, respectively. In regard to the thermodynamic properties, the exothermic peak of mealworm chitin was similar to that of commercial chitin. In the FT-IR spectrum, a band was observed in mealworm chitin corresponding to the C=O of the NHCOCH3 group at 1645 cm-1, but this band showed low-intensity C=O in the mealworm chitosan due to deacetylation. Collectively, mealworm chitosan shows almost similar physical and chemical properties to commercial chitosan. Therefore, it is shown that an eco-friendly process can be introduced into chitosan production by using enzyme-extracted mealworms for chitin/chitosan production.

Extraction of chitin from edible crab shells of Callinectes sapidus and comparison with market purchased chitin / Extração de quitina de cascas de caranguejo comestíveis de Callinectes sapidus e comparação com quitina adquirida no mercado

Chitin and its derived products have immense economic value due to their vital role in various biological activities as well as biomedical and industrial application. Insects, microorganism and crustaceans are the main supply of chitin but the crustaceans shell like shrimp, krill, lobsters and crabs are the main commercial sources. Chitin content of an individual varies depending on the structures possessing the polymer and the species. In this study edible crabs’ shells (Callinectes sapidus) were demineralized and deproteinized resulting in 13.8% (dry weight) chitin recovery from chitin wastes. FTIR and XRD analyses of the experimental crude as well as purified chitins revealed that both were much comparable to the commercially purchased controls. The acid pretreatment ceded 54g of colloidal chitin that resulted in 1080% of the crude chitin. The colloidal chitin was exploited for isolation of eighty five chitinolytic bacterial isolates from different sources. Zone of clearance was displayed by the thirty five isolates (41.17%) succeeding their growth at pH 7 on colloidal chitin agar medium. Maximum chitinolytic activity i.e. 301.55 U/ml was exhibited by isolate JF70 when cultivated in extracted chitin containing both carbon and nitrogen. The study showed wastes of blue crabs can be utilized for extraction of chitin and isolation of chitinolytic bacteria that can be used to degrade chitin waste, resolve environmental pollution as well as industrial purpose.

A quitina e seus produtos derivados têm imenso valor econômico devido ao seu papel vital em várias atividades biológicas, bem como em aplicações biomédicas e industriais. Insetos, microrganismos e crustáceos são o principal suprimento de quitina, mas a casca dos crustáceos como camarão, krill, lagosta e caranguejo são as principais fontes comerciais. O conteúdo de quitina de um indivíduo varia dependendo das estruturas que possuem o polímero e da espécie. Neste estudo, as cascas de caranguejos comestíveis (Callinectes sapidus) foram desmineralizadas e desproteinizadas, resultando em 13,8% (peso seco) de recuperação de quitina a partir de resíduos de quitina. As análises de FTIR e XRD do bruto experimental, bem como das quitinas purificadas, revelaram que ambas eram muito comparáveis aos controles adquiridos comercialmente. O pré-tratamento com ácido cedeu 54 g de quitina coloidal que resultou em 1.080% da quitina bruta. A quitina coloidal foi analisada para isolamento de 85 isolados bacterianos quitinolíticos de diferentes fontes. A zona de eliminação foi exibida pelos 35 isolados (41,17%) que sucederam seu crescimento a pH 7 em meio de ágar de quitina coloidal. A atividade quitinolítica máxima, ou seja, 301,55 U / ml, foi exibida pelo isolado JF70 quando cultivado em quitina extraída contendo carbono e nitrogênio. O estudo mostrou que resíduos de caranguejos azuis podem ser utilizados para extração de quitina e isolamento de bactérias quitinolíticas que podem ser usadas para degradar resíduos de quitina, resolver a poluição ambiental e também para fins industriais.

The impact of the different types of acid solution on the extraction and adsorption performance of chitin from shrimp shell waste.

The properties of chitin-based adsorbents varied among studies since they are influenced by different factors, such as the types of base and acid used to extract the chitin. Therefore, this works aimed to investigate the impact of four different acid solutions on the extraction and properties of chitin from shrimp shell waste, and to evaluate the adsorption performance of the obtained chitin on removing dye from an aqueous solution. The result showed that H2SO4, HCl, and HNO3 could remove high minerals from the shrimp shell, while the effect of CH3COOH was inferior. The Fourier Transform Infrared (FTIR) and X-ray diffraction (XRD) indicated that the extracted chitin was α-amorphous structure, regardless of the type of acid solution. However, the type of acid solution influenced the crystallinity index of the extracted chitin. The Scanning Electron Microscope (SEM) showed both fibrillar material and porous structures. In addition, the chitin extracted through demineralization using H2SO4 was more effective in removing RBBR dye from aqueous solution, followed by HCl, HNO3, and the last, CH3COOH treatment. The performances of chitin-based adsorbent could be attributed to the strength of acid solution used to remove mineral during the extraction process and the obtained pore structures.

A Comparative Analysis of Conventional and Deep Eutectic Solvent (DES)-Mediated Strategies for the Extraction of Chitin from Marine Crustacean Shells.

Chitin, the second most abundant biopolymer on earth, is utilised in a wide range of applications including wastewater treatment, drug delivery, wound healing, tissue engineering, and stem cell technology among others. This review compares the most prevalent strategies for the extraction of chitin from crustacean sources including chemical methods that involve the use of harsh solvents and emerging methods using deep eutectic solvents (DES). In recent years, a significant amount of research has been carried out to identify and develop environmentally friendly processes which might facilitate the replacement of problematic chemicals utilised in conventional chemical extraction strategies with DES. This article provides an overview of different experimental parameters used in the DES-mediated extraction of chitin while also comparing the purity and yields of associated extracts with conventional methods. As part of this review, we compare the relative proportions of chitin and extraneous materials in different marine crustaceans. We show the importance of the species of crustacean shell in relation to chitin purity and discuss the significance of varying process parameters associated with different extraction strategies. The review also describes some recent applications associated with chitin. Following on from this review, we suggest recommendations for further investigation into chitin extraction, especially for experimental research pertaining to the enhancement of the "environmentally friendly" nature of the process. It is hoped that this article will provide researchers with a platform to better understand the benefits and limitations of DES-mediated extractions thereby further promoting knowledge in this area.

Eco-friendly isolation and characterization of nanochitin from different origins by microwave irradiation: Optimization using response surface methodology.

The extraction of nanochitin from marine waste has attracted great industrial interest due to its unique properties, namely biodegradability, biocompatibility and as a functional reinforcing agent. Conventional acid hydrolysis isolation of nanochitin requires high temperatures and acid concentration, time and energy. Herein, for the first time, microwave irradiation method was used as an eco-friendly approach to isolate nanochitin from different sources. The isolation conditions were optimized through an experimental Box-Behnken design using surface response methodology. The data showed optimal conditions of 1 M HCl, 10.00 min and 124.75 W to obtain lobster nanocrystals; 1 M HCl, 14.34 min and 50.21 W to obtain shrimp nanocrystals; and 1 M HCl, 29.08 min and 54.08 W to obtain squid pen nanofibres, reducing time and HCl concentration. The obtained isolation yields where of 85.30, 79.92 and 80.59 % for lobster, shrimp and squid, respectively. The morphology of the nanochitins was dependent of the chitin origin, and the lengths of the nanochitins were of 314.74, 386.12 and > 900 nm for lobster, shrimp and squid pen, respectively. The thermal stability of the ensuing nanochitins was maintained after treatment. The results showed that nanochitin could be obtained by using an eco-friendly approach like microwave irradiation.

Valorization of food waste to produce intelligent nanofibrous ß-chitin films.

The efficient use of waste from food processing industry is one of the innovative approaches within sustainable development, because it can be transferred into added value products, which could improve economic, energetic and environmental sectors. In this context, the squid pen waste from seafood industry was used as raw material to obtain nanofibrous ß-chitin films. In order to extend functionality of obtained films, elderberry extract obtained from biomass was added at different concentrations. The tensile strength of chitin-elderberry extract films was improved by 52%, elongation at break by 153% and water vapor barrier by 65%. The obtained material showed distinct color change when subjected to acidic or basic solutions. It was proven by CIELab color analysis that all color changes could be easily perceived visually. In addition, the obtained nanofibrous film was successfully used to monitor the freshness of Hake fish. Namely, when the film was introduced in a package that contained fresh fish, its color was efficiently changed within the time during the storage at 4 °C. The obtained results demonstrated that food processing waste could be efficiently valorized, and could give sustainable food package design as a spoilage indicator of high protein food.

Utilization of Marine Waste to Obtain ß-Chitin Nanofibers and Films from Giant Humboldt Squid Dosidicus gigas.

ß-chitin was isolated from marine waste, giant Humboldt squid Dosidicus gigas, and further converted to nanofibers by use of a collider machine under acidic conditions (pH 3). The FTIR, TGA, and NMR analysis confirmed the efficient extraction of ß-chitin. The SEM, TEM, and XRD characterization results verified that ß-chitin crystalline structure were maintained after mechanical treatment. The mean particle size of ß-chitin nanofibers was in the range between 10 and 15 nm, according to the TEM analysis. In addition, the ß-chitin nanofibers were converted into films by the simple solvent-casting and drying process at 60 °C. The obtained films had high lightness, which was evidenced by the CIELAB color test. Moreover, the films showed the medium swelling degree (250-290%) in aqueous solutions of different pH and good mechanical resistance in the range between 4 and 17 MPa, depending on film thickness. The results obtained in this work show that marine waste can be efficiently converted to biomaterial by use of mild extractive conditions and simple mechanical treatment, offering great potential for the future development of sustainable multifunctional materials for various industrial applications such as food packaging, agriculture, and/or wound dressing.

Chemical Proprieties of Biopolymers (Chitin/Chitosan) and Their Synergic Effects with Endophytic Bacillus Species: Unlimited Applications in Agriculture.

Over the past decade, reckless usage of synthetic pesticides and fertilizers in agriculture has made the environment and human health progressively vulnerable. This setting leads to the pursuit of other environmentally friendly interventions. Amongst the suggested solutions, the use of chitin and chitosan came about, whether alone or in combination with endophytic bacterial strains. In the framework of this research, we reported an assortment of studies on the physico-chemical properties and potential applications in the agricultural field of two biopolymers extracted from shrimp shells (chitin and chitosan), in addition to their uses as biofertilizers and biostimulators in combination with bacterial strains of the genus Bacillus sp. (having biochemical and enzymatic properties).

Establishment of successive co-fermentation by Bacillus subtilis and Acetobacter pasteurianus for extracting chitin from shrimp shells.

To simplify the process of chitin bio-extraction from shrimp shells powder (SSP), successive co-fermentation using Bacillus subtilis and Acetobacter pasteurianus was explored in this work. Among three protease-producer (B. licheniformis, B. subtilis, and B. cereus), only B. subtilis exhibited high compatibility with A. pasteurianus in co-culture. Successive co-fermentation was constructed as follows: deproteinization was performed for 3 d by culturing B. subtilis in the medium containing 50 g·L-1 SSP, 50 g·L-1 glucose, and 1 g·L-1 yeast extracts; After feeding 5 g·L-1 KH2PO4 and 6 % (v/v) ethanol, A. pasteurianus was cultured for another 2 d without replacing and re-sterilizing medium. Through 5 d of fermentation, the final deproteinization, demineralization efficiency, and chitin yield reached 94.5 %, 92.0 %, and 18.0 %, respectively. This purified chitin had lower molecular weight (12.8 kDa) and higher deacetylation degree (19.6 %) compared with commercial chitin (18.5 kDa, 6.7 %), and showed excellent structural characterization of FESEM and FT-IR analysis.

Extraction and characterization of fungal chitin nanofibers from Mucor indicus cultured in optimized medium conditions.

Chitin nanofibers (ChNFs) were extracted from Mucor indicus by a purification method followed by a mechanical treatment, cultivated under obtained optimum culture medium conditions to improve fungal chitin production rate. A semi synthetic media containing 50 g/l glucose was used for cultivation of the fungus in shake flasks. The cell wall analysis showed that N-acetyl glucosamine (GlcNAc) content, which is an indication of chitin content, was maximum in presence of 2.5 g/l H3PO4, 2.5 g/l of NaOH, 1 g/l of yeast extract, 1 mg/l of plant hormones, and 1 ml/l of trace metals. The chemical characterizations demonstrated that the isolated fibers had a degree of deacetylation lower than of 10%, and were phosphate free. The FTIR results revealed successful removal of different materials during the purification step. The FE-SEM of fibrillated chitin illustrated an average diameter of 28 nm. Finally, X-ray diffraction results showed that the crystallinity index of nanofibers was 82%.

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.

Preparation and characterization of various chitin-glucan complexes derived from white button mushroom using a deep eutectic solvent-based ecofriendly method.

Deep eutectic solvents (DESs) have gained great interests as ecofriendly and safe solvents in diverse areas. Herein, various chitin-glucan complexes (CGCs) were prepared from white button mushroom (Agaricus bisporus) using DESs. Ultrasonication of mushroom in five DESs yielded two types of CGCs from each DES, one from the DES-insoluble residue (DES_P) and another from the DES-soluble extract (DES_S). The ten resulting CGCs with varying chitin-to-ß-glucan ratios were compared with alkali-insoluble matter (AIM), chemically prepared using NaOH. BU_S and BU_P, prepared using BU comprising betaine and urea, were obtained in the highest yields with reasonably low protein and mineral contents. Despite different acetylation degrees (77.3% and 57.3%, respectively), BU_S and BU_P both degraded at 318 °C and showed remarkably low crystallinity (32.0% and 37.0% for BU_S and BU_P, respectively) compared to AIM, commercial chitin, and the reported CGCs. The surface of BU_S and BU_P was very porous and rough compared with AIM as a result of reduced H-bonds and lowered crystallinity. The DES-based method can potentially enable the preparation of advanced biomaterials from mushrooms under mild and ecofriendly conditions.

Obtaining chitin, chitosan and their melanin complexes from insects.

Interest in insects as a source of valuable biologically active substances has significantly increased over the past few years. Insects serve as an alternative source of chitin, which forms up to 40% of their exoskeleton. Chitosan, a deacetylated derivative of chitin, attracts the attention of scientists due to its unique properties (sorption, antimicrobial, film-forming, wound healing). Furthermore, some insect species are unique and can be used to obtain chitin- and chitosan-melanin complexes in the later stages of ontogenesis. Due to the synergistic effect, chitosan and melanin can enhance each other's biological activity, providing a wide range of potential applications.

Chitosan: A review of sources and preparation methods.

Chitosan, derived from chitin, has many desirable biomedical attributes. This review aims to explore different sources of chitin and methods of chitosan production with industrial consideration. This article first discussed different sources of chitin for industrial scale production, with considerations given to both their environmental impacts and commercialization potential. Secondly, this article reviews the two categories of chitosan preparation - chemical methods and biological methods - based on existing publications which used lobster by-products as a feedstock source. The mechanisms of the chemical methods are firstly summarized, and then the different chemical agents and reaction parameters used are discussed. Next, both enzymatic and fermentation-based approaches are reviewed under biological methods and compared with chemical methodologies, with lactic fermentation methods as the major focus. This article concludes that lobster cephalothorax could be an ideal source for chitosan preparation on an industrial scale; and chemical methods involve simpler processing overall, while producing chitosan with stronger bioactivities because of the lower molecular weight (MW) and higher degree of deacetylation (DD) achieved by the products. Moreover, due to biological methods inevitably necessitating further chemical processing, an approach involving some unconventional chemical methods has been regarded as a more suitable strategy for industrial scale chitosan production.

Characteristics of chitin extracted from black soldier fly in different life stages.

Chitin was collected and extracted along different lifecycle stages of the Black Soldier Fly (BSF) (larvae, prepupae, pupae, flies, shedding & cocoons). The chitin content in the collected biomass ranged between 8% and 24%, with sheddings and cocoons being most rich in chitin. Purified chitin was subjected to a physicochemical evaluation based on FTIR, XRD, and TGA as well as a deacetylation step. The data indicated that BSF chitin was α-chitin with FTIR profiles matching closely to shrimp chitin and showing some differences compared to squid pen chitin (ß-chitin). Small physicochemical differences were observed among the different BSF samples. Prepupae and cocoon chitin was more crystalline while chitin from larvae and sheddings had a lower thermal degradation temperature. In addition, sheddings were more difficult to purify. Further processing to chitosan showed that a deacetylation degree of 89% could be obtained for all samples after 3 h, although sheddings were found to be less reactive in the deacetylation process. Overall, the small differences in physicochemical properties that were detected between the BSF chitin samples did not prevent further processing of chitin to chitosan with the same degree of deacetylation via the same treatment.

Chitin from Antarctic krill shell: Eco-preparation, detection, and characterization.

Antarctic krill is a nutrient-rich crustacean that is one of the main species in the Antarctic ecosystem. Antarctic krill shell (AKS) can be used as raw materials to prepare chitin. In this study, lactic acid and dispase were used to prepare Antarctic krill chitin (AKC-1). Amino-monosaccharide contents of chitin samples were detected by pre-column PMP-HPLC method. Analytical instruments were conducted to determine characteristics of chitin samples. Results showed that the amino-monosaccharide content of AKS was 4.62 g/100 g (measured in D-glucosamine). The yield of AKC-1 was 5.49 g/100 g, and the amino-monosaccharide content was 80.90 g/100 g. AKC-1 showed smooth flakes, a porous surface, and α-chitin structural characteristics. The maximum degradation temperature (DTGmax) was 318.3 °C. The yield of deacetylated chitin (AKC-2) was 4.74 g/100 g, with deacetylation degree of 80.8%, viscosity average molecular weight of approximately 145.7 kDa, and amino-monosaccharide content of 97.06 g/100 g. The surface morphology of AKC-2 was similar to that of AKC-1, and the DTGmax was 311.5 °C. A mild, eco-friendly chitin preparation method and an amino-monosaccharide content detection method of raw material before chitin preparation are described in this study, which can provide technical support for comprehensive utilization of Antarctic krill resources.

Purification, biochemical/biophysical characterization and chitooligosaccharide binding to BGL24, a new PP2-type phloem exudate lectin from bottle gourd (Lagenaria siceraria).

Phloem Protein 2 (PP2), highly abundant in the sieve elements of plants, plays a significant role in wound sealing and anti-pathogenic responses. In this study, we report the purification and characterization of a new PP2-type lectin, BGL24 from the phloem exudate of bottle gourd (Lagenaria siceraria). BGL24 is a homodimer with a subunit mass of ~24 kDa and exhibits high specificity for chitooligosaccharides. The isoelectric point of BGL24 was estimated from zeta potential measurements as 5.95. Partial amino acid sequence obtained by mass spectrometric studies indicated that BGL24 exhibits extensive homology with other PP2-type phloem exudate lectins. CD spectroscopic measurements revealed that the lectin contains predominantly ß-sheets, with low α-helical content. CD spectroscopic and DSC studies showed that BGL24 exhibits high thermal stability with an unfolding temperature of ~82 °C, and that its secondary structure is essentially unaltered between pH 3.0 and 8.0. Fluorescence titrations employing 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitotrioside as an indicator ligand revealed that the association constants for BGL24-chitooligosaccharide interaction increase considerably when the ligand size is increased from chitotriose to chitotetraose, whereas only marginal increase was observed for chitopentaose and chitohexaose. BGL24 exhibited moderate cytotoxicity against MDA-MB-231 breast cancer cells, whereas its effect on normal splenocytes was marginal.