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.

Integrated extraction, structural characterization, and activity assessment of squid pen protein hydrolysates and ß-chitin with different protease hydrolysis.

Squid pen (SP) is a valuable source of protein and ß-chitin. However, current research has primarily focused on extracting ß-chitin from SP. This study innovatively extracted both SP protein hydrolysates (SPPHs) and SP ß-chitin (SPC) simultaneously using protease hydrolysis. The effects of different proteases on their structural characteristics and bioactivity were evaluated. The results showed that SP alcalase ß-chitin (SPAC) had the highest degree of deproteinization (DP, 98.19 %) and SP alcalase hydrolysates (SPAH) had a degree of hydrolysis (DH) of 24.47 %. The analysis of amino acid composition suggested that aromatic amino acids accounted for 17.44 % in SPAH. Structural characterization revealed that SP flavourzyme hydrolysates (SPFH) had the sparsest structure. SPC exhibited an excellent crystallinity index (CI, over 60 %) and degree of acetylation (DA, over 70 %). During simulated gastrointestinal digestion (SGD), the hydroxyl radical scavenging activity, ABTS radical scavenging activity, Fe2+ chelating activity, and reducing power of the SPPHs remained stable or increased significantly. Additionally, SPFC exhibited substantial inhibitory effects on Staphylococcus aureus and Escherichia coli (S. aureus and E. coli), with inhibition circle diameters measuring 2.4 cm and 2.1 cm. These findings supported the potential use of SPPHs as natural antioxidant alternatives and suggested that SPC could serve as a potential antibacterial supplement.

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.

Melt electrowritten poly-lactic acid /nanodiamond scaffolds towards wound-healing patches.

Multifunctional wound dressings, enriched with biologically active agents for preventing or treating infections and promoting wound healing, along with cell delivery capability, are highly needed. To address this issue, composite scaffolds with potential in wound dressing applications were fabricated in this study. The poly-lactic acid/nanodiamonds (PLA/ND) scaffolds were first printed using melt electrowriting (MEW) and then coated with quaternized ß-chitin (QßC). The NDs were well-dispersed in the printed filaments and worked as fillers and bioactive additions to PLA material. Additionally, they improved coating effectiveness due to the interaction between their negative charges (from NDs) and positive charges (from QßC). NDs not only increased the thermal stability of PLA but also benefitted cellular behavior and inhibited the growth of bacteria. Scaffolds coated with QßC increased the effect of bacteria growth inhibition and facilitated the proliferation of human dermal fibroblasts. Additionally, we have observed rapid extracellular matrix (ECM) remodeling on QßC-coated PLA/NDs scaffolds. The scaffolds provided support for cell adhesion and could serve as a valuable tool for delivering cells to chronic wound sites. The proposed PLA/ND scaffold coated with QßC holds great potential for achieving fast healing in various types of wounds.

Chitin nanofibrils assisted 3D printing all-chitin hydrogels for wound dressing.

The direct ink writing technique used in 3D printing technology is generally applied to designing biomedical hydrogels. Herein, we proposed a strategy for preparing all-chitin-based inks for wound dressing via direct ink writing technique. The ß-chitin nanofibers (MACNF) with a high aspect ratio were applied as a nanofiller to modulate the rheological properties of the alkaline dissolved chitin solution. The printing fidelity significantly depends on the MACNF introduction amount to the composite ink. 5-10 wt% MACNF ratio showed superior printing performance. The printed scaffold showed a uniform micron-sized pore structure and a woven network of nanofibers. Due to the good biocompatibility of chitin and the stereoscopic spatial skeleton, this scaffold showed excellent performance as a wound dressing, which can promote cell proliferation, collagen deposition and the angiogenesis of wounds, demonstrating its potential in biomedical applications. This approach successfully balanced the chitinous printability and biofunctions.

Green preparation of ß-chitins from squid pens by using alkaline deep eutectic solvents.

Based on the assumption that protein could be removed by the combined mechanism of alkaline induced degradation and strong hydrogen bond interactions of deep eutectic solvents (DESs), ß-chitins were successfully prepared from squid pens by using alkaline DESs formed by potassium carbonate and glycerol. The chemical structures of the DESs were investigated by 1H nuclear magnetic resonance (1H NMR), attenuated total reflection Fourier transform infrared (ATR-FTIR) and molecular modeling, and the physicochemical property of the prepared ß-chitins were characterized. The preparation yields was about 32 %, and DESs with K2CO3/glycerol of 1/10 could be reused for three times while maintaining high preparation yields (31 %-32 %) and degree of deacetylation of 66.9 %-76.9 %. The mechanisms of deproteinization and demineralization by the alkaline DESs were proposed to follow the degradation and dissolution steps, and proteins and minerals were removed from squid pens through the synergistic actions of alkaline degradation and hydrogen bonding interactions. This alkaline DESs are promising to be used as a green and efficient approach for commercial production of ß-chitin.

Thixotropic ß-Chitin Nanofiber Hydrogel with a Living Body-Responsive Self-Hardening Property and Its Application as a Sprayable Antiadhesion Barrier.

Considering recent advances in surgical techniques, sprayable antiadhesion barriers that are compatible with minimally invasive procedures are needed. However, the relatively low mechanical stiffness of the current thixotropic reversible sol-to-gel transition hydrogels has hindered their medical application. Herein, we show a thixotropic sprayable ß-chitin nanofiber hydrogel that spontaneously lost the thixotropic property in response to the environments within the living body. Furthermore, interactions between hydrogels and the biological environment result in a significant increase in mechanical stiffness. Due to these advantageous properties, ß-chitin nanofiber hydrogels administered by spray prevent postoperative abdominal adhesions and are thus promising sprayable antiadhesion barriers.

Adipose-derived mesenchymal stem cell-loaded ß-chitin nanofiber hydrogel activates the AldoA/HIF-1α pathway to promote diabetic wound healing.

OBJECTIVES: To identify the effect of adipose-derived mesenchymal stem cell-loaded ß-chitin nanofiber (ADSC-loaded ß-ChNF) hydrogel on diabetic wound healing and clarify its mechanism of action. METHODS: We prepared the ADSC-loaded ß-ChNF hydrogel to repair wounds of db/db diabetic mice. Wound healing rate, histopathology, enzyme-linked immunosorbent assay, and western blot were used to confirm its role and mechanism in promoting diabetic wound healing. RESULTS: The ADSC-loaded ß-ChNF hydrogel accelerated wound healing in db/db diabetic mice, as indicated by increased cell proliferation, epithelization, and tissue granulation in the skin. Moreover, expression of vascular endothelial growth factor (VEGF) and its receptor (VEGFR), matrix metalloproteinase 9 (MMP9), and TIMP metallopeptidase inhibitor 1 (TIMP1) were upregulated. These results demonstrate the beneficial effects of this ADSC-loaded ß-ChNF hydrogel on diabetic wound healing. Furthermore, we show that the ADSC-loaded ß-ChNF hydrogel activated aldolase A (AldoA)/hypoxia-inducible factor 1α (HIF-1α) signaling. An inhibitor of HIF-1α markedly decreased the promotive effects of the ADSC-loaded ß-ChNF hydrogel on wound healing and reduced expression of VEGF, VEGFR, MMP9, and TIMP1. CONCLUSIONS: Our findings suggest that the ADSC-loaded ß-ChNF hydrogel activated the HIF-1α/MMP9 axis through AldoA feedback to promote diabetic wound healing.

Novel Chitin Deacetylase from Thalassiosira weissflogii Highlights the Potential for Chitin Derivative Production.

ß-Chitin is an important carbon fixation product of diatoms, and is the most abundant nitrogen-containing polysaccharide in the ocean. It has potential for widespread application, but the characterization of chitin-related enzymes from ß-chitin producers has rarely been reported. In this study, a chitin deacetylase (TwCDA) was retrieved from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) database and was heterologously expressed in vitro for functional analysis. The results showed that both the full-length sequence (TwCDA) and the N-terminal truncated sequence (TwCDA-S) had chitin deacetylase and chitinolytic activities after expression in Escherichia coli. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) indicated that TwCDA and TwCDA-S could catalyze the deacetylation of oligosaccharide (GlcNAc)5. TwCDA had higher deacetylase activity, and also catalyzed the deacetylation of the ß-chitin polymer. A dinitrosalicylic acid (DNS) assay showed that TwCDA-S had high chitinolytic activity for (GlcNAc)5, and the optimal reaction temperature was 35 °C. Liquid chromatography combined with time-of-flight mass spectrometry (LC-coTOF-MS) detected the formation of a N-acetylglucosamine monomer (C8H15NO6) in the reaction mixture. Altogether, we isolated a chitin deacetylase from a marine diatom, which can catalyze the deacetylation and degradation of chitin and chitin oligosaccharides. The relevant results lay a foundation for the internal regulation mechanism of chitin metabolism in diatoms and provide a candidate enzyme for the green industrial preparation of chitosan and chitin oligosaccharides.

Tailoring nanohole sizes through the deacetylation process in chitosan powders obtained from squid pens.

An experimental study on the evolution of the physicochemical, thermal and nanostructural properties of chitosan samples obtained from squid pens as the deacetylation treatment proceeds is presented. To this aim, potentiometric titration, capillary viscosimetry, infrared spectroscopy, differential scanning calorimetry and positron annihilation lifetime spectroscopy were used. The results obtained are discussed in terms of the influence of the deacetylation time on the deacetylation degree, average molecular weight, thermal parameters and average free nanohole size of the different samples. A way of preparing chitosan matrices with tailored nanostructural characteristics for specific applications through the deacetylation process is explored.

Evaluating Non-Conventional Chitosan Sources for Controlled Release of Risperidone.

In this work, two chitosan samples from cuttlebone and squid pen are produced and characterized. We studied the formation of thermoresponsive hydrogels with ß-glycerol phosphate and found proper formulations that form the hydrogels at 37 °C. Gel formation depended on the chitosan source being possible to produce the thermoresponsive hydrogels at chitosan concentration of 1% with cuttlebone chitosan but 1.5% was needed for squid pen. For the first time, these non-commercial chitosan sources have been used in combination with ß-glycerol phosphate to prepare risperidone formulations for controlled drug delivery. Three types of formulations for risperidone-controlled release have been developed, in-situ gelling formulations, hydrogels and xerogels. The release profiles show that in-situ gelling formulations and particularly hydrogels allow an extended control release of risperidone while xerogels are not appropriate formulations for this end since risperidone was completely released in 48 h.

Heterointerface Engineering of ß-Chitin/Carbon Nano-Onions/Ni-P Composites with Boosted Maxwell-Wagner-Sillars Effect for Highly Efficient Electromagnetic Wave Response and Thermal Management.

The rational construction of microstructure and composition with enhanced Maxwell-Wagner-Sillars effect (MWSE) is still a challenging direction for reinforcing electromagnetic wave (EMW) absorption performance, and the related EMW attenuation mechanism has rarely been elucidated. Herein, MWSE boosted ß-chitin/carbon nano-onions/Ni-P composites is prepared according to the heterointerface engineering strategy via facile layer-by-layer electrostatic assembly and electroless plating techniques. The heterogeneous interface is reinforced from the aspect of porous skeleton, nanomaterials and multilayer construction. The composites exhibit competitive EMW response mechanism between the conductive loss and the polarization/magnetic loss, as describing like the story of "The Hare and the Tortoise". As a result, the composites not only achieve a minimum reflection loss (RLmin) of - 50.83 dB and an effective bandwidth of 6.8 GHz, but also present remarkable EMW interference shielding effectiveness of 66.66 dB. In addition, diverse functions such as good thermal insulation, infrared shielding and photothermal performance were also achieved in the hybrid composites as a result of intrinsic morphology and chemicophysics properties. Therefore, we believe that the boosted MWSE open up a novel orientation toward developing multifunctional composites with high-efficient EMW response and thermal management.

Valorization of ß-Chitin Extraction Byproduct from Cuttlefish Bone and Its Application in Food Wastewater Treatment.

The nontoxicity, worldwide availability and low production cost of cuttlefish bone products qualify them an excellent biocoagulant to treat food industry wastewater. In this study, cuttlefish bone liquid waste from the deproteinization step was used as a biocoagulant to treat food industry wastewater. This work concerns a waste that has never before been investigated. The objectives of this work were: the recovery of waste resulting from cuttlefish bone deproteinization, the replacementof chemical coagulants with natural ones to preserve the environment, and the enhancement ofthe value of fishery byproducts. A quantitative characterization of the industrial effluents of a Moroccan food processing plant was performed. The physicochemical properties of the raw cuttlefish bone powder and the deproteinization liquid extract were determined using specific analysis techniques: SEM/EDX, FTIR, XRD and 1H-NMR. The protein content of the deproteinization liquid was determined by OPA fluorescent assay. The zeta potential of the liquid extract was also determined. The obtained analytical results showed that the deproteinization liquid waste contained an adequate amount of soluble chitin fractions that could be used in food wastewater treatment. The effects of the coagulant dose and pH on the food industrial effluents were studied to confirm the effectiveness of the deproteinization liquid extract. Under optimal conditions, the coagulant showed satisfactory results. Process optimization was performed using the Box-Behnken design and response surface methodology. Thus, the optimal removal efficiencies predicted using this model for turbidity (99.68%), BOD5 (97.76%), and COD (82.92%) were obtained at a dosage of 8 mL biocoagulant in 0.5 L of food processing wastewater at an alkaline pH of 11.

Application of adipose mesenchymal stem cell-derived exosomes-loaded ß-chitin nanofiber hydrogel for wound healing.

INTRODUCTION: Clarifying the role and mechanism of exosome gel in wound repair can provide a new effective strategy for wound treatment. MATERIALS AND METHODS: The cellular responses of adipose mesenchymal stem cell-derived exosomes (AMSC-exos) and the wound healing ability of AMSC-exos-loaded ß-chitin nanofiber (ß-ChNF) hydrogel were studied in vitro in mouse fibroblasts cells (L929) and in vivo in rat skin injury model. The transcriptome and proteome of rat skin were studied with the use of sequenator and LC-MS/MS, respectively. RESULTS: 80 and 160 µg/mL AMSC-exos could promote the proliferation and migration of mouse fibroblastic cells. Furthermore, AMSC-exos-loaded ß-ChNF hydrogel resulted in a significant acceleration rate of wound closure, notably, acceleration of re-epithelialization, and increased collagen expression based on the rat full-thickness skin injury model. The transcriptomics and proteomics studies revealed the changes of the expression of 18 genes, 516 transcripts and 250 proteins. The metabolic pathways, tight junction, NF-κB signaling pathways were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway. Complement factor D (CFD) and downstream Aldolase A (Aldoa) and Actn2 proteins in rats treated with AMSC-exos-loaded ß-ChNF hydrogel were noticed and further confirmed by ELISA and Western blot. CONCLUSION: These findings suggested that AMSC-exos-loaded ß-ChNF hydrogel could promote wound healing with the mechanism which is related to the effect of AMSC-exos on CFD and downstream proteins.

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.

Fluorescent Amphiphilic Quaternized ß-Chitin: Antibacterial Mechanism and Cell Imaging.

Fluorescent materials span multiple applications from biological probes and chemical sensing to optoelectronic systems. Although great efforts have been made toward developing classes of fluorescent materials, 100,000+ traditional fluorescent dyes still suffer from the obstacle of aggregation-caused quenching (ACQ). Thus, designing fluorescent materials with excellent optical performance from ACQ luminogens remains challenging. In this work, we prepared fluorescent amphiphilic quaternized ß-chitin (QC-F) via nucleophilic addition between the amino groups of QC and isothiocyanate groups of fluorescein isothiocyanate (FITC). Due to the covalent anchoring of the QC backbone, steric hindrance of the bulky acetamido groups, electrostatic repulsion of the quaternary ammonium groups, and homogeneous distribution of FITC units, the FITC units were spatially and electronically isolated, and the QC-F series exhibited unique fluorescent behaviors. The QC-F series could be used to observe their interactions with microbial cells through fluorescence imaging to gain insights into the QC antibacterial mechanism. Moreover, with their favorable cytocompatibility, the QC-F series are also suitable for cell imaging. Thus, the present work will broaden the applications of chitin and conventional ACQ luminogens.

Thermal degradation and lifetime of ß-chitin from Dosidicus gigas squid pen: Effect of impact at 9.7 GPa and a comparative study with α-chitin.

The kinetics of thermal degradation of ß-chitin extracted from Dosidicus gigas squid pen, was studied at normal conditions as well as after being subjected to the action of high-pressure impact of 9.7 GPa. The integral iso-conversional procedure of Kissinger-Akahira-Sunose (KAS) recommended by the ICTAC kinetics committee was applied to the non-isothermal data obtained from thermogravimetry (TGA). Lifetimes were predicted without assumption of any reaction model. Heating rates of ß = 10, 15, 20 and 25 °C/min under nitrogen atmosphere were used from room temperature to 1300 °C. A comparative study with α-chitin was performed. All the samples were structurally and chemically characterized by several techniques. The extracted ß-chitin was found to be in the monohydrate form; while with the action of high-pressure impact, it was transformed into ß-chitin dehydrate showing slightly higher stability. Reliable prediction for lifetimes considering working temperatures over 425 K was found for α and ß-chitin.

Innovation inspired by nature: Biocompatible self-healing injectable hydrogels based on modified-ß-chitin for wound healing.

Remarkable properties of hydrogels are compromised by failure to recover from damage, bringing their intended functions to an end. To address this, hydrogels can be functionalized with self-healing property to enable them to restore themselves after damage, thus, extending their lifetime. Herein, hydrogels were prepared by cross-linking acrylamide-modified ß-chitin (Am-ß-Chn) with alginate dialdehyde (ADA) to form Schiff base, showing IR characteristic peak at 1650 cm-1, attributed to the stretching vibration of CN. The dynamic Schiff base and H-bond rendered the double crosslinked hydrogels self-healing as demonstrated by continuous step strain rheology. Characterization of the hydrogels revealed excellent biocompatibility, biodegradability, injectability and self-healing properties. Furthermore, the wound healing property of the hydrogels was investigated in vivo using zebrafish as a model system. Indirect application of Am-ß-Chn/ADA hydrogel remarkably led to ~87% wound healing as compared to control which gave ~50%, suggesting that hydrogels are effective in accelerating wound healing. However, a clear understanding of the exact mechanism of its wound healing property remains to be investigated. To the best of our knowledge, this is the first innovation of developing novel double crosslinked Am-ß-Chn/ADA hydrogels with both self-healing and accelerated wound healing properties, directly from marine-food wastes.

Natural ß-chitin-protein complex film obtained from waste razor shells for transdermal capsaicin carrier.

In the literature, the produced ß-chitin samples are in powder or flake forms but there is no natural ß-chitin based film. Also, the commercially available transdermal patches are produced from synthetic polymers. In this regard, we produced natural ß-chitin-protein complex (CPC) film from the waste shells of Ensis spp. The obtained natural film was characterized by FTIR, TGA and SEM. Additionally, swelling, thickness, contact angle and antioxidant tests were done to learn more about the films. After production and characterization of the film, capsaicin, which is commonly used for pain relief was loaded into the film. The loading capacity was recorded as 5.79%. The kinetic models were studied in three different pH, then the results were fitted with Higuchi model with high correlation at pH 7.4. After considering all the obtained results, the capsaicin loaded CPC film may be an alternative candidate for transdermal patch instead of the synthetic ones.

Versatile synthesis, characterization and properties of ß-chitin derivatives from aqueous KOH/urea solution.

Chitin is the second most abundant natural polysaccharide with biocompatibility, biodegradability, and bioactivity. Homogeneous modification of chitin is an efficient way to improve or to impart new properties to chitin. Here, amide-modified ß-chitin (AMC), hydroxyethyl ß-chitin (HEC), and hydroxybutyl ß-chitin (HBC) through Michael addition, Williamson reaction, and ring-opening addition, were homogeneously synthesized from aqueous KOH/urea solution, respectively, with controlled structures and uniform properties. The reactions mainly occur at the hydroxyl groups of C-6 positions of ß-chitin chains due to mild conditions, and the obtained ß-chitin derivatives with high DS values are water-soluble. AMC could transform from sol to gel at acidic condition or upon adding Fe3+ due to the presence of partial carboxylate groups. As a specific highlight, HEC and HBC could thermally form smart hydrogels at physiological temperature, with cytocompatibility and blood biocompatibility, which is very useful as drug/ cell carriers for biomedical applications. The remarkably mild and "green" condition of aqueous KOH/urea solution for the synthesis of chitin derivatives has pioneered a better way to exploit its great diversity of the natural, sustainable polysaccharide.