Phosphorylated chitin from shrimp shell waste: A robust solution for cadmium remediation.

In this work, chitin (CT) was isolated from shrimp shell waste (SSW) and was then phosphorylated using diammonium hydrogen phosphate (DAP) as a phosphorylating agent in the presence of urea. The prepared samples were characterized using Scanning Electron Microscopy (SEM) and EDX-element mapping, Fourier Transform Infrared Spectroscopy (ATR-FTIR), X-Ray Diffraction (XRD), Thermogravimetric Analysis (TGA/DTG), conductometric titration, Degree of Substitution (DS) and contact angle measurements. The results of characterization techniques reveal the successful extraction and phosphorylation of chitin. The charge content of the phosphorylated chitin (P-CT) was 1.510 mmol·kg-1, the degree of substitution of phosphorus groups grafted on the CT surface achieved the value of 0.33. The adsorption mechanisms appeared to involve electrostatic attachment, specific adsorption (CdO or hydroxyl binding), and ion exchange. Regarding the adsorption of Cd2+, the effect of the adsorbent mass, initial concentration of Cd2+, contact time, pH, and temperature were studied in batch experiments, and optimum values for each parameter were identified. The experimental results revealed that P-CT enhanced the Cd2+ removal capacity by 17.5 %. The kinetic analyses favored the pseudo-second-order model over the pseudo-first-order model for describing the adsorption process accurately. Langmuir model aptly represented the adsorption isotherms, suggesting unimolecular layer adsorption with a maximum capacity of 62.71 mg·g-1 under optimal conditions of 30 °C, 120 min, pH 8, and a P-CT dose of 3 g·L-1. Regeneration experiments evidenced that P-CT can be used for 6 cycles without significant removal capacity loss. Consequently, P-CT presents an efficient and cost-effective potential biosorbent for Cd2+ removal in wastewater treatment applications.

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.

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%.

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.

Using Flammulina velutipes derived chitin-glucan nanofibrils to stabilize palm oil emulsion:A novel food grade Pickering emulsifier.

We herein report chitin-glucan nanofibrils from edible mushroom Flammulina velutipes (CGNFs) as a novel stabilizer for palm oil Pickering emulsion (o/w, 30:70, v:v). Generally, these CGNFs being composed of glucose and glucosamine, are threadlike with 4.9 ± 1.2 nm wide and 222.6 ± 91.9 nm long. They were easily absorbed on the oil-water interface to form a compact layer around the oil droplets referring to Pickering emulsion. This emulsion presented shear-thinning and gel-like behaviors, wherein CGNFs concentration had a profound influence on the emulsion volume, droplet size, and stabilization index. Moreover, CGNFs showed an ability to stabilize the emulsion with a minimum of surface coverage approximately 30%. It indicated that moderate concentration of NaCl improved the emulsification effect, and the emulsion were stable in a large range of pH. These CGNFs are easy to prepare, eco-friendly and sustainable, which provides a potential for large-scale application of Pickering emulsion in food and nutraceuticals fields.

Chitin, Characteristic, Sources, and Biomedical Application.

BACKGROUND: Chitin stands at second, after cellulose, as the most abundant polysaccharide in the world. Chitin is found naturally in marine environments as it is a crucial structural component of various marine organisms. METHODS: Different amounts of waste chitin and chitosan can be discovered in the environment. Chitinase producing microbes help to hydrolyze chitin waste to play an essential function for the removal of chitin pollution in the Marine Atmosphere. Chitin can be converted by using chemical and biological methods into prominent derivate chitosan. Numerous bacteria naturally have chitin degrading ability. RESULTS: Chitin shows promise in terms of biocompatibility, low toxicity, complete biodegradability, nontoxicity, and film-forming capability. The application of these polymers in the different sectors of biomedical, food, agriculture, cosmetics, pharmaceuticals could be lucrative. Moreover, the most recent achievement in nanotechnology is based on chitin and chitosan-based materials. CONCLUSION: In this review, we examine chitin in terms of its natural sources and different extraction methods, chitinase producing microbes and chitin, chitosan together with its derivatives for use in biomedical and agricultural applications.

Hyaluronan biofilms reinforced with partially deacetylated chitin nanowhiskers: Extraction, fabrication, in-vitro and antibacterial properties of advanced nanocomposites.

Fabrication of nanocomposite biofilms with enhanced mechanical and antibacterial properties was successfully achieved from hyaluronan (HA) and partially deacetylated chitin nanowhiskers (ChNWs) by a casting-evaporation method. The hydrolysis process of chitin showed an important role in the dimensions, stability, and the crystallinity of extracted ChNWs in a time-dependent manner. The volume fraction of ChNWs nanofiller varying from (0.001 to 0.5) exhibited a great influence on the mechanical properties of the biofilms (young modulus, strength) was enhanced by the high load-bearing capacity of NWs compared with net HA film. The antibacterial activity of the nanocomposite biofilms exhibited significant bactericidal activity against different types of bacteria (-/+ gram). HA/ChNWs Nanocomposite biofilms did not show any toxicity against normal human dermal fibroblasts (NHDF) and human primary osteogenic sarcoma (Saos-2) cell lines. The new biofilms with unique properties like edibleness, environmental friendliness, high mechanical properties, antibacterial performance, and non-cytotoxicity that could be used in skin tissue regenerations, and drug delivery applications.

Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species.

Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758) (Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae)) for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%, 10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614 °C to 748 °C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales. Elemental analysis has been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.

Study on the "Glutamic Acid-Enzymolysis" Process for Extracting Chitin from Crab Shell Waste and its By-Product Recovery.

Chitin is the second-most abundant bioresource and widely used in the food, agricultural, biomedicine, and other industries. However, under the mutual restriction of extraction cost and environmental protection, it is relatively difficult to prepare chitin from natural sources by pure separation. The aim of this study is to extract chitin from fresh crab shell waste by decalcification (DC) and deproteinization (DP) using glutamic acid and alkaline protease. The optimum technological conditions for DC and DP were as follows: (1) 5% (w/v) glutamic acid solution was used as decalcifying agent, the ratio of material to liquid was 1:10 (m/v), and the ash content in chitin was 0.83 ± 0.027% after decalcification at 75° C for 12 h. (2) Using alkaline protease as enzymatic hydrolyzer, 1500 U of alkaline protease was added per gram of crab shell. Under the conditions of material-liquid ratio of 1:10 (m/v) and pH value of hydrolysate of 9.0, N content in chitin was 6.63 ± 0.10% after 6 h of enzymatic hydrolysis at 55° C. And the extraction rate of chitin was 92.25 ± 0.51%. As a decalcifying agent, glutamic acid could be recycled with a recovery rate of 77.42 ± 2.16%. Calcium carbonate in crab shell was converted into calcium hydrogen phosphate by calcium glutamate, and protein into amino acids and polypeptides, which could be used as feed additives. The "glutamic acid-enzymolysis" for extracting chitin from crab shell is a relatively closed process, which has the advantages of mild reaction, greatly reducing the discharge of three wastes and high comprehensive utilization rate of raw materials.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Otimização e padronização de processos para obtenção de quitosana purificada para uso farmacêutico e alimentício / Optimization and standardization of processes to obtain purified chitosan for pharmaceutical and food use

Quitosana é um biopolímero encontrado principalmente na parede celular de crustáceos e é obtida pela desacetilação da quitina. Como biopolímero a quitosana é utilizada como excipiente para medicamentos e composição de alimentos. No entanto a quitosana devidamente purificada para uso farmacêutico ou alimentício tem custo financeiro elevado. Outro fator que contribui para o uso limitado é a falta de procedimento padronizado para desacetilação, o que resulta em materiais com diferentes graus de qualidade, dificultando suas aplicações e controle de qualidade de matéria prima e produto. Este trabalho tem como principal objetivo estabelecer procedimento reprodutível para a extração da quitina e da quitosana, por meio da aplicação dos conceitos de Quality by Design e planejamento de experimentos. A quitosana foi obtida pela desacetilação da quitina de crustáceos pelas etapas de desmineralização, desproteinização e despigmentação. O procedimento técnico para purificação da quitosana foi definido a partir de planejamento fatorial com ponto central para as etapas otimizadas, por meio da aplicação dos conceitos de Quality by Design e planejamento de experimentos. O projeto definiu um procedimento padronizado para purificação da quitosana que pode ser empregado em escala industrial, e financeiramente vantajoso para produção de medicamentos ou alimentos

Chitosan is a biopolymer found mainly in the cell wall of crustaceans and is obtained by the deacetylation of chitin. As biopolymer chitosan is used as excipient for medicaments and food composition. However, chitosan duly purified for pharmaceutical or food use has a high financial cost. Another factor that contributes to the limited use is the lack of standardized procedure for deacetylation, which results in materials with different grades of quality, hindering their applications and quality control of raw material and product. This work has as main objective to establish a reproducible procedure for the extraction of chitin and chitosan, through the application of the concepts of Quality by Design and planning of experiments. Chitosan was obtained by the deacetylation of chitin from crustaceans through the demineralization, deproteinization and depigmentation stages. The technical procedure for purification of chitosan was defined from a factorial planning with a central point for the optimized steps, through the application of the concepts of Quality by Design and planning of experiments. The project defined a standard procedure for the purification of chitosan that can be used on industrial scale and financially advantageous for the production of medicines or foods

Effect of Nanocrystalline Particles of Chitin on Blood Components in Humans and Experimental Animals.

Nanocrystalline particles of chitin in the form of hydrosol in a concentration of 0.63 mg/ml have no effect on aggregation of human platelets and clotting time of platelet-poor plasma in coagulation tests. ADP-induced aggregation of human platelets was inhibited by these nanoparticles in concentrations of 0.63 and 1.00 mg/ml in comparison with the control. Intravenous administration of nanoparticles in a dose of 1 mg/kg to guinea pigs produced no anticoagulant effect. The nanocrystalline particles of chitin can be of interest as potential drug delivery systems.

Chitin and chitosan preparation from shrimp shells Penaeus monodon and its human ovarian cancer cell line, PA-1.

In the present study, chitin and chitosan preparation from shrimp shells Penaeus mondon and its ovarian cancer cell line (PA-1). FTIR spectrum sharp absorption peak at 1655cm-1 is assigned to ketone C=O (α) unsaturated with chitosan. X-ray diffraction showed the presence of chitin and chitosan were strongest peak at 18.91° (ß) and 29.75° (α) characters. SEM observations of chitin and chitosan surface morphologies of P. monodon showed that microfibril and porous structures. Anticancer activity of chitin and chitosan against human ovarian cancer cell line showed that chitosan an exhibited notable higher activity than chitin. Anticancer activity of aquacultural waste of shrimp shells mediated chitosan, which was proved to be good novel pharmaceutical industries.

Towards the Shell Biorefinery: Sustainable Synthesis of the Anticancer Alkaloid Proximicin†A from Chitin.

A shell biorefinery would involve fractionation of crustacean shells and incorporation of the components into value-added products, particularly those that contain nitrogen. In a proof-of-concept study that validates this concept, the anticancer alkaloid proximicin A has been synthesized from the chitin-derived platform chemical 3-acetamido-5-acetylfuran (3A5AF). This study accentuates the leading role chitin is likely to play in the sustainable production of nitrogen-containing fine chemicals that are not directly attainable from lignocellulose.

Chitin and chitosan from the Norway lobster by-products: Antimicrobial and anti-proliferative activities.

Chitin was recovered through enzymatic deproteinization of the Norway lobster (Nephrops norvegicus) processing by-products. The obtained chitin was characterized and converted into chitosan by N-deacetylation, the acid-soluble form of chitin. Chitosan samples were then characterized by Fourier transform infrared spectroscopy (FTIR) and 13 Cross polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy. The antimicrobial activity and anti-proliferative capacity of chitosan were evaluated. Antimicrobial activity assays indicated that prepared chitosan exhibited marked inhibitory activity against the bacterial and fungal strains tested. Further, cytotoxic effects of chitosan samples on human colon carcinoma cells HCT116 was evaluated using the MTT assay. Chitosan showed the antiproliferative capacity against the colon-cancer-cell HCT116 in a dose dependent manner with IC50 of 4.6mg/ml. Indeed, HCT116 cell proliferation was significantly inhibited (p<0.05) between 13.5 and 67.5% at 0.5-6mg/mL of chitosan after 24h of cell treatment. The chitosan showed high antitumor activity which seemed to be dependent on its characteristics such as acetylation degree.

Advances in preparation, analysis and biological activities of single chitooligosaccharides.

Chitooligosaccharides (COS), as a source of potential bioactive material, has been reported to possess diverse bioactivities. These bioactivities of COS are often tested using relatively poorly characterized oligomer mixtures during past few decades, resulting in difficult identification of COS molecules responsible for biological effects. Therefore, a new interest has recently been emerged on highly purified COS of defined size. Several technological approaches have been used to produce single COS and new improvements were introduced to their characterization in order to understand the unrevealed structure-function relationship. Here we provide an overview of techniques that were used to prepare and analyze reasonably well-defined COS fractions. Based on the latest reports, several applications of single COS for plants and animals, are also presented, including antitumor, immunostimulatory, antioxidant, antimicrobial, elicitors of plant defence and neural activity.

Preparation, physicochemical and pharmaceutical characterization of chitosan from Catharsius molossus residue.

Polypeptide from Catharsius molossus L. is an active ingredient in the treatment of benign prostatic hyperplasia. The residue after extraction is harmful to the environment and is also a waste of resources. Chitosan was extracted from C. molossus L. residue with chemical methods and with an improved intermittent heating method. Physicochemical and pharmaceutical characteristics of chitosan from C. molossus L. and shrimp were mainly measured by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM). The results showed chitosan from C. molossus L. was superior to commercial medical-grade chitosan from shrimp in the aspects of degree of deacetylation, crystallinity, heavy metal content, viscosity, protein residue, ash content, and in vitro adhesion. In addition, properties of chitosan membrane were studied, including water vapor permeability, light transmittance, enzymatic hydrolysis, swelling behavior, mechanical properties, and SEM images. It was found that the membrane of chitosan from C. molossus L. had better performance. This preliminary result shows chitosan from C. molossus L. is more suitable than shrimp's as a pharmaceutical excipient in colonic adhesive drug delivery system.

Chitin and chitosan preparation from marine sources. Structure, properties and applications.

This review describes the most common methods for recovery of chitin from marine organisms. In depth, both enzymatic and chemical treatments for the step of deproteinization are compared, as well as different conditions for demineralization. The conditions of chitosan preparation are also discussed, since they significantly impact the synthesis of chitosan with varying degree of acetylation (DA) and molecular weight (MW). In addition, the main characterization techniques applied for chitin and chitosan are recalled, pointing out the role of their solubility in relation with the chemical structure (mainly the acetyl group distribution along the backbone). Biological activities are also presented, such as: antibacterial, antifungal, antitumor and antioxidant. Interestingly, the relationship between chemical structure and biological activity is demonstrated for chitosan molecules with different DA and MW and homogeneous distribution of acetyl groups for the first time. In the end, several selected pharmaceutical and biomedical applications are presented, in which chitin and chitosan are recognized as new biomaterials taking advantage of their biocompatibility and biodegradability.

Isolation & identification of bacteria for the treatment of brown crab (Cancer pagurus) waste to produce chitinous material.

AIMS: To isolate bacteria from soil for microbial pretreatment of brown crab (Cancer pagurus) shell waste and the production of chitin. METHODS AND RESULTS: Isolates were screened for protease enzymes and acid production in order to facilitate the removal of protein and calcium carbonate fractions from brown crab shell to yield a chitinous material. Selected isolates were applied in various combinations in successive, two-step fermentations with brown crab shell waste. These isolates were identified as: Exiguobacterium spp. (GenBank accession number: KP050496), Bacillus cereus (GenBank accession number: KP050499), B. subtilis (GenBank accession number: KP050498), Bacillus licheniformis (GenBank accession number: KP050497), Pseudomonas migulae (GenBank accession number: KP050501), Pseudomonas spp. (GenBank accession number: KP050500), Pseudomonas spp. (GenBank accession number: KP050502), Arthrobacter luteolus (GenBank accession number: KP050503), Lactobacillus spp. (GenBank accession number: KP072000) and Enterococcus spp. (GenBank accession number: KP071999). CONCLUSIONS: Successive two-step fermentations with isolates in certain combinations resulted in a demineralization of >94% and the extraction of a crude chitin fraction from brown crab processing waste. The highest demineralization, 98·9% was achieved when isolates identified as B. cereus and Pseudomonas spp. were used in combination. The transfer of fermentations to a larger scale requires further research for optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful application of these isolates in successive two-step fermentation of brown crab shell waste to extract chitin means with further research into optimization and scale up, this chitin extraction process may be applied on an industrial scale and provide further commercial value from brown crab shell waste.

Preparation of melanin from Catharsius molossus L. and preliminary study on its chemical structure.

A great deal of melanin was found in the waste alkali liquor produced by extraction of chitin from Catharsius molossus L. Discarding the lye could harm the environment and cause waste of resources. In this paper, melanin from C. molossus L. was recovered through acid precipitation and purified by pepsin and so on. The purity, chemical composition and structure of the prepared melanin were explored by UV-visible absorption spectroscopy, Fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, high resolution (13)C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy pyrolysis gas chromatography mass spectrometry, X ray diffraction, X ray fluorescence, matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry, thermal analysis, and so on. The results showed that the purity of the prepared melanin was higher than the commercial standard melanin and it was a kind of nanoaggregates composed of a large quantity of 5,6-dihydroxyindole eumelanin and a small amount of phaeomelanin. In addition, the prepared melanin was irregular in shape and its structure could be divided into three levels: advanced structure maintained by polypeptides, substructure maintained by the ferric ion and microstructure. In particular, the smallest structural unit showed the graphite-like layered structure containing five layers linked by non-covalent bonds and each layer mainly consisted of 5,6-dihydroxyindole and its derivatives, which might be connected to each other through various chemical bonds.