High Degree Of Deacetylation Research Articles

Effects of chitosan molecular weight and deacetylation degree on the properties of collagen‐chitosan composite films for food packaging

AbstractComposite films based on collagen and chitosan with different molecular weights (MWs) and deacetylation degree (DD) are prepared for food packaging. Fourier‐transform infrared analysis indicates that more hydrogen bonds have formed between collagen molecules and chitosan with larger MW or higher DD due to their more reactive groups for interactions. The light barrier capacity, mechanical, and thermal properties of the composite films enhanced with increasing the MW or DD of chitosan. However, composite film with the lowest MW of 50 kDa possesses the best antioxidative and antibacterial activities. Among all the films, composite films containing chitosan with low MW of 50 kDa and high DD of 95% exhibit better preservation effect in pork. The shelf life of pork wrapped with film containing chitosan of 50 kDa has been extended to 3 days with the lowest total volatile basic nitrogen level (12.13 mg/100 g), thiobarbituric acid reactive substance value (0.541 mg malondialdehyde/kg) and total viable count (5.64 log colony forming unit/g). These results indicate that MW and DD of chitosan are two important factors that should be considered when designing collagen‐chitosan composite films for food packaging.

Influence of Deacetylation Degree of Chitosan on the Anticorrosive Properties of Carbon Steel Coatings

AbstractThis study outlines the improvement on anticorrosion behavior of chitosan‐based sol‐gel coatings on carbon steel surface obtained for high deacetylation degree values of this natural biopolymer. Important economic losses in major industries represent a problem due to corrosion of metals in aggressive medium, such as carbon steel, which is widely used due to its hardness, durability, ductility, low cost, handiness. Therefore, non‐toxic, cost‐effective, and eco‐friendly strategies are needed to protect metallic surface of steel. Against this background, the anticorrosion coating performance of chitosan, from three different commercial sources, as a single component, on carbon steel in chloride solutions is investigated using electrochemical impedance spectroscopy technique. The present study is performed to evaluate the corrosion protective abilities of chitosan in order to determine the relationship with its deacetylation degree (DDA), evaluated by potentiometric titration, a simple and reliable method. Depending on the DDA, the experimental results indicate that there is a remarkable difference between different chitosan samples regarding the value of the charge transfer resistance of coating on steel.

Antibacterial Bio-Nanocomposite Textile Material Produced from Natural Resources.

Growing demand for sustainable and green technologies has turned industries and research toward the more efficient utilization of natural and renewable resources. In an effort to tackle this issue, we developed an antibacterial textile nanocomposite material based on cotton and peat fibers with immobilized Cu-based nanostructures. In order to overcome poor wettability and affinity for Cu2+-ions, the substrate was activated by corona discharge and coated with the biopolymer chitosan before the in situ synthesis of nanostructures. Field emission scanning electron microscopy (FESEM) images show that the application of gallic or ascorbic acid as green reducing agents resulted in the formation of Cu-based nanosheets and mostly spherical nanoparticles, respectively. X-ray photoelectron spectroscopy (XPS) analysis revealed that the formed nanostructures consisted of Cu2O and CuO. A higher-concentration precursor solution led to higher copper content in the nanocomposites, independent of the reducing agent and chitosan deacetylation degree. Most of the synthesized nanocomposites provided maximum reduction of the bacteria Escherichia coli and Staphylococcus aureus. A combined modification using chitosan with a higher deacetylation degree, a 1 mM solution of CuSO4 solution, and gallic acid resulted in an optimal textile nanocomposite with strong antibacterial activity and moderate Cu2+-ion release in physiological solutions. Finally, the Cu-based nanostructures partially suppressed the biodegradation of the textile nanocomposite in soil.

Positive effects and mechanism of ultrasound on chitin preparation from shrimp shells by co-fermentation

The objective of this study is to explore the effect and mechanism of ultrasound on chitin extraction from shrimp shells powder (SSP) by the co-fermentation of Bacillus subtilis and Acetobacter pasteurianus. After pre-treating the SSP with high-intensity ultrasound (HIU) at 800 W, the protease activity in the fermentation solution reached 96.9 U/mL on day 3, which was significantly higher than for SSP that had not been pre-treated with ultrasound (81.8 U/mL). The fermentation time of the chitin extraction process was 5.0 d without ultrasound pre-treatment, while it was shortened to 4.5 d when using ultrasound at 800 W to treat SSP. However, there were no obvious differences when we applied ultrasound at low power (200 W, 400 W). Furthermore, chitin purified from shrimp shells pre-treated with HIU at 800 W exhibited lower molecular weight (11.2 kDa), higher chitin purity (89.8%), and a higher degree of deacetylation (21.1%) compared to SSP with no ultrasound pre-treatment (13.5 kDa, 86.6%, 18.5%). Results indicate that HIU peels off the protein/CaCO3 matrix that covers the SSP surface. About 9.1% of protein and 4.7% of Ca2+ were released from SSP pre-treated with HIU at 800 W. These figures were both higher than with no ultrasound pre-treatment (4.5%, 3.2%). Additionally, the amount of soluble protein extracted from SSP through HIU at 800 W was 50% higher than for the control sample. SDS-PAGE analysis indicated that the soluble protein was degraded to the micromolecule. It also revealed that HIU (600, 800 W) induced the secondary and tertiary structure destruction of protein extracted from SSP. In conclusion, HIU-induced degradation and structural damage of protein enhances the protein/CaCO3 matrix to be peeled off from SSP. Also, in the co-fermentation process, an increase of protease activity further accelerates deproteinization.

Effect of molecular weight of chitosan on the physicochemical, morphological, and biological properties of polyplex nanoparticles intended for gene delivery

The use of viral vectors in gene therapy has many disadvantages that have been widely described. The development of new nonviral vectors using safe polymers such as chitosan provides a viable alternative. Chitosan molecular weight and nitrogen/phosphorus (N/P) ratio are key factors that determine the shape, size, and surface charge of polyplex nanoparticles (NPs) assembled by complex coacervation for gene therapy; and at the same time, their transfection efficiency is affected by these characteristics, besides the cell line used for transfection. However, there are some inconsistencies in previous reports about the effect of chitosan molecular weight on all these parameters. Enhanced Green Fluorescent Protein plasmid (pEGFP-N1) and two molecular weight (MW) chitosan (20.6 and 57.5 kDa) with a high degree of deacetylation (≥85%) were used to design and assembly nanoparticles by complex coacervation with N/P ratios of 43 and 8. NPs were characterized determining morphology by Scanning Electron Microscopy (SEM), size by Dynamic Light Scattering (DLS) and zeta-potential by Laser-Doppler velocimetry (LDV). Also, biological functionality was assessed by in vitro transfection assays. NPs assembled using the lowest MW chitosan (20.6NPs) and N/P ratio = 8 had spherical shape, smaller mean size and polydispersity index (119 ± 20 nm; 0.257, respectively), and lower zeta-potential (17.65 ± 1.21 mV). Moreover, no significant difference was observed in size, polydispersity index and zeta-potential after 30 days of storage, in contrast with NPs assembled using the 57.5 kDa chitosan (57.5NPs). In vitro transfection assays using two cervical cancer cell lines (HeLa and SiHa) showed that 20.6NPs leaded to a higher expression of Green Fluorescent Protein (GFP) in contrast to 57.5NPs for both cell lines, and expression in HeLa cells was higher than in SiHa cell line. It was confirmed that transfection efficiency depends on all these physical parameters given by MW of chitosan, cell physiology, and the addition of fetal bovine serum in the transfection formulation. These results indicate that low MW chitosan and a low N/P ratio are more suitable to design chitosan-based nonviral vectors for gene therapy, given that physicochemical and biological properties, so as the stability of these NPs are better that those formulated with chitosan of higher MW.

Making a meal out of bugs

Making a meal out of bugs

Green synthesis of chitosan by Cunninghamella elegans UCP 1306 using sustainable substrates mediated morphological changes

Currently, the commercial production of chitosan takes place through the thermochemical deacetylation of crustacean shells, which process requires the use of chemical substances, such as strong alkaline solutions, which are important sources of environment pollution. We present a method for the production of chitosan by Cunninghamella elegans UCP 1306 using a green metabolic conversion of agro-industrial residues, based on a 22 Factorial Design and describe the main morphological changes observed in the specie. The highest yield of biomass (6.375 g/L) and chitosan (101.7 mg/g) were observed in the assay 2 (Corn steep liquor -CSL 4%, Cassava wastewater-CWW 4%), where the specie presented loose and branched hyphae with the presence of septations, pellets of 0.4 to 0.5 mm and clumps of up to 0.1 mm. Statistical analysis showed that higher concentration of CSL contributed significantly to the growth of the species. The bands determined in Fourier transform infrared spectroscopy confirmed the degree of deacetylation of 84.61% of the chitosan obtained. This research showed that the use of CSL and CWW in association was very promising, being able to be used as sustainable sources of nitrogen and carbon in the production of fungal chitosan with a high degree of deacetylation and can be applied to the industrial process.

Impact of Deacetylation Degree on Properties of Chitosan for Formation of Electrosprayed Nanoparticles

Biopolymer of natural origin as chitosan has been studied and applied widely in practice. In the pharmaceutical field, especially, chitosan nanoparticles have been researched for a variety of drug delivery systems. There are many factors influencing the success of the chitosan nanoparticle delivery system. Therein, the specific parameters to the physicochemical nature of chitosan greatly determine the efficiency of its drugs carrier. The degree of deacetylation (DD) of chitosan is one of those parameters. In this study, the influence of DD on chitosan properties was clarified to facilitate the preparation of nanoparticles by the electrospraying method. DD can affect the solubility, crystallinity, and surface tension of chitosan, but it cannot strongly impact the viscosity of chitosan solution as much as the molecular weight (Mv). From these results, M3 chitosan, owning a high DD of 86.70%, and crystalline index of 44%, was dissolved in acetic acid for the collection of electrosprayed nanoparticles. The M3 solution having low viscosity of under 50 mm2/s displayed the easy adjustment of the stable Taylor-cone droplet at the nozzle tip. Particularly, the M3 chitosan solution with a concentration of 1.5 wt.% in acetic acid of 90 wt.% concentration operated at the working condition of 12 kV voltage, a distance between the two electrodes of 10 cm created spherical particles with an average diameter of 338 nm, narrow size distribution. These chitosan nanoparticles can obtain the initial requirement for application as injectable drugs carrier.

Biological Characterization of Different Samples of Chitin and Chitosan for their Application on Biomedical Field

In this study, the degree of acetylation (DA) and degree of deacetylation (DDA) were determined by Fourier Transform Infrared Spectroscopy (FT-IR) analysis, and their viability was evaluated by the cytocompatibility test with L132 epithelial cells, from samples of chitin and chitosan extracted of Cryphiops caementarius. FT-IR measurements resulted in the successful determination of DA and DDA. Particularly the results showed that, the chitosan CHT-C has the highest DDA (93%), while viability tests employed to determine the cytotoxicity of chitin and chitosan showed that the cell survival rate of CH-C and CHT-C are 130% and 75% respectively while commercial CHT has a rate of 80% approximately, demonstrating that the CH-C (chitin) a favorable behavior against L132 cell.

Enhanced antimycotic activity of nanoconjugates from fungal chitosan and Saussurea costus extract against resistant pathogenic Candida strains

Targeting the control of pathogenic Candida spp., especially the fungicides resistant strains from C albicans and C glabrata, nanoconjugates from the biopolymer (chitosan) and costus root extract (Saussurea costus) was synthesized and characterized. Chitosan was extracted from the grown mycelia of Aspergillus niger and characterized with high deacetylation degree of 91.2% and moderate molecular weight of 106.8 kDa. Synthesis of nanoconjugates from fungal chitosan/costus extract (NCt/CE) was conducted using ionic gelation technique; the resulted NCt/CE particles were characterized with mean diameter of 48 nm, positive zeta potentiality (+3.28 mV) and high stability. The infra‐red spectra of synthesized nanoconjugates indicated their strong biochemical cross‐linkage. The antimycotic activities, of the synthesized NCt, CE and their nanocomposite, were evaluated against standard and antibiotic‐resistant strains from C albicans and C. glabrata and revealed that the entire agents had notable antimycotic potentiality against all examined strains; the NCt/CE nanoconjugates had significantly stronger antimicrobial action. The scanning microscope imaging, of exposed resistant strains to NCt/CE, indicated their vigorous structural and morphological alterations and confirmed the antimycotic activity of the nanocomposite. NCt/CE nanoconjugates' synthesis could be exceedingly recommended as a natural, biodegradable and effectual antimycotic agent to control resistant pathogenic yeast strains.

Characterization of chitin and chitosan derived from Hermetia illucens, a further step in a circular economy process

Due to their properties and applications, the growing demand for chitin and chitosan has stimulated the market to find more sustainable alternatives to the current commercial source (crustaceans). Bioconverter insects, such as Hermetia illucens, are the appropriate candidates, as chitin is a side stream of insect farms for feed applications. This is the first report on production and characterization of chitin and chitosan from different biomasses derived from H. illucens, valorizing the overproduced larvae in feed applications, the pupal exuviae and the dead adults. Pupal exuviae are the best biomass, both for chitin and chitosan yields and for their abundance and easy supply from insect farms. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscope analysis revealed the similarity of insect-derived polymers to commercial ones in terms of purity and structural morphology, and therefore their suitability for industrial and biomedical applications. Its fibrillary nature makes H. illucens chitin suitable for producing fibrous manufacts after conversion to chitin nanofibrils, particularly adults-derived chitin, because of its high crystallinity. A great versatility emerged from the evaluation of the physicochemical properties of chitosan obtained from H. illucens, which presented a lower viscosity-average molecular weight and a high deacetylation degree, fostering its putative antimicrobial properties.

Interactions of water-soluble myofibrillar protein with chitosan: Phase behavior, microstructure and rheological properties

This study investigated the electrostatic interaction between water-soluble myofibrillar protein (MP) and chitosan (CH) for further developing muscle protein-based functional foods. Effects of pH (3.0–7.5), protein/polysaccharide mixing ratio (20:1–1:1), ionic strength (0.05–0.6 M KCl) and deacetylation degree (DD) of CH (70–95%) on the interactions were studied by turbidity analysis, phase behavior, zeta-potential, particle size, microstructure and rheological properties. Lower mixing ratios (5:1 and 1:1) improved the colloid stability of MP in mildly acidic environments (pH < 6.5). KCl (0.05–0.6 M) destroyed the colloidal stability and promoted phase separation. A high DD of CH (95%) promoted complexation by increasing the available cationic groups. Rheology displayed that the maximum viscoelasticity for each factor was occurred at a MP/CH ratio of 1:1, pH 6.5 and 95% DD of CH due to the enhanced intermolecular interactions. Fourier transform infrared spectroscopy (FTIR) confirmed that the electrostatic interactions contributed to complex formation, while hydrogen bonds also participated in. This research provided great insight for understanding the interaction of water-soluble MP with CH to further exploit low-salt meat protein-based foods.

Impact of Chitosan on the Mechanical Stability of Soils.

Chitosan is becoming increasingly applied in agriculture, mostly as a powder, however little is known about its effect on soil mechanical properties. Uniaxial compression test was performed for cylindrical soil aggregates prepared from four soils of various properties (very acidic Podzol, acidic Arenosol, neutral Fluvisol and alkaline Umbrisol) containing different proportions of two kinds of chitosan (CS1 of higher molecular mass and lower deacetylation degree, and CS2 of lower molecular mass and higher deacetylation degree), pretreated with 1 and 10 wetting–drying cycles. In most cases increasing chitosan rates successively decreased the mechanical stability of soils that was accompanied by a tendential increase in soil porosity. In one case (Fluvisol treated with CS2) the porosity decreased and mechanical stability increased with increasing chitosan dose. The behavior of acidic soils (Podzol and Arenosol) treated with CS2, differed from the other soils: after an initial decrease, the strength of aggregates increased with increasing chitosan amendment, despite the porosity consequently decreasing. After 10 wetting–drying cycles, the strength of the aggregates of acidic soils appeared to increase while it decreased for neutral and alkaline soils. Possible mechanisms of soil–chitosan interactions affecting mechanical strength are discussed and linked with soil water stability and wettability.

Pickering emulsions stabilized with chitosan/gum Arabic particles: Effect of chitosan degree of deacetylation on the physicochemical properties and cannabidiol (CBD) topical delivery

Pickering emulsions (PEs) have recently gained increasing attention as green carriers of bioactive agents due to their surfactant-free and eco-friendly nature. Herein, the effect of the degree of deacetylation (DDA) of chitosan on the properties of chitosan/gum Arabic (CH/GA) particles and PEs was investigated. CH/GA particles with a DDA of 96% (high DDA) and 78% (low DDA) were prepared. The contact angle of water drop for the CH/GA particles prepared with high and low DDA chitosan was 84.2° and 77.5°, respectively. The dynamic interfacial tension of the particles has shown lower values with high DDA chitosan. The high DDA PE formulation exhibited higher stability after two months of storage than its counterpart prepared with low DDA chitosan. The mean emulsion droplet size was 15.6 ± 4.3 µm and 21.1 ± 2.6 µm for PEs prepared with high and low DDA chitosan, respectively. Both PE formulations demonstrated shear thinning and elastic gel-like properties. The amount of cannabidiol absorbed by the stratum corneum, viable epidermis and dermis was significantly higher in both PE formulations than the permeated amount, but no significant difference was observed between the amounts absorbed from both formulations.

Effects of different degrees of deacetylation and levels of chitosan on performance, egg traits and serum biochemistry of laying hens

ABSTRACT The present study was conducted to evaluate whether the deacetylation degree of chitosan (low: 70% vs. high: 90%) and its dietary level (0, 200, 400, 800, 1600 mg/kg diet) would affect laying performance, faeces viscosity, egg quality, egg and serum biochemistry of layers. For the experimental feeding period of 8 weeks, 140 four weeks old Hisex Brown layers were divided into 10 treatment groups, comprising 14 birds each. The birds were housed in individual cages in a complete randomised design. Performance was assessed by recording feed intake, egg weight, daily egg production, egg quality and egg biochemistry. Serum biochemistry parameters were determined at the beginning and end of the experiment and faeces viscosity at the end of the experiment. Feed conversion ratio and faeces viscosity were deteriorated by increased level of chitosan. Lightness of egg yolk was significantly increased in animals receiving high-degree deacetylated chitosan compared to low-degree deacetylated chitosan. Yellowness of egg yolk was affected by interaction of deacetylation degree and level of chitosan. Yolk cholesterol concentration was lower in groups receiving high deacetylated chitosan by increasing chitosan level, while laying hens fed low deacetylated chitosan had a higher level of yolk cholesterol. A significant interaction between degree of deacetylation and chitosan level was determined for serum glucose and calcium concentration. Serum total antioxidant content increased with higher levels of dietary chitosan. In conclusion, dietary level or different degrees of deacetylated chitosan may reduce yolk cholesterol and improve serum antioxidant status. However, feed conversion ratio and faeces viscosity were impaired by increasing levels of chitosan supplementation, and lightness of yolk was increased by supplementation of chitosan with a high degree of deacetylation.

Scaffolds from medical grade chitosan: A good choice for 3D cultivation of mesenchymal stem cells.

Chitosan has high biocompatibility, supports proliferation of many cells, and can be a good carrier for various growth factors. However, low attachment ratio and spheroid formation of several stem cell types on plain chitosan scaffolds/films is still a problem. In this study, it was aimed to obtain 3D scaffolds using medical grade chitosan (MC) with a high deacetylation degree (DD ≥ 92.6%) to overcome the spheroid formation of rat adipose tissue derived mesenchymal stem cells (rAdMSCs) on control chitosan (C, DD = 75%-85%) scaffolds. Genipin was used as a biological chemical crosslinker, and glycerol phosphate salt was used both as a pH adjusting agent and physical crosslinker. MTT and SEM analyses and live/dead staining indicated the increase in the attachment, cell viability, and proliferation of rAdMSCs on MC scaffolds with or without crosslinking when compared to the cells in spheroid formation on control scaffolds. Moreover, filamentous actin protein organization of rAdMSCs was found to be triggered on the crosslinked MC scaffolds. In conclusion, plain medical grade chitosan scaffolds with or without crosslinking prevented spheroid formation, supported the attachment, proliferation, and organization of rAdMSCs indicating that medical grade type of chitosan scaffolds with high DD can be a very good candidate as 3D carriers in stem cell cultivation.

Comparison of Chitosan Characterization from Mussel Shell Waste Using Varying Concentration of Solvents

Massive amounts of mussel shell waste are generated and wasted from the aquaculture processing sectors, resulting in environmental pollution. This material contains chitosan as a valuable compound characterized as a non-toxic structural component with several food processing applications or medicinal applications. In this research, mussel shells were processed using different solvents concentrations in several stages: demineralization, deproteination, decolourization, and deacetylation. Our result showed that the C2 samples gained a high degree of deacetylation (31.8±0.21%) with low moisture and ash content and medium weight of yield. Further research is recommended to purify chitosan using various instrumentation and assess its bioactivity.

Impact of Chitosan on Water Stability and Wettability of Soils.

Chitosan has become increasingly applied in agriculture worldwide, thus entering the soil environment. We hypothesized that chitosan should affect the water stability of soil. Since this problem has not been studied to date, we examined, for the first time, the influence of chitosan on the water stability and wettability of soil aggregates. The aggregates were prepared from four soils with various properties amended with different amounts of two kinds of powdered chitosan, and subjected to 1 and/or 10 wetting–drying cycles. The water stability was measured by monitoring air bubbling after aggregate immersion in water, and the wettability was measured by a water drop penetration test. The biopolymer with a lower molecular mass, lower viscosity, and higher degree of deacetylation was more effective in increasing the water stability of the soil than the biopolymer with a higher molecular mass, higher viscosity, and lower deacetylation degree. After a single wetting-drying cycle, the water stability of the soil aggregates containing chitosan with a higher molecular mass was generally lower than that of the soil; after ten wetting–drying cycles, the water stability increased 1.5 to 20 times depending on the soil. The addition of low-molecular-mass chitosan after a single wetting-drying cycle caused the water stability to become one to two hundred times higher than that of the soil. A trial to find out which soil properties (pH, C and N content, bulk density, porosity, and particle size distribution) are responsible for the effectiveness of chitosan action was not successful, and this will be the objective of further studies.

Optimisation of conditions for deacetylation of chitin-containing raw materials

The paper describes the differences between chitosan and chitin, and reviews works by foreign scientists on obtaining chitosan from various raw materials. Methods of modifying chitosan and obtaining combined sorbents have been analysed. It has been studied whether chitosan is applicable in the technology of wines and alcoholic beverages as a sorbent. The purpose of the study was determining the optimal conditions of the deacetylation stage to obtain chitosan with the best sorption properties from Aspergillus niger biomass. A three-factor experiment has been carried out. It involved obtaining 27 samples of chitosan using sequential four-step acid-base hydrolysis under various conditions of the deacetylation stage. The deacetylation process was optimised under alkaline conditions depending on the alkali concentration, processing temperature, and exposure. For each of the samples obtained, the adsorption activity, specific surface area, and distribution coefficient in the sorbent–sorbate system have been determined. The degrees of deacetylation of all chitosan samples have been determined by potentiometric titration. The study has resulted in determining the optimal conditions for the deacetylation stage: processing temperature 110–130°C, sodium hydroxide concentration 27–36 g/dm3, exposure 45 to 65 minutes. The sample deacetylated at the temperature 120 °C, alkali concentration 30 g/dm3, and exposure 45 minutes has shown the best adsorption activity values: the adsorption activity for methyl orange 347.96 mg/g, the specific surface area of the sorbent samples 0.52·105 m2/g, the distribution coefficient in the sorbent–sorbate system 3.29·10-3 ml/g. This sample had the highest degree of deacetylation, 43.6%. The sample has been analysed using IR spectroscopy, and its main characteristic frequencies have been studied. It has been concluded that the sample obtained was equivalent to the reference chitosan

Positive Effects and Mechanism of Ultrasound on Chitin Preparation from Shrimp Shells by Co-Fermentation

The objective of this study is to explore the effect and mechanism of ultrasound on chitin extraction from shrimp shells (SSP) by the co-fermentation of Bacillus subtilis and Acetobacter pasteurianus. After pre-treating the SSP with high-intensity ultrasound (HIU, 800 W), the protease activity in the fermentation solution reached 96.9 U/mL on Day 3, which was significantly higher than for SSP that had not been pre-treated with ultrasound (81.8 U/mL). The fermentation time of the chitin extraction process was 5.0 days without ultrasound pre-treatment, while it was shortened to 4.5 days when using 800 W ultrasound to treat SSP. However, there were no obvious differences when we applied low-intensity ultrasound (200 W, 400 W) treatment. Furthermore, chitin purified from shrimp shells pre-treated with HIU exhibited lower molecular weight (11.2 kDa), higher chitin purity (89.8%), and a higher degree of deacetylation (21.1%) compared to SSP with no ultrasound pre-treatment (13.5 kDa, 86.6%, 18.5%). Results indicate that HIU peels off the protein-CaCO3 matrix that covers the SSP surface. About 9.1% of protein and 4.7% of Ca2+ were released from SSP pre-treated with 800 W ultrasound. These figures are both higher than with no ultrasound pre-treatment (4.5%, 3.2%). Additionally, the amount of soluble protein extracted from SSP through HIU was 50% higher than for the control sample. SDS-PAGE analysis indicated that the soluble protein was degraded to the micromolecule. It also revealed that HIU induced the secondary and tertiary structure destruction of protein extracted from SSP. In conclusion, HIU-induced degradation and structural damage of protein enhances the protein-CaCO3 matrix to be peeled off from SSP. Also, in the co-fermentation process, an increase of protease activity further accelerates deproteinization.