Green Foaming of Biologically Extracted Chitin Hydrogels Using Supercritical Carbon Dioxide for Scaffolding of Human Osteoblasts.

Chitin is a structural polysaccharide abundant in the biosphere. Chitin possesses a highly ordered crystalline structure that makes its processing a challenge. In this study, chitin hydrogels and methanogels, prepared by dissolution in calcium chloride/methanol, were subjected to supercritical carbon dioxide (scCO2) to produce porous materials for use as scaffolds for osteoblasts. The control of the morphology, porosity, and physicochemical properties of the produced materials was performed according to the operational conditions, as well as the co-solvent addition. The dissolution of CO2 in methanol co-solvent improved the sorption of the compressed fluid into the hydrogel, rendering highly porous chitin scaffolds. The chitin crystallinity index significantly decreased after processing the hydrogel in supercritical conditions, with a significant effect on its swelling capacity. The use of scCO2 with methanol co-solvent resulted in chitin scaffolds with characteristics adequate to the adhesion and proliferation of osteoblasts.

Antioxidant and antimicrobial material by grafting of L-arginine onto enzymatic poly(gallic acid).

Microwave-mediated grafting of L-Arg onto naturally derived and stable multiradical poly(gallic acid) (PGAL) in aqueous media has been successfully achieved. This polymeric material has no adverse effect in human cells as there is no hemolytic activity upon MTT and Neutral Red assays. The analytical and computational characterization studies carried out in this study describe a helical molecular structure with random incorporation of L-Arginine pendant groups from PGAL's backbone. The antioxidant properties of the precursor polymer are preserved as proved by the elimination of stable DPPH and hydroxyl radical scavenging, as well as the FRAP and ORAC assays. Regarding the latter, the oxygen radical inhibition is enhanced compared to PGAL, which is attributed to the guanidyl moieties. PGAL-g-L-Arg displays antimicrobial activity against Gram (+) Listeria monocytogenes and Staphylococcus aureus strains with a MIC of 0.8 g/L and a bacteriostatic effect against Gram (-) Escherichia coli. Additionally, scanning electron and confocal fluorescence microscopies as well as crystal violet colorimetric assay demonstrate that the mechanism involved in the bacterial inhibition is related to the formation of porous channels on the membrane, which is discussed according to the helical secondary structure of the polymer and the amino acid guanidyl moieties interacting to bacterial membranes.

Copper removal from wastewater by a chitosan-based biodegradable composite.

This work demonstrates that a biodegradable chitosan-based biocomposite packed in mini-reactors successfully removes copper ions from aqueous solutions. The chitosan is obtained by deacetylation of biological chitin, which is extracted from shrimp wastes by lactic acid fermentation. The polysaccharide is embedded in a biodegradable prepolymer matrix before extrusion to produce porous cylindrical pellets of 2 × 80 mm. The highest copper ion removal is 62.5 mg Cu2+ per g of the biodegradable adsorbent. Additionally, the adsorption capacity of the material, below its saturation, allows several cycles of reuse with a hydraulic retention time reduction of 1 h. This chitosan-based material is advantageous when compared with other approaches using non-biodegradable materials or costly commercial adsorbents for removing heavy metal ions in wastewater effluents as well as a filter component in water purification devices.

Novel transglycosylation activity of ß-N-acetylglucosaminidase of Lecanicillium lecanii produced by submerged culture.

N-acetylglucosaminidase produced from Lecanicillium lecanii on submerged culture displayed hydrolytic and transglycosylation activities. The highest specific activity for the enzyme was 1.87 U/mg after 120 h of culture. The chromatographic purification for a single protein fraction showed a molecular weight of 50.4 kDa and hydrolytic N-acetylglucosaminidase activity of 17.59 U/mg at 37 °C and pH 6. This enzyme was able to transglycosylate and to synthesize oligosaccharides from 2 to 6 units with a degree of acetylation between 100 and 26% employing glucose, mannose, N-acetyl-D-glucosamine and N-acetyl-D-lactosamine as donor substrates. Optimal conditions of temperature and pH were determined for both types of enzymatic activities.

The Radical-Scavenging Activity of a Purified and Sequenced Peptide from Lactic Acid Fermentation of Thunnus albacares By-Products.

Yellowfin tuna by-products (Thunnus albacares) were processed to produce radical-scavenging peptides from hydrolysis by lactic acid fermentation (LAF) with Lactobacillus plantarum, papaya fruit (Carica papaya), and molasses as a carbon source for 72 h. A 15-kDa peptide was purified; after de novo sequencing, it was determined that fragments are rich in hydrophobic and neutral amino acids. The results suggest this effect is mainly to the hydrophobicity of the amino acids in their sequence. Further work is on progress to assess the ability of peptides to provide stability in lipids or in other types of samples sensitive to the action of free radicals.

Cross-Linking Chitosan into Hydroxypropylmethylcellulose for the Preparation of Neem Oil Coating for Postharvest Storage of Pitaya (Stenocereus pruinosus).

The market trend for pitaya is increasing, although the preservation of the quality of this fruit after the harvest is challenging due to microbial decay, dehydration, and oxidation. In this work, the application of antimicrobial chitosan-based coatings achieved successful postharvest preservation of pitaya (Stenocereus pruinosus) during storage at 10 ± 2 °C with a relative humidity of 80 ± 5%. The solution of cross-linked chitosan with hydroxypropylmethylcellulose with entrapped Neem oil (16 g·L-1) displayed the best postharvest fruit characteristics. The reduction of physiological weight loss and fungal contamination, with an increased redness index and release of azadirachtin from the microencapsulated oil, resulted in up to a 15 day shelf life for this fruit. This postharvest procedure has the potential to increase commercial exploitation of fresh pitaya, owing to its good taste and high content of antioxidants.

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles.

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1 µg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe3O4@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

Effect of bio-chemical chitosan and gallic acid into rheology and physicochemical properties of ternary edible films.

The ternary edible films based on commercial or bio-chemical chitosan (CCh or BCh) and starch (S) with different concentration of gallic acid (GA) were produced by casting. This work analyzed the effect of different type of Ch and GA concentrations (1.25 mg/gTB and 2.5 mg/gTB) on the rheological properties of the film-forming solutions, as well as physicochemical (thickness, moisture content, swelling index, water vapor permeability (WVP), tensile strength, microstructure, opacity, and color) and antioxidant properties of the edible films themselves. The chemical cross-linking between the GA and Ch into the edible films of biopolymers could form of ester linkages between the carboxyl groups of GA and the hydroxyl groups of Ch. Results showed that the ternary edible films of BCh-S-GA5.0 resulted with a positive effect on their rheological (η0, λ, and p), physicochemical (WVP, swelling, tensile strength, microstructure) and antioxidant properties. The greatest incorporation of polyphenol into the BCh-S-GA5.0 films greatly reduced WVP and swelling values in approximately 61.01% and 23%, respectively also the tensile strength value increased to 37.372 MPa. The ternary edible films developed in this study showed many desirable characteristics, which could potentially be used as bioactive packaging films for food applications.

Polyelectrolyte complex of Aloe vera, chitosan, and alginate produced fibroblast and lymphocyte viabilities and migration.

Chitosan, sodium alginate and gel of Aloe vera (Aloe barbadensis Miller) were employed for the preparation of polyelectrolyte complexes at pH 4 and 6. FT-IR spectroscopy analysis showed evidence on complexes formation and incorporation of the Aloe vera gel. The ζ potential determination of the polyelectrolyte complexes revealed the presence of surface charges in the range of -20 to -24 mV, which results in stable systems. The dynamic moduli exhibited a high dependence on angular frequency, which is commonly found in solutions of macromolecules. The materials showed human fibroblast and lymphocyte viabilities up to 90% in agreement with null cytotoxicity. The polyelectrolyte complexes at pH 6 with Ca2+ were stable, showed high water absorption, satisfactory morphology, pore size and rigidity, characteristics that allowed significant human fibroblast migration in wound closure in vitro assays.

Polycyclic aromatic hydrocarbon-emulsifier protein produced by Aspergillus brasiliensis (niger) in an airlift bioreactor following an electrochemical pretreatment.

An emulsifier protein (EP) was produced and easily separated from oil-contaminated water as an economical substrate when Aspergillus brasiliensis, pretreated in a solid state culture with a controlled electric field, was used in an airlift bioreactor. The hydrocarbon-EP comprised 19.5% of the total protein, its purification enhanced the specific emulsifying activity (EA) seven times. The influence of operational conditions (pH and salt concentration) on the EA were assessed to characterise the emulsion stability. The EA was increased by 19% in alkaline environments (pH 7-11), but it was not affected by the presence of salt (0-35 g L-1). On the other hand, preheating the EP samples (60 °C) enhanced the EA by 2.5 times. Based on analysis of its EA, this EP can be applied as a bioremediation enhancer in contaminated soils.

Production and characterization of a nanocomposite of highly crystalline nanowhiskers from biologically extracted chitin in enzymatic poly(ε-caprolactone).

A nano-composite from biologically obtained chitin nanofillers homogenously dispersed in a poly(ε-caprolactone) matrix was successfully achieved by an ultrasonication-assisted non-toxic and non-aqueous methodology. For this purpose, biological chitin was obtained from lactic acid fermentation of shrimp wastes and converted into chitin whiskers by acidic hydrolysis in a novel process at low temperature (4°C) that enhanced the distribution and yield. Additionally, the polyester matrix was enzymatically produced in a non-toxic compressed fluid (1,1,1,2-tetrafluoroethane at 25bar and 65°C) medium. The homogeneous distribution of the nanofiller in the matrix was corroborated by confocal and atomic force microscopies. Films of the nanocomposite were physicochemically characterized to assess its adequate properties. Additionally, the qualitative viability of human fibroblasts and osteoblasts cells was studied on the produced nanocomposite films showing good biocompatibility.

Hydroxyapatite crystallization in shrimp cephalothorax wastes during subcritical water treatment for chitin extraction.

The extraction of calcareous chitin from shrimp cephalothorax was successfully achieved using a subcritical water treatment to attain a deproteinization up to 96%. The treatments also increased the crystalline domain size in the α-chitin fibers. An experimental design of Taguchi allowed the optimization of experiments. The macroelements identified in all samples were Ca, P, S, K, Cl and Al, whereas Cr, Mn, Fe, Ni, Cu, Zn, Br and Sr were also detected as microelements. The assigned crystalline phases by XRD were α-chitin, calcite, HAP and traces of quartz. The presence of these phases was corroborated by ATR-FTIR and SEM-EDS analyses. The highest content of α-chitin (82.2wt%) was obtained for the 0.17 chitin:dH2O (wt/wt) ratio for 30min treatment at 260°C. Noteworthy, this treatment promotes the crystallization of both minerals as microcrystals of calcite and nanocrystals of hydroxyapatite with needle and flake shapes as well as intermediate morphologies.

Fungal biocatalyst activated by an electric field: Improved mass transfer and non-specificity for hydrocarbon degradation in an airlift bioreactor.

The combination of biological and electrochemical techniques enhances the bioremediation efficiency of treating oil-contaminated water. In this study a non-growing fungal whole cell biocatalyst (BC; Aspergillus brasiliensis attached to perlite) pretreated with an electric field (EF), was used to degrade a hydrocarbon blend (hexadecane-phenanthrene-pyrene; 100:1:1w/w) in an airlift bioreactor (ALB). During hydrocarbon degradation, all mass transfer resistances (internal and external) and sorption capacity were experimentally quantified. Internal mass transfer resistances were evaluated through BC effectiveness factor analysis as a function of the Thiele modulus (using first order reaction kinetics, assuming a spherical BC, five particle diameters). External (interfacial) mass transfer resistances were evaluated by kLa determination. EF pretreatment during BC production promoted surface changes in BC and production of an emulsifier protein in the ALB. The BC surface modifications enhanced the affinity for hydrocarbons, improving hydrocarbon uptake by direct contact. The resulting emulsion was associated with decreased internal and external mass transfer resistances. EF pretreatment effects can be summarized as: a combined uptake mechanism (direct contact dominant followed by emulsified form dominant) diminishing mass transfer limitations, resulting in a non-specific hydrocarbon degradation in blend. The pretreated BC is a good applicant for oil-contaminated water remediation.

Inhibition of Listeria monocytogenes in Fresh Cheese Using Chitosan-Grafted Lactic Acid Packaging.

A chitosan from biologically obtained chitin was successfully grafted with d,l-lactic acid (LA) in aqueous media using p-toluenesulfonic acid as catalyst to obtain a non-toxic, biodegradable packaging material that was characterized using scanning electron microscopy, water vapor permeability, and relative humidity (RH) losses. Additionally, the grafting in chitosan with LA produced films with improved mechanical properties. This material successfully extended the shelf life of fresh cheese and inhibited the growth of Listeria monocytogenes during 14 days at 4 °C and 22% RH, whereby inoculated samples with chitosan-g-LA packaging presented full bacterial inhibition. The results were compared to control samples and commercial low-density polyethylene packaging.

Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5.

Suppression of the tert-butylhydroquinone toxicity by its grafting onto chitosan and further cross-linking to agavin toward a novel antioxidant and prebiotic material.

The enzyme-mediated grafting of tert-butylhydroquinone (TBHQ) onto chitosan and further crosslinking to agave inulin (agavin) has been successfully achieved in a mild and non-toxic two-step route. The resulting products were characterized by nuclear magnetic resonance (NMR) and Infra-red spectroscopies to assess the molecular structure. The study of acute oral toxicity in mice revealed no adverse short-term effects of consumption in the synthesized materials with non-toxicity evidence until 2000 mg/kg through an oral acute administration. Importantly, this study proves that the compound maintains the radical scavenging capacity of the phenolic antioxidant upon ferric-reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays with a measured half-maximal inhibitory concentration (IC50) for the best case of 1.54 g/L based on inhibition of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid diammonium salt (ABTS). Additionally, the novel compound presented high prebiotic activities as ascertained in the presence of lactic acid bacteria (LAB).

Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii.

In this study, chitin oligosaccharides have been successfully produced using chitinases from submerged fermentation of Lecanicillium lecanii. The highest Hex, Chit and Prot production was 0.14, 0.26 and 2.05 U/mg of protein, respectively, which were attained varying pH from 5 to 8 after 96 h. Culture conditions conducted at constant pH of 6 resulted in significantly lower enzyme production. The crude enzyme was partially purified by salting out with (NH4)2SO4 followed by size exclusion chromatography to isolate the chitinase mixture for further chitin hydrolysis assays. In this regard, chitin substrates were pretreated with sonication and steam explosion prior to enzymatic reaction. Structural changes were observed with steam explosion with 11.28% reduction of the crystallinity index attained with the lowest chitin/water ratio (0.1g/mL). Pretreated chitins reached the highest production of reducing sugars (0.37 mg/mL) and GlcNAc (0.59 mg/mL) in 23.6% yield.

Activity of chitin deacetylase from Colletotrichum gloeosporioides on chitinous substrates.

Production of chitin deacetylases from the phytopathogenic fungus Colletotrichum gloeosporioides was successfully achieved by submerged fermentation. The highest specific activity of 0.018 U mg(-1) of protein was obtained after 96 h of cultivation at pH 6 and 28°C. Two bands with molecular weights of 35 kDa and 170 kDa determined with SDS-PAGE displayed deacetylase activities as detected in the zymograms. Reacetylated commercial chitosan (52% acetylation degree) was used as substrate for the extracellular crude extract in order to estimate the kinetic parameters of acetate production as undirected deacetylation measurement. The highest acetate production of 12.8 µmol mL(-1) was obtained using 7.5 mg mL(-1) of substrate. The produced enzyme from C. gloeosporioides achieved up to 25% deacetylation of a chitin substrate (hydrolyzed biological chitin) having 80% degree of acetylation, MW of 102×10(3) g mol(-1) and a crystallinity index of ca. 60%.

Lipase-catalyzed synthesis of hyperbranched poly-L-lactide in an ionic liquid.

Hyperbranched poly-L-lactides have been synthesized by eROP in [C4MIM][PF6] media. The bis(hydroxymethyl)butyric acid molecule was used as the AB2 core co-monomer and immobilized lipase B from Candida antarctica as biocatalyst. The degree of branching could be controlled by the reaction conditions, with the maximum achieved being 0.21. The successful achievement of the hyperbranched structure is attributed to the high solvent power of substrates and products in the ionic liquid besides sustained lipase activity.

Morphological changes, chitinolytic enzymes and hydrophobin-like proteins as responses of Lecanicillium lecanii during growth with hydrocarbon.

Lecanicillium lecanii, Verticillium chlamydosporium, V. fungicola var flavidum and Beauveria bassiana were evaluated on their growth with pure n-hexane, toluene and n-hexane:toluene 17:83 (v:v) mixture. Another set of treatments were conducted with colloidal chitin as additional carbon source. All the strains of Lecanicillium were able to grow using hydrocarbons with or without the addition of chitin, although the presence of hydrocarbons showed significant inhibition evidenced by measured biomass, radial growth and microscopic analyses. Degradation of n-hexane ranged within 43 and 62 % and it was higher than that with toluene. The strains L460, L157 and L2149, which presented the highest growth, were further selected for determinations of hydrocarbon consumptions in microcosms. Strain L157 showed the highest consumption of n-hexane (55.6 %) and toluene (52.9 %) as sole carbon source and it also displayed activities of endochitinases, N-acetylhexosaminidase and production of hydrophobins class I and II.