Surimi wash water treatment for protein recovery: effect of chitosan-alginate complex concentration and treatment time on protein adsorption

Chitosan (Chi), a protein recovery agent for the treatment of aqueous food processing streams, appears to work by mechanical entrapment and electrostatic interaction of chitosan amino groups with anionic groups on proteins. Chitosan effectiveness for recovering soluble proteins from surimi wash water (SWW) is increased by complexation with alginate (Alg) and by adjusting complex concentration and treatment time. Flocculation at 20 degrees C with Chi-Alg at a 0.2 mixing ratio added as 20, 40, 100 and 150 mg/L SWW was aided by 5 min agitation at 130 rpm and then held at the same temperature for 30 min, 1 and 24 h. Turbidity measurements, protein determinations and qualitative FTIR analysis confirmed SWW protein adsorption which depended on Chi-Alg concentration and reaction time while turbidity reduction was affected by concentration only. No differences (p < 0.05) in protein adsorption were found between 1 and 24 h. Using 100 mg Chi-Alg complex/L SWW for 1 h achieved 83% protein adsorption and 97% turbidity reduction.     

Successful removal of p-quinone with chitosan in an aqueous phase in relation to degree of deacetylation

Phenol oxidant is successfully removed by using chitosan particles in the aqueous phase. Removal of p-quinone by chitosan from crab shells was investigated kinetically from molecular weight (MW) of chitosan, deacetylation degree (DD) and reaction temperature. The rate constant assuming first-ordered reaction on removal of p-quinone in aqueous phase primarily depended on the MW of chitosan, not on the DD. Quantities of chitosan exceeding 5 x 10(5) MW are able to obtain a sufficiently high rate constant (10(-3) s(-1)). At higher temperatures, higher rate constants were obtained in the entire experimental MW and DD. The activation energy obtained was 43.8 kJ x mol(-1).     

Chitosan-based coagulating agents for treatment of cheddar cheese whey

Chitosan-Polyanion (Chi-Pol) complexes were used as coagulating agents for treating Cheddar cheese whey. Complexation and coagulation time played a significant role in adsorption, whereas polymer concentration was significant only for chitosan-alginate complexes. Complexes of chitosan with alginate (ALG), pectin (PEC), and carrageenan (CAR) used at 30 mg complex/L whey showed turbidity reductions of 40-43% and 65-72% after 1 and 39 h, respectively. At 10 mg/L, the percent reduction in turbidity after 1 and 39 h were 35-39% and 61-64%, respectively. No significant differences in turbidity reduction (P > 0.05) were observed when using complexes at different Chi-Pol monomeric mixing ratios (MR) except for Chi-Alg at 30 mg/L, wherein reduction at 0.2 was higher than 0.8 MR. Also, UV-vis spectroscopy suggested the preference of this complex for the absorption of specific whey protein fractions. This study successfully demonstrated the effectiveness of Chi-Pol complexes in flocculation of suspended solid wastes in cheese whey with over 70% protein recovery.     

Bioconversion of industrial wastewater and wastewater sludge into Bacillus thuringiensis based biopesticides in pilot fermentor

Starch industry wastewater (SWW), slaughterhouse wastewater (SHWW) and secondary sludges from three different wastewater treatment plants (Jonquière--JQS, Communauté Urbaine de Québec--CUQS and Black lake-BLS) were used as raw materials for the production of Bacillus thuringiensis (Bt) based biopesticides in a pilot scale fermentor (100 L working volume). The slaughterhouse wastewater exhibited the lowest Bt growth and entomotoxcity (Tx) potential (measured against spruce budworm) due to low availability of carbon, nitrogen and other nutrients. Performance variation (growth, sporulation, proteolytic activity and Tx potential) within the three types of sludges was directly related to the availability of nitrogen and carbohydrates, which could change with sludge origin and methods employed for its generation. The Tx potential of Bt obtained in different secondary sludges (JQS: 12 x 10(9) SBU/L; CUQS: 13 x 10(9) SBU/L and BLS: 16 x 10(9) SBU/L) and SWW (18 x 10(9) SBU/L) was higher than the soybean based synthetic medium (10 x 10(9) SBU/L). The maximum protease activity was obtained in CUQ secondary sludge (4.1 IU/mL) due to its high complex protein concentration. Nevertheless, high carbohydrate concentration in SWW repressed enzyme production. The secondary sludges and SWW were found to be suitable raw materials for high potency Bt biopesticide production.     

Determination of the Mark-Houwink equation for chitosans with different degrees of deacetylation.

The values of k and alpha in the Mark-Houwink equation have been determined for chitosans with different degrees of deacetylation (DD) (69, 84, 91 and 100% respectively), in 0.2 M CH3COOH/0.1 M CH3COONa aqueous solution at 30 degrees C by the light scattering method. It was shown that the values of alpha decreased from 1.12 to 0.81 and the values of k increased from 0.104 x 10(-3) to 16.80 x 10(-3) ml/g, when the DD varied from 69 to 100%. This is due to a reduction of rigidity of the molecular chain and an increase of the electrostatic repulsion force of the ionic groups along the polyelectrolyte chain in chitosan solution, when the DD of chitosan increases gradually.     

The influence of the degree of cross-linking on the adsorption properties of chitosan beads

The influence of the degree of cross-linking (DCL) on chitosan beads was studied. Chitosan was prepared from the exoskeleton of Cape rock-lobsters, collected from the surroundings of Cape Town, South Africa. The chitosan beads were characterized; the beads water contents and pKa varied in the range of 90-96% and 4.3-6.0, respectively, and were found to decrease with increasing DCL (0.0-34.0%). A pH-model, which described the reversibility of the metal adsorbed onto the beads, was used to predict the equilibrium properties of copper adsorption onto the cross-linked beads. The model accounts for the effect of pH and the important model parameters, the equilibrium adsorption constant (Kads) and to a lesser extent the adsorbent adsorption capacity (qmax) showed to decrease with the DCL. The adsorbent capacity and the adsorption constant were determined as 3.8-5.0mmol/g chitosan and (9-90)x10(-4), respectively. The adsorption kinetics could be described using a shrinking core model and the effective diffusion coefficient (Deff) was determined as (8.0-25.8)x10(-11)m2/s. It was found that Deff decreases with the DCL mainly due to the decreased in water content of the beads at high DCL.     

Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction

A new method to determine the degree of deacetylation (DD) of alpha-chitin and chitosan in the range of 17-94% DD using X-ray powder diffraction (XRD) is proposed. The results were calibrated using (1)H NMR spectroscopy for chitosan and FTIR for chitin, in comparison with the potentiometric titration method. The results showed a good linear correlation between the CrI020 from XRD and the calibrated DD value. This method is found to be simple, rapid and nondestructive to the sample.     

Synthesis and characterization of soluble concanavalin A oligomer

Concanavalin A, (Con A, MW 26,500/monomer unit) was crosslinked with glutaraldehyde to form soluble, high-molecular-weight (larger than MW 300,000) Con A Oligomers. After filtration to remove insoluble and low-molecular-weight portions (below 300,000 daltons), the size and molecular-weight distribution were characterized by laser light scattering and gel-filtration chromatography. The molecular-size determined by laser light scattering ranged from 870 to 4070 A, while the molecular weight determined by gel chromatography ranged from 6 x 10(5) to higher than 2 x 10(6) daltons. The affinity and kinetics of Con A oligomer binding to polysaccharide (glycogen) were evaluated by precipitation test and turbidity development, respectively. The binding with glycogen was strongest at neutral pH and showed similar activity to unmodified Con A molecules. The binding constants of alpha-D-glucose and succinyl-aminophenyl alpha-D glucopyranoside-insulin to Con A oligomer were 1.0 x 10(3)M(-1) and 4.5 x 10(4)M(-1), respectively and the binding capacity of the oligomer was nearly 85% to 95% of monomeric Con A. The complexes of saccharides and soluble Con A oligomer were stable for at least 7 days. (c) 1993 Wiley & Sons, Inc.     

Study of the interpolyelectrolyte reaction between chitosan and alginate: influence of alginate composition and chitosan molecular weight

The interpolyelectrolyte reaction between chitosan (CHI) and alginate (ALG) was followed by conductimetry and potentiometry. Five chitosan samples, all with almost the same degree of N-acetylation (DA approximately 0.20) and molecular weights ranging from 5 x 10(3) to 2.5 x 10(5) Da were used. The polyelectrolyte complex was formed using alginate samples with three different M/G values (0.44, 1.31 and 1.96). The composition of the complex, Z (Z = [CHI]/[ALG]) resulted 0.70 +/- 0.02, independently of the molecular weight of chitosan and the composition of the alginate used. The degree of complexation was 0.51 with no dependence on the alginate composition.     

Purification of ferrichrome synthetase from Aspergillus quadricinctus and characterisation as a phosphopantetheine containing multienzyme complex

Aspergillus quadricinctus was grown under iron limitation to induce the enzymes for ferrichrome biosynthesis. The mycelium was disintegrated by ultraturrax homogenization, and ferrichrome synthetase was purified by column chromatography on DEAE cellulose, hydroxyapatite and Bio-Gel A-5m. The enzyme was almost homogeneous in single fractions as shown in gel electrophoresis under non-denaturating conditions. By fast-protein liquid chromatography on Superose 6, the purified ferrichrome synthetase (molecular weight 9.6.10(5) dissociated partly into an enzyme complex with reduced ferrichrome synthetase activity of 8 x 10(5) Da, one acetylhydroxyornithine (AHO) activating protein of 5.5 x 10(5) Da and one glycine activating protein of 4 x 10(5) Da. After SDS treatment the AHO activating protein dissociated into subunits of 9 x 10(4) Da, while the glycine activating protein dissociated into subunits of 5 x 10(4) Da and 4 x 10(4) Da in a molar ratio of 6:1. No subunits were found after SDS treatment of the larger of the two ferrichrome synthetizing enzyme complexes. Pantetheine was detected in protein bands of defined molecular weights (4 x 10(4), 9 x 10(4) and greater than 3.4 x 10(5) after SDS polyacrylamide gel electrophoresis. Gel slices were cut out, and the growth factor activity for Lactobacillus plantarum ATCC 8014 was analyzed. The calculated content was 2 mol of pantetheine per mol of ferrichrome synthetase of 9.6 x 10(5) Da.     

Chitosan-Mediated siRNA Delivery <Emphasis Type="Italic">In Vitro</Emphasis>: Effect of Polymer Molecular Weight,Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.     

Renin inhibition activity by chitooligosaccharides

Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity.     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.     

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.     

Fungal chitosan production and its characterization

AIMS: The objective of this investigation was to evaluate the chitosans produced by several species of fungi. METHODS AND RESULTS: Representatives of four species of filamentous fungi, Aspergillus niger, Rhizopus oryzae, Lentinus edodes and Pleurotus sajo-caju, and two yeast strains, Zygosaccharomyces rouxii TISTR5058 and Candida albicans TISTR5239, were investigated for their ability to produce chitosan in complex media. Fungal chitosan was produced at 10-140 mg g-1 cell dry weight, had a degree of deacetylation of 84-90% and a molecular weight of 2.7 x 104-1.9 x 105 Da with a viscosity of 3.1-6.2 centipoises (cP). CONCLUSIONS: Rhizopus oryzae TISTR3189 was found to be the producer of the highest amounts of chitosan. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial chitosan could be obtained from Rhizopus mycelia and would have potential applications for medical and agricultural uses.     

Impact of pulsed Nd:YAG laser on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora: significance of physiological status

The impact of pulsed Nd:YAG (neodymium-doped yttrium/aluminium garnet) laser irradiation on the marine biofilm-forming bacteria Pseudoalteromonas carrageenovora during two growth stages (log phase and stationary phase) and under two stresses (reduced temperature and nutrient limitation) was investigated. Bacteria were exposed to a laser fluence of 0.1 J x cm(-2) for 5, 10, and 15 min with a peak power of 20 MW x cm(-2), a pulse width of 5 ns, and an average power of 1 W x cm(-2) with a repetition rate of 10 Hz. The mortality of bacteria immediately after the irradiation as well as after a set period of time was determined. Mortality was higher among log-phase bacteria (72%) than bacteria in the stationary phase (51%) and those grown under nutrient limitation (51%). Bacteria grown at reduced temperature had a mortality of 49%. However, the differences in cell density of log-phase, stationary-phase, nutrient-limited, and low-temperature irradiated samples compared with controls after 5 h of incubation were 96, 93, 94, and 86%, respectively. The mortality values suggest that the same laser fluence has different degrees of effectiveness, depending on the physiological state of the bacteria.     

Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.     

Interaction of low-molecular-weight chitosan with mimic membrane studied by electrochemical methods and surface plasmon resonance

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed to be in a concentration-dependent manner.     

Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.