Chitosan Hydrolysate Research Articles

Cellulase degradation of shrimp chitosan for the preparation of a water-soluble hydrolysate with immunoactivity

ABSTRACT: Samples of colloidal chitin and chitosans with deacetylation degrees (DD) of 50, 65, 75 and 95% (DD50, DD65, DD75 and DD95, respectively) were prepared from shrimp chitin. The hydrolytic activity of cellulase on these samples increased with the increasing DD of chitosan, with DD95 being the most easily hydrolyzed. Colloidal chitin was hardly hydrolyzed. The optimal reaction temperature and pH for cellulase digestion of chitosan were 55°C and pH 5.2, respectively. During cellulase digestion of chitosan, the 9-h hydrolysate had the highest enhancing effect on the proliferation of a human hybridoma cell, HB4C5. This hydrolysate is composed of low-molecular-weight chitosan (LMWC), with a molecular mass of 20.0 kDa, and chitooligosaccharides, which are composed of sugars with a degree of polymerization of 1–6. In vitro, the 9-h hydrolysate increased both cell proliferation and immunoglobulin (Ig)M secretion of HB4C5 because of the presence of chitooligosaccharides for the former activity and LMWC for the latter activity. In vivo, samples of the chitosan hydrolysate, chitooligosaccharide mixture and LMWC significantly increased the levels of serum IgG and IgM, and enhanced concanavalin A- and lipopolysaccharide-induced proliferation of mouse lymphocytes.

Induced resistance against Sclerotinia sclerotiorum in carrots treated with enzymatically hydrolysed chitosan

We have measured the susceptibility of carrots to the storage pathogen Sclerotinia sclerotiorum after roots were treated postharvest with a 0.2% (w/v) chitosan hydrolysate (number average degree of polymerisation = 7) prepared using Streptomyces N-174 chitosanase. Whereas the hydrolysate did not affect radial growth of S. sclerotiorum colonies on potato dextrose agar plates, it reduced the frequency and size of rot compared to untreated controls when applied to carrots 3 days before inoculation with S. sclerotiorum. When carrots were treated at time zero with either chitosan hydrolysate or high molecular weight chitosan then inoculated at intervals over the next 5 days, there was a decline in Sclerotinia infection, with the hydrolysate showing a greater effect than the high molecular weight chitosan. Our results suggest that the chitosan treatments induced host resistance to the pathogens.

Antimicrobial Activity of a Low-Molecular-Weight Chitosan Obtained from Cellulase Digestion of Chitosan

A water-soluble chitosan hydrolysate with high activity against Escherichia coli was obtained during cellulase digestion of chitosan for 18 h. This 18-h hydrolysate is composed of low-molecular-weight chitosan (LMWC), with a molecular weight of 12.0 kDa, and chitooligosaccharides, which are composed of sugars with a degree of polymerization of 1 to 8. LMWC has a strong activity at 100 ppm against many pathogens and yeast species, including Bacillus cereus, E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, and Saccharomyces cerevisiae, while the chitooligosaccharides have much weaker antimicrobial activity than does LMWC. Accordingly, the antimicrobial activity against E. coli in the 18-h hydrolysate proved to come mainly from the presence of LMWC.

Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for the enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55 degrees C and the enzyme to substrate ratio of 1:400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol: distilled water) yielded fractions with molecular weights in the range 3.2-26.4 kDa.

Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods.

The objective of this study was to determine whether chitosan (poly-beta-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7 degrees C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of hummus, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7 degrees C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.

Elicitor-Induced Cinnamyl Alcohol Dehydrogenase Activity in Lignifying Wheat (Triticum aestivum L.) Leaves.

The substrate-specific induction of wheat (Triticum aestivum L. cv Fenman) leaf cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) was examined in relation to its role in regulating the composition of defensive lignin induced at wound margins. Treatment of wounds with a partially acetylated chitosan hydrolysate or spores of the nonpathogen Botrytis cinerea elicited lignification at wound margins and invoked significant increases in phenylalanine ammonia-lyase (EC 4.3.1.5), peroxidase (EC 1.11.1.7), and CAD activities. The substrate-specific induction of CAD with time was determined in elicitor-treated leaves and in excised lignifying wounds. In whole leaf extracts no significant increases in p-cou-maryl and coniferyl alcohol dehydrogenase activities were detectable, but a significant 5-fold increase in sinapyl alcohol dehydrogenase activity was evident 32 h after elicitor treatment. Similarly, fungal challenge resulted in elevated levels of only sinapyl alcohol dehydrogenase in whole-leaf extracts. In excised lignifying tissues p-coumaryl alcohol dehydrogenase levels were similar to those observed in healthy tissue. A small yet significant increase in coniferyl alcohol dehydrogenase was apparent, but the most dramatic increase occurred in sinapyl alcohol dehydrogenase activity, which increased to values approximately 10 times higher than the untreated controls. Our results show for the first time that CAD induction in lignifying tissues of wheat is predominantly attributable to highly localized increases in sinapyl alcohol dehydrogenase activity.

Controlled release of indomethacin by chitosan-polyelectrolyte complex: optimization and in vivo/in vitro evaluation

Chitosan gel beads containing indomethacin, an acidic drug, were prepared by a polyelectrolyte complexation of sodium tripolyphosphate and chitosan. The effects of the molecular weights of chitosan hydrolysates on the release and absorption rates of indomethacin from gel beads were examined. The release rates of indomethacin decreased with increasing of molecular weight and indomethacin content. A negative correlation was observed between the molecular weight and release rate constant ( r = −0.983). The release of indomethacin depended upon the dispersion of the indomethacin solid particles in the beads, as well as the porosity, tortuosity and surface area of the matrix. The plasma concentrations ofindomethacin after oral administration of chitosan gel beads to beagle dogs exhibited the sustained-release pattern. With increasing molecular weight of chitosan, the AUC and C max were decreased, while MAT was increased. A good correlation was observed between the molecular weight of chitosan or dissolution rate constant and the MAT or AUC. The results revealed that the chitosan gel beads composed of chitosan hydrolysate, of molecular weight 25 000, might be the best for the sustained-release preparation of indomethacin. It was suggested that the absorption of other drugs could be controlled by the proper selection of chitosan hydrolysate of optimal molecular weight and predicted from in vitro dissolution tests.

Effects of chitosan hydrolyzates on lipid absorption and on serum and liver lipid concentration in rats

The enzymatic hydrolysates of chitosan with average molecular weights range from 5000 to 20000 and with low viscosity interfered with intestinal absorption of cholesterol in lymph fistulated rats and increased fecal excretion of neutral steroids in rats fed a cholesterol-enriched diet. The hydrolysates exerted a cholesterol-lowering activity, and it was more marked in the liver than in the serum. They also reduced liver triglyceride significantly. Chitosan hydrolysates with average molecular weights of 10000-20000 were as effective as high-viscous chitosan with a molecular weight of 50000. The results indicate usability of low-viscosity chitosan hydrolysate as a hypocholesterolemic agent.

Chitin oligosaccharides elicit lignification in wounded wheat leaves

Oligomers of chitin (monomer to hexamer) and their deacetylated derivatives (monomer to tetramer) were purified from acid hydrolysates of chitin and chitosan, respectively, and assayed for lignification-eliciting activity in wounded wheat leaves. At 40 μg per wound, the chitin monomer and dimer were inactive, whist the chitin trimer possessed very slight activity. In contrast, the chitin tetramer, pentamer and hexamer all possessed significant elicitor activity. Dose-response curves indicated that the activities of the elicitor-active chitin oligomers were similar, and that their activity rapidly diminished at lower doses. Digestion of the tetramer with chitinase abolished elicitor activity. None of the deacetylated oligomers tested (monomer to tetramer) possessed any significant elicitor activity, confirming the requirement of acetyl groups for elicitor activity. The activity of the chitin tetramer was entirely dependent on the inclusion of a pre-wounding step in the assay, which explains why a previous study failed to identify an elicitor-active oligomer. Prior wounding also increased the activity of a number of other known lignification elicitors.