The antioxidant activity of chitosan before micronization (BMC, average particle size of 1850 ± 26.3 μm) and after micronization (AMC, average particle size of 1.37 ± 0.2 μm) in grape juice was studied. Antioxidant activity was determined, including that of DPPH radicals, hydrogen peroxide and superoxide anion radicals, as well as ABTS radicals of BMC or AMC in grape juice. AMC exhibits stronger scavenging activity toward DPPH radicals, superoxide anion radicals and hydrogen peroxide than BMC. At a concentration of 1.0 mg/mL, AMC in grape juice exhibited 90.0%, 97.3% and 88.7% scavenging activities toward DPPH radicals, hydrogen peroxide and superoxide anion radicals, respectively. The TEAC (Trolox Equivalent Antioxidant Capacity) values of AMC (3.94 ± 0.19) greatly exceeded those of BMC (2.21 ± 0.10) in grape juice. The in vitro results in this investigation suggest the possibility that AMC can increase the antioxidant activity in grape juice. However, comprehensive studies must be performed to ascertain the in vivo safety of AMC in experimental animal models.
Chitosan (β-(1-4)-2-amino-2-deoxy-D-glucose) is a linear hydrophilic polysaccharide polymer of d-glucosamine. It is abundant in nature and is present in the exoskeleton of crustaceans, including crabs and shrimp [
In recent years, various researchers have evaluated the antioxidant activity of chitosan derivatives. For example, Xie et al. [
Crab-shell chitosan with 86.70% N-deacetylation as a powder were obtained from VA & G Bioscience Inc (Taoyuan, Taiwan) and micronzied using a high-speed planetary ball-mill (PM100, Retsch, Germany), by the approach of that was proposed by Chau et al. [
The antioxidant activities of micronized individual chitosans were evaluated in grape juice.
In the preparation of 1.0 L of 0.1% - 1.0% chitosan solutions, 0.1, 0.5 and 1.0 g of chitosan were dispersed in 900 ml of distilled water, to which 50 ml of glacial acetic acid was added to dissolve the chitosan. The pH of each solution was adjusted to 5.0 by adding 0.1 M NaOH and each solution was made up to 1.0 L. An acid solution without chitosan at pH 5.0 was used as a control.
Clear, UTH-treated, shelf-stable grape juice that contained no added preservatives and was packed in laminated, was purchased from a local retailer. To 45 mL of this grape juice in a 250 mL Erlenmeyer flask was added 5 mL of micronized chitosan solution. To the control flask was added 5 mL of water, rather than chitosan solution. The used chitosan concentrations ranged from 0.1 to 1.0 g/L of juice.
The effect of chitosans on DPPH radicals was examined using the modified method of Shimada et al. [
The activity of chitosan in scavenging hydrogen peroxide was measured by a modified version of the method that was proposed by Yen and Chang [
The superoxide scavenging capacity of chitosans was assayed using the method of Robak and Gryglewski [
Total antioxidant capacity was evaluated using the ABTS modified assay [12-14]. In the most recent version of the trolox equivalent antioxidant capacity (TEAC) assay, an antioxidant is added to a pre-formed ABTS radical solution and, after a fixed period, the remaining ABTS●+ is quantified spectrophotometrically [
ABTS●+ was formed by reacting 2.45 mM ABTS salt, of potassium persulfate (K2S2O8), was reacted with 7 mM ABTS salt in 0.01 M phosphate-buffered saline, pH7.4, for 15 h at room temperature in the dark. The resulting ABTS●+ radical cations were diluted with 0.01 M phosphate-buffered saline at pH 7.4 to yield an absorbance of approximately 0.70 at 734 nm. The standard or sample was diluted by a factor of 100 using the ABTS●+ solution to a total volume of 1mL and allowed to react for 6 min. Absorbance was measured spectrophotometrically at various times. A blank (without a standard or sample) was used as a control and 990 μL of PBS was added to the control samples instead. The absorption peak of ABTS●+ was at 734 nm. The addition of antioxidant reduced ABTS●+ to its colorless form. The extent of decolorization as a percentage of inhibition of ABTS●+ is determined as a function of concentration and calculated relative to the reactivity of Trolox, a water-soluble analog of vitamin E (α-tocopherol).
In this investigation, three analyses of each sample were performed and each experiment was conducted in triplicate (n = 3). The mean value and its standard deviation were calculated from the obtained data. These data were then compared with each other using Duncan’s multiple range method.
The DPPH, a compound with a proton free radical and a characteristic absorption at 517 nm, is decreased greatly upon exposure to proton radical scavengers [
Superoxides are radicals whose unpaired electrons are on oxygen. While they are relatively weak oxidants, superoxides exhibit limited chemical reactivity, but can form more dangerous species, including singlet oxygen and hydroxyl radicals, which cause the peroxidation of lipids [