Comparison in antioxidant action between α-chitosan and β-chitosan at a wide range of molecular weight and chitosan concentration
Graphical abstract
Introduction
Antioxidant activity is one of the well-known functionalities of chitosan. Many studies have shown that chitosan inhibit the reactive oxygen species (ROS) and prevent the lipid oxidation in food and biological systems. Several mechanisms about the antioxidant action of chitosan have been proposed.1 Chitosan can scavenge free radicals or chelate metal ions from the donation of a hydrogen or the lone pairs of electrons.2, 3 The interaction of chitosan with metal ions could involve several complex actions including adsorption, ion-exchange, and chelation.4 The hydroxyl groups (OH) and amino groups (NH2) in chitosan are the key functional groups for its antioxidant activity, but can be difficult to be dissociated due to the semi-crystalline structure of chitosan with strong hydrogen bonds.2 Chitin has two forms, named α- and β-chitin, in which α-chitin is very stable with intra-chain, intra-sheet, and inter-sheet hydrogen bonds from the antiparallel sheets along with c axis in orthorhombic cell, while β-chitin has no hydrogen bonds between two inter-sheets owing to their parallel directions.5, 6, 7 Also, the initial crystallinity index (CI) of α- and β-chitin are different, 28.3% for α-chitin and 20.8% for β-chitin. Through deacetylation, CI of α-chitosan was slightly decreased, while CI of β-chitosan exhibited large reduction.7, 8, 9 Similarly, Kurita et al. (1993) reported that α-chitin is rigid and can be less susceptible to deacetylation compared to β-chitin.10 Therefore, the polymeric structures (e.g., CI) of chitosan deacetylated from different forms of chitin may not be identical and β-chitosan can have higher solubility with less crystallinity, thus providing better functionalities than α-chitosan in similar Mw and DDA. For this reason, we hypothesized that the form of chitosan (FC) may be a significant factor determining the antioxidant activity of chitosan. However, little study has compared the difference and/or similarity between α- and β-chitosan in their antioxidant activity.
Molecular weight (Mw) of chitosan is one of the most important factors affecting its antioxidant activity. Je et al. (2004) indicated that 1–5 kDa chitosan with 90% degree of deacetylation (DDA) has the highest radical scavenging activity.11 Sun et al. (2007) reported that chitosan oligomers with low Mw (2.30, 3.27, and 6.12 kDa) have better antioxidant activity than that of higher Mw oligosaccharides (15.25 kDa).12 Tomida et al. (2009) also showed that low Mw chitosan (2.8, 17.0, and 33.5 kDa) inhibits the oxidation of serum albumin, resulting in reduction of oxidative stress in uremia in comparison with higher Mw chitosan (62.6–931 kDa).13 In the study of antioxidant effect of chitosan on salmon at Mw of 30, 90 and 120 kDa,1 the lowest Mw of chitosan (30 kDa) showed the strongest antioxidant activity, resulting in approximately 85% scavenging activity for free radicals. Chien et al. (2007) also found that lower Mw (12 kDa) chitosan increases antioxidant activity in apple juice, compared to higher Mw chitosan (95 and 318 kDa).14 Also, some studies reported that Mw is dependent on its crystallinity. Kumar et al. (2004) reported decreased CI in lower Mw,15 whereas Ogawa found increased CI in lower Mw.16 Liu et al. (2006) reported increased crystallinity in high DDA and low Mw.17 In respect to the effect of DDA, most studies reported that antioxidant property is enhanced with higher DDA.11, 18, 19
This study was aimed to investigate the antioxidant action of α- and β-chitosan obtained from shrimp shells and jumbo squid pens, respectively, at a wide range of Mw and chitosan concentration (CS). DDA effect was also considered. Different antioxidant measurements including DPPH radical scavenging activity, reducing ability, chelating ability, and hydroxyl radical scavenging were conducted to verify predominant antioxidant action in chitosan.
Section snippets
Materials
Dried jumbo squid (Dosidicus gigas) pens were provided by Dosidicus LLC (USA). Commercial α-chitosan from shrimp shells was purchased from Primax (Iceland) with Mw of 300 kDa and DDA of 88%, determined in this study. NaOH, NaCl, ascorbic acid, and trichloroacetic acid were purchased from Mallinckrodt Chemicals Co. (USA). 2-Thiobarbituric acid and ferric chloride were from Sigma Chemical Co. (USA) and ammonium thiocyanate and deoxyribose from Alfa Aesar (USA). 1,1-Dephenyl-2-picrylhydrzyl (DPPH),
Solubility
Changes of transmittance (T, %) in different Mw of α- and β-chitosan solutions at the pH range of 3–11 are reported in Figure 1, including a β-chitosan sample with Mw of 15 kDa and 86% DDA for evaluating the possible effect of different DDAs between α-chitosan (88%) and β-chitosan (97%) samples on the measured antioxidant activity. T (%) was not changed at pH 3–11 in both α- and β-chitosan samples with Mw of 4–5 kDa and 22–30 kDa except 22–30 kDa α-chitosan where T (%) decreased when pH was over 9.
Discussion
The key compounds contributing to the antioxidant activity in chitosan is oxygen and hydrogen from hydroxyl groups, and nitrogen and hydrogen from positively charged amino groups. Hydrogen or the lone pair of electrons can scavenge free radicals, and the lone pair of electron in oxygen and nitrogen chelates metal ions, forming chitosan–metal ion complex since those functional groups act as ligands.14, 27, 28, 29 FC, Mw, and CS were considered as main factors affecting antioxidant activity and
Conclusion
This study demonstrated the higher reducing ability and hydroxyl radical scavenging activity in β-chitosan than those in α-chitosan at high Mw. DPPH radical scavenging activity, reducing ability, and hydroxyl radical scavenging activity were higher in 22–30 kDa α- and β-chitosan samples, but chelating ability was the highest in 4–5 kDa chitosan. There was no significant difference between α- and β-chitosan in DPPH radical scavenging, but EC50 of β-chitosan was extremely lower than that of
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