**3. Antioxidant activity**

Seafood is considered as excellent sources of functional foods for balanced nutrition favorable for promoting good health. The beneficial health effects of marine foods are ascribed to their lipids, mainly the long-chain omega-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Newton, 2001). However, these valuable compounds in seafood are highly sensitive to oxidative reactions and development of offflavor even during cold storage (Cadwalladar & Shahidi, 2001). It has been proposed that lipid oxidation in fish and shellfish may be initiated and propagated by a number of mechanisms, namely autooxidation, photosentized oxidation, lipoxygenase, peroxidase and microsomal enzymes (Hsieh & Kinsella, 1989). Additionally, fish and shellfish muscles contain protein-bound iron compounds, for example, myoglobin, hemoglobin, ferritin, transferrin and haemosiderin, as well as other metals. This is a factor that plays an important role in initiating lipid oxidation in marine-based products (Decker & Hultin, 1992). Castell, Maclean and Moore (1965) showed that the relative pro-oxidant activity of metal ions in fish and shellfish muscles decreased in the order of Cu+2> Fe+2> Cd+2> Li+2> Mg+2> Zn+2> Ca+2> Ba+2. The iron-bound proteins and other metal ions may be released during the storage period and can thus activate and/or initiate lipid oxidative reactions (Decker & Hultin, 1992).

Along with the growing consumer demand for seafood devoid of synthetic antioxidants, chitosan has been a booming antioxidant agent in fish and shellfish. The antioxidant activity of chitosans of different viscosities (360, 50 and 14 cP) in cooked, comminuted flesh of herring (*Clupea harengus*) was investigated (Kamil, Jeon and Shahidi, 2002). The oxidative stability of fish flesh during cold storage at 4 C with the addition of chitosan at concentrations of 50, 100 and 200 ppm was compared with that of fish treated with conventional antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) and *tert*-butylhydoquinone (THQ) (all at 200 ppm). Among the three chitosan samples tested, the 14 cP chitosan was the most effective in preventing lipid oxidation. The formation of thiobarbituric acid test reactive substances (TBARS) in herring samples containing 200 ppm of 14 cP chitosan was reduced by 52% as after 8 days of storage compared with that the control sample without chitosan. At a chitosan concentration of 200 ppm, the 14 cP chitosan exerted an antioxidant effect similar to that of commercial antioxidants in reducing TBARS values in comminuted herring flesh. A study by Kamil, Jeon and Shahidi (2002) indicated that the antioxidant capacity of chitosan added to fish muscle depends on its molecular weight (MW) and concentration in the product. Similarly, Kim and Thomas (2007) observed that the antioxidative effects of chitosan in salmon depended on its molecular weight (tested at MW = 30, 90 and 120 kDa) and concentration (evaluated at 0.2%, 0.5% and 1% w/w). The authors reporteda that the 30 kDa chitosan showed the highest radical-scavenging activity. The scavenging activities of chitosan werer increased by increasing its concentration. However, varying the concentration showed no significant effects when 120 KDa chitosan was used. Ahn and Lee (1992) studied the preservative effect of chitosan film on the quality of highly salted and dried horse mackerel. The product was prepared by soaking the fresh horse mackerel in 15% salt solution for 30 min, coating with or without (control) chitosan, and drying for 3h at 40 °C in a hot air dryer. During cold storage at a temperature of 5 °C for 20 days, the chitosan-coated samples had lower TBARS and peroxide values (PV) than the control samples. Similarly, Lopez-Caballero et al. (2005) also found that coating codfish patties with a chitosan-gelatin blend considerably lowered lipid oxidation. However, being non soluble in powder form at neutral pH, chitosan had no effect on the prevention of lipid oxidation. Shahidi, Kamil and Jeon (2002) reported that chitosans with different molecular weights and viscosities (14, 57 and 360 cP) were effective in controlling the oxidation of lipids in comminuted cod (*Gadus morhua*) following cooking. Both PV and TBARS values were reduced as a result of treatment of the fish prior to cooking with 50, 100 and 200 ppm of 14, 57, and 360 cP chitosans. Inhibition of the oxidation was concentration-dependent and was the highest for the 14 cP chitosan. The authors stated that the antioxidant activity of chitosans of different viscosity in cooked comminuted cod may be attributed to their metal-chelating capacities. Chitosans with different viscosities were found to protect cooked cod from oxidation at various levels. The observed differences were presumably due to differing degrees of deacetylation and molecular weights of the chitosan molecules. In the study reported by Qin (1993), it has been indicated that the ion-chelating ability of chitosan is strongly affected by the degree of deacetylation. The highly acetylated chitosan has very little chelating activity. In addition, high molecular weight chitosan has a compact structure and the effect of intramolecular hydrogen bonding is stronger, which weakens the activities of the hydroxyl and amino groups. As result, the probability of the exposure of these active moieties may be restricted, resulting in less radical scavenging activity. Obviously, low molecular weight chitosan exhibits higher hydroxyl radical scavenging activity, which is partially attributable to its metal chelating ability. The Fe+2 chelating ability of chitosan is mainly attributed to the presence of amino groups, which contain free electron pairs that contribute to form chitosan/Fe+2 complex. The Fe+2 chelating ability of low molecular weight chitosan is more pronounced than that of high molecular weight chitosan. Consequently, the amino groups in chitosan can react with free radicals to form additional stable macroradicals. Therefore, the active hydroxyl and amino groups in the polymer chains are the origin of the scavenging ability of chitosan (Jeon, Shahidi & Kim, 2000; Feng, Du, Li, Hu & Kennedy, 2008; No et al., 2007). Kamil et al. (2002) explained that in the charged state (protonated amino groups), the cationic groups of chitosans impart intramolecular electric repulsive forces. This

414 The Complex World of Polysaccharides

**3. Antioxidant activity** 

(Decker & Hultin, 1992).

trout (*Oncorhynchus mykiss*) during 16-day cold storage.

20 days, whereas it was only 8 days for the control sample, 10 days for the ozonated samples, and 14 days for the chitosan-treated samples. Duan, Cherian and Zhao (2009) indicated that fish oil incorporated in chitosan coatings lowered significantly the total and psychrotrophic counts in frozen lingcod fillets over three months cold storage. Ojagh, Rezaei, Razavi and Hosseini (2010) also reported that chitosan coatings enriched with cinnamon oil decreased effectively total viable counts and psychrotropic bacteria in rainbow

Seafood is considered as excellent sources of functional foods for balanced nutrition favorable for promoting good health. The beneficial health effects of marine foods are ascribed to their lipids, mainly the long-chain omega-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Newton, 2001). However, these valuable compounds in seafood are highly sensitive to oxidative reactions and development of offflavor even during cold storage (Cadwalladar & Shahidi, 2001). It has been proposed that lipid oxidation in fish and shellfish may be initiated and propagated by a number of mechanisms, namely autooxidation, photosentized oxidation, lipoxygenase, peroxidase and microsomal enzymes (Hsieh & Kinsella, 1989). Additionally, fish and shellfish muscles contain protein-bound iron compounds, for example, myoglobin, hemoglobin, ferritin, transferrin and haemosiderin, as well as other metals. This is a factor that plays an important role in initiating lipid oxidation in marine-based products (Decker & Hultin, 1992). Castell, Maclean and Moore (1965) showed that the relative pro-oxidant activity of metal ions in fish and shellfish muscles decreased in the order of Cu+2> Fe+2> Cd+2> Li+2> Mg+2> Zn+2> Ca+2> Ba+2. The iron-bound proteins and other metal ions may be released during the storage period and can thus activate and/or initiate lipid oxidative reactions

Along with the growing consumer demand for seafood devoid of synthetic antioxidants, chitosan has been a booming antioxidant agent in fish and shellfish. The antioxidant activity of chitosans of different viscosities (360, 50 and 14 cP) in cooked, comminuted flesh of herring (*Clupea harengus*) was investigated (Kamil, Jeon and Shahidi, 2002). The oxidative stability of fish flesh during cold storage at 4 C with the addition of chitosan at concentrations of 50, 100 and 200 ppm was compared with that of fish treated with conventional antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) and *tert*-butylhydoquinone (THQ) (all at 200 ppm). Among the three chitosan samples tested, the 14 cP chitosan was the most effective in preventing lipid oxidation. The formation of thiobarbituric acid test reactive substances (TBARS) in herring samples containing 200 ppm of 14 cP chitosan was reduced by 52% as after 8 days of storage compared with that the control sample without chitosan. At a chitosan concentration of 200 ppm, the 14 cP chitosan exerted an antioxidant effect similar to that of commercial antioxidants in reducing TBARS values in comminuted herring flesh. A study by Kamil, Jeon and Shahidi (2002) indicated that the antioxidant capacity of chitosan added to fish phenomenon may be responsible for lesser chelating ability of high viscosity (high Mw) chitosans. Jeon et al. (2002) demonstrated that the antioxidant activity of chitosan is also effective when it is applied as a protective film. In this kind of application, it retards lipid oxidation by acting as a barrier against oxygen penetration. Sathivel, Liu, Huang and Prinyawiwatkul (2007) showed that the TBARS value of coated pink salmon (*O. gorbuscha*) fillets glazed with chitosan (1.3 mg MDA(malondialdehyde)/kg sample) was significantly lower than that of fillets glazed with lactic acid (3 mg MDA/kg sample) or distilled water (1.8 mg MDA/kg sample). The results indicated that chitosan glazing was more effective at reducing lipid oxidation among the studied alternatives. Sathivel (2005) also reported that the TBARS value of pink salmon fillets coated with 1% and 2% chitosan was significantly lower than in both the control sample and protein-coated product after 3 months of frozen storage. The author stated that a higher the concentration of chitosan, resulted in a lower TBARS value. The latter implies that the antioxidant effect of chitosan in the coating state is highly correlated to the coating material thickness, thereby hindering the entrance of oxygen into pink salmon fillet and initiating the oxidative process. Moreover, the primary amino groups of chitosan would form a stable fluorosphere with volatile aldehydes such as malondialdehyde which are derived from breakdown of fats during lipid oxidation (Weist & Karel, 1992).

Duan et al. (2009; 2010) showed that the combination of chitosan with modified atmosphere packaging enhanced the lipid stability of lingcod (*O. elongates*) within 21 days of cold storage. When applied on the surface of lingcod fillets, chitosan coatings may act as a barrier between the fillet and the surrounding atmosphere. This is mainly due to the good oxygen barrier properties of chitosan films, which slow down the diffusion of oxygen from the surrounding air to the surface of fillet and retard lipid oxidation (Aider, 2010). Additionally, Ojagh et al. (2010) reported that chitosan coatings enriched with cinnamon oil could suitably delay lipid oxidation in the refrigerated rainbow trout during 16 days of storage and markedly reduced the TBARS and PV values as compared with the control product. Mao and Wu (2007) showed that lipid oxidation of kamabako gel from grass carp (*Ctenopharyngodon idellus*) significantly decreased when a 1% chitosan solution was added.
