**1. Introduction**

Since people are increasingly conscious of the relationship between diet and health, the consumption of marine-based foods has been growing continuously. Consumers identified seafoods as nutritious and complete foods. Hence, they are perceived them as an excellent source of high quality proteins, valuable lipids with high amounts of PUFA. These compounds are well known to contribute to the enhancement of human health by different alternatives such as reducing the risk of cardiovascular disease, coronary disease and hypertension. Additionally, marine-based food products are easily digested and constitute excellent source of essential minerals. Recently, seafoods have been recognized as nutraceuticals or functional foods. Functional foods, first evolved in Japan in 1980, are defined as foods demonstrating beneficial effect on one or more targeted functions on human organism (Ross, 2000). Marine-based functional foods or nutraceuticals, include omega-3 fatty acids, chitin and chitosan, fish protein hydrolysates, algal constituents, carotenoids, antioxidants, fish bone, shark cartilage, taurine and bioactive compounds (Kadam & Prabhasankar, 2010).

Despite the aforementioned desirable properties, seafood products are highly susceptible to quality deterioration, mainly due to the lipid oxidative reactions, particularly involving polyunsaturated fatty acids (PUFAs). These reactions are enhanced (catalyzed) by the presence of high concentrations of heme and non-heme proteins. These proteins are known to contain iron and other metal ions in their structures (Decker & Hultin, 1992). Moreover, marine-based food quality is highly influenced by autolysis, bacterial contamination and loss of protein functionality (Jeon, Kamil & Shahidi, 2002). More recently, pollution of seafood with different hazardous materials such as refinery, industrial wastes and heavy metals has resulted in elevated concern about the consumption of seafood (Kadam & Prabhasankar, 2010). Additionally, aquaculture industry has increasingly attracted much

© 2012 Alishahi, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

attention for the intensive farming of fish and shellfish, mainly due to the depleting of wild fish and shellfish stocks worldwide. However, this intensive farming entails several difficulties such as stress, which is the most important factor affecting the immunity system of fish (Ledger, Tucker & Walker, 2002). To address the aforementioned problems, the use of chitosan as protective material appears to be a potential alternative.

In nature, chitosan is found in the cell walls of fungi of the class *Zygomycetes*, in the green algae *Chlorella sp.*, yeast and protozoa as well as insect cuticles and especially in the exoskeleton of crustaceans. Chitosan is a deacetylated derivative of chitin, the second abundant polysaccharide in nature after cellulose. In 1811, the French scientist, Henri Braconnot first discovered chitin in mushroom. In 1820, chitin was isolated from insect cuticles (Bhatnagar & Sillanpa, 2009). In 1859, Rouget reported finding chitosan after boiling chitin in potassium hydroxide (KOH). This treatment rendered the material soluble in organic acids. Hoppe-Seyler named it chitosan in 1894 (Khor, 2001). Chemically, chitosan is a high molecular weight, linear, polycationic heteropolysaccharide consisting of two monosaccharides: N-acetyl-glucosamine and D-glucosamine. They are linked by β-(1→4) glycosidic bonds. The relative amount of these two monosaccharides in chitosan vary considerably, yielding chitosans of different degrees of deacetylation varying from 75% to 95%, molecular weight in the range of 50-2000 KDa, different viscosities and pKa values (Tharanathan & Kittur, 2003). In addition, chitosan has three functional moieties on its backbone; the amino group on the C2, the primary and secondary hydroxyl groups on the C3 and C6 positions, respectively. These functional groups play important roles in different functionalities of chitosan. The amino group is the most important among the other moieties, especially in acidic conditions, due to the protonation phenomenon, rendering it able to interact with negatively charged molecules (or sites). Additionally, chitosan polymer interacts with the metal cations through the amino groups, hydroxyl ions and coordination bonds.

Commercially, chitosan is produced from chitin by exhaustive alkaline deacetylation, involving boiling chitin in concentrated alkali for several hours. Because this Ndeacetylation is almost never complete, chitosan is classified as a partially N-deacetylated derivative of chitin (Kumar, 2000). From a practical point of view, many of commercial interests and applications of chitosan and its derivatives originate from the fact that this polymer combines several features, such as biocompatibility, biodegradability, nontoxicity and bioadhesion, making it as valuable compound for pharmaceutical (Dias, Queiroz, Nascimento & Lima, 2008), cosmetics (Pittermann, Horner & Wachter, 1997), medical (Carlson, Taffs, Davison & Steward, 2008), food (Shahidi, Kamil & Jeon, 1999; No, Meyers, Prinyawiwatkul & Xu, 2007; Kumar, 2000), textile (El Tahlawy, Bendary, El Henhawy & Hudson, 2005), waste water treatment (Che & Cheng, 2006), paper finishing, photographic paper (Kumar, 2000), and agricultural applications (Hirano, 1996).

Although there have been several prior reviews on the use of chitosan in food applications (No et al., 2007; Shahidi et al., 1999), the use of chitosan in seafood applications, especially its novel application in the form of nanocarriers for bioactive compounds for shelf life extention, has not yet been reported. Recently, a study was published on the use of chitosan nanoparticle for stability enhancement of vitamin C in rainbow trout diet (Alishahi, Mirvaghefi, Rafie-Tehrani, Farahmand, Shojaosadati, Dorkoosh & Elsabee, 2011). Hence, this chapter attempts to survey the applications of chitosan in various fields of marine-based products.

## **2. Antibacterial activity**

410 The Complex World of Polysaccharides

attention for the intensive farming of fish and shellfish, mainly due to the depleting of wild fish and shellfish stocks worldwide. However, this intensive farming entails several difficulties such as stress, which is the most important factor affecting the immunity system of fish (Ledger, Tucker & Walker, 2002). To address the aforementioned problems, the use of

In nature, chitosan is found in the cell walls of fungi of the class *Zygomycetes*, in the green algae *Chlorella sp.*, yeast and protozoa as well as insect cuticles and especially in the exoskeleton of crustaceans. Chitosan is a deacetylated derivative of chitin, the second abundant polysaccharide in nature after cellulose. In 1811, the French scientist, Henri Braconnot first discovered chitin in mushroom. In 1820, chitin was isolated from insect cuticles (Bhatnagar & Sillanpa, 2009). In 1859, Rouget reported finding chitosan after boiling chitin in potassium hydroxide (KOH). This treatment rendered the material soluble in organic acids. Hoppe-Seyler named it chitosan in 1894 (Khor, 2001). Chemically, chitosan is a high molecular weight, linear, polycationic heteropolysaccharide consisting of two monosaccharides: N-acetyl-glucosamine and D-glucosamine. They are linked by β-(1→4) glycosidic bonds. The relative amount of these two monosaccharides in chitosan vary considerably, yielding chitosans of different degrees of deacetylation varying from 75% to 95%, molecular weight in the range of 50-2000 KDa, different viscosities and pKa values (Tharanathan & Kittur, 2003). In addition, chitosan has three functional moieties on its backbone; the amino group on the C2, the primary and secondary hydroxyl groups on the C3 and C6 positions, respectively. These functional groups play important roles in different functionalities of chitosan. The amino group is the most important among the other moieties, especially in acidic conditions, due to the protonation phenomenon, rendering it able to interact with negatively charged molecules (or sites). Additionally, chitosan polymer interacts with the metal cations through the amino groups, hydroxyl ions and coordination bonds.

Commercially, chitosan is produced from chitin by exhaustive alkaline deacetylation, involving boiling chitin in concentrated alkali for several hours. Because this Ndeacetylation is almost never complete, chitosan is classified as a partially N-deacetylated derivative of chitin (Kumar, 2000). From a practical point of view, many of commercial interests and applications of chitosan and its derivatives originate from the fact that this polymer combines several features, such as biocompatibility, biodegradability, nontoxicity and bioadhesion, making it as valuable compound for pharmaceutical (Dias, Queiroz, Nascimento & Lima, 2008), cosmetics (Pittermann, Horner & Wachter, 1997), medical (Carlson, Taffs, Davison & Steward, 2008), food (Shahidi, Kamil & Jeon, 1999; No, Meyers, Prinyawiwatkul & Xu, 2007; Kumar, 2000), textile (El Tahlawy, Bendary, El Henhawy & Hudson, 2005), waste water treatment (Che & Cheng, 2006), paper finishing, photographic

Although there have been several prior reviews on the use of chitosan in food applications (No et al., 2007; Shahidi et al., 1999), the use of chitosan in seafood applications, especially its novel application in the form of nanocarriers for bioactive compounds for shelf life extention, has not yet been reported. Recently, a study was published on the use of chitosan

paper (Kumar, 2000), and agricultural applications (Hirano, 1996).

chitosan as protective material appears to be a potential alternative.

The modern era of chitosan research was heralded by publications in the 1990s, that described the antimicrobial potentials of chitosan and its derivatives, exhibiting a wide spectrum of activities against human pathogens and food-borne microorganisms (Chen, Xing & Park & Kong, 2010; No, Park, Lee & Meyers, 2002; Rabea, Badway, Stevens, Smagghe & Steurbaut, 2003: Raafat, Bargen, Haas & Sahl, 2008; Raafat & Sahl, 2009). , The first study reporting antibacterial properties was reported by Allan & Hardwiger (1979). They reported that chitosan showed a broad range of activities and a high inactivation rate against both Gram-positive and Gram-negative bacteria, (Allan & Hardwiger, 1979). However, although several studies have been published in this area, the exact mechanism of the antimicrobial activity of chitosan remains ambiguous.

Six major mechanisms have been proposed in the literature, as follows (Kong et al., 2010; Raafat & Sahl, 2009; Rafaat et al., 2008): (1) the interaction between the positively charged chitosan amine groups and the negatively charged microbial cell membranes, leadingto the leakage of proteinaceous and other intracellular constituents; (2) the activation of several defense processes in the host tissue by the chitosan molecule acting as a water-binding agent and inhibiting various enzymes by blocking their active centers; (3) the action of chitosan as a chelating agent, selectively binding metals and then inhibiting the production of toxins and microbial growth; (4) the formation, generally by high molecular weight chitosan, of an impervious polymeric layer on the surface of the cell, thereby altering cell permeability and blocking the entry of nutrients into the cell; (5) the penetration of mainly low-molecular weight chitosan into the cystosol of the microorganism to bind DNA, resulting in interference with the synthesis of mRNA and proteins; and (6) the adsorption and flocculation of electronegative substances in the cell by chitosan, distributing the physiological activities of the microorganisms, causing their death.. However, it is very important to mention that chitosan is soluble only in acidic media and therefore, the effect of pH on microorganisms must be considered together with the effect of chitosan. Thus, the synergetic effect of chitosan/pH together is probably the most evident explanation of the antimicrobial effect of chitosan.

Complicating the issue, a number of studies aimed at determining the antibacterial activities of chitosan on Gram-positive and Gram-negative bacteria have been reported antithetical outcomes, making their interpretation difficult. More recently, Kong et al. (2010) showed that chitosan and its derivatives are more powerful antibacterial agent against Gramnegative bacteria than against Gram-positive microorganism. Conversely, Raafat and Sahl (2009) reported a study in which they demonstrated that Gram-positive bacteria are more

sensitive to the antibacterial effect of chitosan than Gram-negative bacteria. Therefore, the interpretation of the sensitivity of bacteria to chitosan is quite difficult.

To address this problem, Kong et al. (2010) proposed that the variation in the bactericidal efficacy of chitosan arises from different parameters that must be considered when evaluating the antibacterial activity of chitosan. These factors can be categorized into four classes as follows: (1) intrinsic microbial factors, including microbial species and cell age, (2) intrinsic factors of chitosan molecules, namely, the positive charge density, protonation level of the amine group, the chitosan molecular weight and concentration, hydrophilic/hydrophobic characteristics and chelating capacity of the chitosan molecule; (3) its physical state, i.e., either water soluble or solid chitosan; and (4) environmental factors including the ionic strength of the testing medium, pH, temperature and contact time between chitosan and bacterial cells. In addition, it is worth noting that, despite the widely reported antimicrobial properties of chitosan in the literature, the results are mainly based on in vitro experiments. In real-world appliactions, it is important to consider that most foods are complex matrices composed of different compounds (proteins, carbohydrates, lipids, minerals, vitamins, salts and others) and many of them may interact with chitosan to varying levels, possibly leading to a loss or enhancement of its antibacterial activity (Devlieghere, Vermeulen, & Debevere, 2004).

Taking into account the above insights about the antibacterial characteristics of chitosan, the following applications of chitosan in seafood products were considered. Due to the high perishability characteristics of marine-based products, there has been an increased interest in the application of chitosan to extend the shelf life of the products. In this context, chitosan has increasingly gained attention as an antibacterial additive in seafood from both seafood processors and consumers, largely due to a desire to reduce the use of synthetic chemicals in seafood preservation. Cao, Xue and Liu (2009) reported that chitosan at 5 g/L extended the shelf life of oyster (*Crossostrea gigas*) from 8-9 days to 14-15 days. They explained that Pseudomonas and Shewanella are the most prolific microorganisms during cold storage of fish and shellfish and these bacteria can easily be reduced or eliminated with the addition of chitosan at this concentration.

Fish balls have a high water activity and are prone to the growth of microorganisms, with a relatively short shelf life of 4-5 days at a storage temperature of approximately 5 °C. Kok and Park (2007) reported that fish ball shelf life has been increased by adding chitosan which maintained both aerobic and yeast counts at < 1 log CFU/g over 21 days of storage. Kok and Park (2007) also reported that physical state of chitosan is an important parameter for its antibacterial activity. In the dissolved state, chitosan showed excellent antibacterial activity and contributed to the extension of the product shelf life. However, in the study reported by Lopez-Caballero et al. (2005a and b), it was demonstrated that the addition of powdered chitosan to fish patties had no effect on bacterial growth. Roller and Corvill (2000) reported that the spoilage flora was inhibited from log 8 CFU/g in the control sample (without chitosan) to log 4 CFU/g over the 4-week study at 5 °C with the use of chitosan combined with acetic acid in shrimp salad. However, it is important to consider the effect of

acetic acid on the ability of chitosan to extend shelf life. Fernandez-Saiz, Soler, Lagaron and Ocio (2010) studied bacterial growth in two conditions. The first one was a fish soup (ANETO ® brand, packaged in TetraBrik ® and fabricated by Jamon Aneto, S. L., Barcelona, Spain). The other medium was a model laboratory growth medium, tryptone soy broth. They reported a significant reduction of the growth of *Listeria monocytogenes*, *Staphylococcus aureus* and *Salmonella spp* when the products were stored in the presence of chitosan. In fish soup and under laboratory conditions, the effect of chitosan in the tested medium at 4, 12, and 37 °C depended significantly on the bacteria type, incubation temperature and food matrix (substrate). The antibacterial effectiveness of chitosan was decreased in the fish soup, suggesting that the constituents of the fish soup had high influence on the antimicrobial efficacy of chitosan. The authors reported that chitosan was probably irreversibly bound by microbial cells or negatively charged compound in the soup and therefore rendered it inactive against the remaining unbound microorganisms. In conclusion, the effect of chitosan-to-cell ratio must be considered. Lopez-Caballero et al. (2005a-b) showed that the addition of chitosan to sausage treated at high pressure yielded a 2-log cycle decrease of total bacterial counts of *Pseudomonas* and *Enterobacteria* at 8-11 days storage. Ye, Neetoo and Chen (2008) stated that chitosan has antibacterial activity that is effectively expressed in aqueous system; however, its antibacterial properties against *L. monocytogenes* in coldsmoked salmon were negligible when chitosan was in the form of an insoluble film. The growth of *L. monocytogenes* in salmon samples wrapped in the chitosan-coated film and plain films was similar. The authors demonstrated that it is possible that chitosan is ineffective in films because it is unable to diffuse through a rigid food matrix such as salmon. Regarding microbial counts, Lopez-Caballero et al. (2005b) showed that chitosan in the soluble state had no significant effect in high-pressure treated cod sausages. However, when chitosan was added in a dry form, higher counts of microorganisms were recorded. This is an indication that chitosan in soluble form contributed to maintaining product safety. The microbial counts in their study were for lactic acid bacteria, *Enterobacteria*, *Pseudomonas* and *Staphylococcus*. Duan, Jiang, Cherian and Zhao (2010) reported that the initial total plate count (TPC) of fresh lingcod was 3.67 log CFU/g, which then rapidly increased to 6.16 and 8.36 log CFU/g on day 6 and day 14, respectively. When chitosan coatings were used, the results showed a 0.15-0.64 reduction in TPC. Moreover, the TPCs of chitosan-coated samples stored under vacuum or modified-atmosphere packaging were significantly lower than those of the control sample during the subsequent cold storage. The combination of chitosan coating and vacuum or modified atmosphere packaging (MAP) resulted in 2.22-4.25 reductions in TPC for the first 14 days of cold storage. The TPCs of chitosan-coated and MPA samples were lower than 105 CFU/g even after 21 days of cold storage. This result indicated a significant delay of microbial spoilage. Qi, Zhang and Lan-Lan (2010) reported that because non-fermenting Gram-negative bacteria are dominant in the initial microbial flora of fish and shellfish sourced from cold seawater, controlling the growth of these Gramnegative bacteria may be important for the preservation of oysters. They demonstrated that combined treatment with chitosan and ozonated water had better antibacterial effect than either treatment alone. When only aerobic plate count was measured, the authors showed that the product shelf life with the combination of chitosan with ozonated water was at least

412 The Complex World of Polysaccharides

(Devlieghere, Vermeulen, & Debevere, 2004).

chitosan at this concentration.

sensitive to the antibacterial effect of chitosan than Gram-negative bacteria. Therefore, the

To address this problem, Kong et al. (2010) proposed that the variation in the bactericidal efficacy of chitosan arises from different parameters that must be considered when evaluating the antibacterial activity of chitosan. These factors can be categorized into four classes as follows: (1) intrinsic microbial factors, including microbial species and cell age, (2) intrinsic factors of chitosan molecules, namely, the positive charge density, protonation level of the amine group, the chitosan molecular weight and concentration, hydrophilic/hydrophobic characteristics and chelating capacity of the chitosan molecule; (3) its physical state, i.e., either water soluble or solid chitosan; and (4) environmental factors including the ionic strength of the testing medium, pH, temperature and contact time between chitosan and bacterial cells. In addition, it is worth noting that, despite the widely reported antimicrobial properties of chitosan in the literature, the results are mainly based on in vitro experiments. In real-world appliactions, it is important to consider that most foods are complex matrices composed of different compounds (proteins, carbohydrates, lipids, minerals, vitamins, salts and others) and many of them may interact with chitosan to varying levels, possibly leading to a loss or enhancement of its antibacterial activity

Taking into account the above insights about the antibacterial characteristics of chitosan, the following applications of chitosan in seafood products were considered. Due to the high perishability characteristics of marine-based products, there has been an increased interest in the application of chitosan to extend the shelf life of the products. In this context, chitosan has increasingly gained attention as an antibacterial additive in seafood from both seafood processors and consumers, largely due to a desire to reduce the use of synthetic chemicals in seafood preservation. Cao, Xue and Liu (2009) reported that chitosan at 5 g/L extended the shelf life of oyster (*Crossostrea gigas*) from 8-9 days to 14-15 days. They explained that Pseudomonas and Shewanella are the most prolific microorganisms during cold storage of fish and shellfish and these bacteria can easily be reduced or eliminated with the addition of

Fish balls have a high water activity and are prone to the growth of microorganisms, with a relatively short shelf life of 4-5 days at a storage temperature of approximately 5 °C. Kok and Park (2007) reported that fish ball shelf life has been increased by adding chitosan which maintained both aerobic and yeast counts at < 1 log CFU/g over 21 days of storage. Kok and Park (2007) also reported that physical state of chitosan is an important parameter for its antibacterial activity. In the dissolved state, chitosan showed excellent antibacterial activity and contributed to the extension of the product shelf life. However, in the study reported by Lopez-Caballero et al. (2005a and b), it was demonstrated that the addition of powdered chitosan to fish patties had no effect on bacterial growth. Roller and Corvill (2000) reported that the spoilage flora was inhibited from log 8 CFU/g in the control sample (without chitosan) to log 4 CFU/g over the 4-week study at 5 °C with the use of chitosan combined with acetic acid in shrimp salad. However, it is important to consider the effect of

interpretation of the sensitivity of bacteria to chitosan is quite difficult.

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