**3. Lipid oxidation in seafood products**

Lipids are one of the important structural and functional components of foods. They provide energy to humans and essential nutrients such as eicosapentaenoic acid; docosahexaenoic acid; and fat-soluble vitamins including, vitamins A, D, E, and K. Lipids are generally defined as "fatty acids and their derivatives, and substances related biosynthetically or functionally to these components" (Christie, 1987). They have been known to significantly affect food quality even though they constitute a minor component of food. Lipids not only impart flavor, odor, texture, and color to foods but also contribute to the feeling of satiety and help in making food products palatable.

However, constant exposure of lipids, particularly unsaturated fatty acids, to air could adversely affect food quality. The susceptibility of lipids to oxidation is one of the main causes of quality deterioration in various types of fresh food products as well as in processed foods. Lipid oxidation is a perennial problem for both the food industry and the consumers. Lipid oxidation has been believed to be one of the factors limiting the shelf life of foods, particularly that of many complex products (Jacobsen, 1999). It is a complex process whereby lipids, particularly polyunsaturated fatty acids, are degraded via free radical formation, causing the deterioration of flavor, texture, color, aroma, taste, consistency, nutritional benefits, and to some extent, the safety of foodstuffs for human consumption. Notably, consumers` preferences for foods are being influenced by such factors. Thus, lipid oxidation is a decisive factor in the useful processing and storage of food products.

Various effects of lipid oxidation on food properties are briefly summarized in Figure 1. Lipid oxidation itself is primarily the formation of reactive compounds like hydroperoxides (HPO) and peroxy radicals. The primary products very often undergo further reactions to form more stable compounds such as hydroxy acids or epoxides. It has been reported that compounds like hydroxy acids can contribute to bitter taste (Grosch et al., 1992). In complex food systems, the interaction of lipid HPO and secondary oxidation products with proteins and other components significantly impact oxidative and flavor stability and texture during processing, cooking, and storage (Erickson, 1992). Oxidized lipids can react with amines, amino acids, and proteins to form brown macromolecular products (Frankel, 1998). Color formation is known to be primarily influenced by the degree of fatty acid unsaturation, water activity, oxygen pressure, and the presence of phenolic compounds (Pan, 2004).

Some of the known factors that promote or inhibit lipid oxidation in foods are shown in Table 1. Metal, metallo-proteins, and enzymes are important factors affecting lipid oxidation in raw materials. Water activity, lipid interactions, proteins, and sugars are important elements affecting the food quality of processed foods.

Fig. 1. Reactions of polyunsaturated fatty acids leading to quality and nutritional changes in foods (Erickson, 1992).


Table 1. Lipid oxidation factors in food (Frankel, 1998).

160 Aquaculture

an antioxidant radical, the antioxidant radical subsequently either combines with other lipid

Several categories of antioxidants can be used for food applications. However, selecting antioxidants for foods is a major concern in the industry because of strict regulations. Generally, the antioxidants involved in food additives must be effective at low doses, must not affect sensory flavor, and must not toxic. Therefore, natural antioxidants are generally

Lipids are one of the important structural and functional components of foods. They provide energy to humans and essential nutrients such as eicosapentaenoic acid; docosahexaenoic acid; and fat-soluble vitamins including, vitamins A, D, E, and K. Lipids are generally defined as "fatty acids and their derivatives, and substances related biosynthetically or functionally to these components" (Christie, 1987). They have been known to significantly affect food quality even though they constitute a minor component of food. Lipids not only impart flavor, odor, texture, and color to foods but also contribute to

However, constant exposure of lipids, particularly unsaturated fatty acids, to air could adversely affect food quality. The susceptibility of lipids to oxidation is one of the main causes of quality deterioration in various types of fresh food products as well as in processed foods. Lipid oxidation is a perennial problem for both the food industry and the consumers. Lipid oxidation has been believed to be one of the factors limiting the shelf life of foods, particularly that of many complex products (Jacobsen, 1999). It is a complex process whereby lipids, particularly polyunsaturated fatty acids, are degraded via free radical formation, causing the deterioration of flavor, texture, color, aroma, taste, consistency, nutritional benefits, and to some extent, the safety of foodstuffs for human consumption. Notably, consumers` preferences for foods are being influenced by such factors. Thus, lipid oxidation is a decisive factor in the useful processing and storage of food

Various effects of lipid oxidation on food properties are briefly summarized in Figure 1. Lipid oxidation itself is primarily the formation of reactive compounds like hydroperoxides (HPO) and peroxy radicals. The primary products very often undergo further reactions to form more stable compounds such as hydroxy acids or epoxides. It has been reported that compounds like hydroxy acids can contribute to bitter taste (Grosch et al., 1992). In complex food systems, the interaction of lipid HPO and secondary oxidation products with proteins and other components significantly impact oxidative and flavor stability and texture during processing, cooking, and storage (Erickson, 1992). Oxidized lipids can react with amines, amino acids, and proteins to form brown macromolecular products (Frankel, 1998). Color formation is known to be primarily influenced by the degree of fatty acid unsaturation, water activity, oxygen pressure, and the presence of phenolic compounds (Pan, 2004).

Some of the known factors that promote or inhibit lipid oxidation in foods are shown in Table 1. Metal, metallo-proteins, and enzymes are important factors affecting lipid oxidation in raw materials. Water activity, lipid interactions, proteins, and sugars are important

elements affecting the food quality of processed foods.

peroxyl radical or another antioxidant radical to terminate the reaction.

the feeling of satiety and help in making food products palatable.

preferred for food applications.

products.

**3. Lipid oxidation in seafood products** 

### **3.1 Control of lipid oxidation by natural antioxidants**

Fish tissue contains many endogenous antioxidants that can contribute to stabilizing the natural color of fish meat by acting against either lipid oxidation or Mb oxidation in the meat. These antioxidants may act directly or indirectly to inhibit the initiation and propagation steps of lipid oxidation. Antioxidants that interfere with the propagation step by converting free radicals to stable compounds are generally referred to as primary antioxidants, and those interfering with the initiation step are considered as secondary antioxidants or preventive inhibitors. Secondary antioxidants include both oxygen scavengers and chelators.

The endogenous antioxidants found in aquatic food products include the tocopherols (Syvaoja & Salminen, 1985), ubiquinol (Petillo et al., 1998), carotenoids (Miki, 1991), ascorbate, organic acids, glutathione peroxidase (Watanabe et al., 1996), peroxide dismutase (Aksnes & Njaa, 1981), catalase, peroxidases (Kanner & Kinsella, 1983), ferroxidases (Kanner et al., 1988), nucleotides, peptides, amino acids (Boldyrev et al., 1987), and phospholipids (Ohshima et al., 1993). These natural antioxidants are usually extracted from fruits, vegetables, and other edible materials in nature.

Mushrooms have been known as a potential source of antioxidants. Numerous studies have shown that certain mushroom extracts (ME) have antioxidative activity *in vitro* and *in vivo* (Mau et al., 2002; Jang et al., 2004; Cheung & Cheung, 2005; Elmastas et al., 2007). Active compounds, including ergothioneine (ERT), certain phenolic compounds, and saccharides, have been found in extracts from different mushroom species (Wasser, 2002; Quang et al., 2006; Dubost et al., 2007).

#### **3.2 Prevention of fish meat discoloration and lipid oxidation by dietary supplementation with ME**

Mushrooms are widely cultivated because of their known uses and health benefits. However, accumulation of industrial waste has been an issue with the increasing production of mushrooms worldwide. Although spent medium is still underutilized, it is normally used as compost or raw material for extracting soluble sugars (Makishima et al., 2006). Among the mushroom species that are cultivated, *Flammulina velutipes* has been known as a medicinal mushroom and has also been cultured as an edible mushroom on a large scale in Japan and other Asian countries (Wasser, 2002).

Jang et al. (2004) reported the antioxidative properties of the *F. velutipes* extracts against the oxidation of cod liver oil in oil-in-water emulsions. In addition, water extracts from this mushroom have been reported to inhibit the oxidation of oxymyoglobin (MbO2) isolated from cattle meats (Ashida & Sato, 2005). The activity of phenolic compounds depends on their structure and is relative to the number and location of the hydroxyl (OH) groups involved (Dziedric & Hudson, 1984). Recently, ERT is another potent antioxidant which usually exists in mushrooms. It has been known as a powerful scavenger of hydroxyl radicals (**.** OH) and an inhibitor of **.** OH generation from hydrogen peroxide; **.** OH generation is catalyzed by iron or copper ions (Akanmu et al., 1991). Arduini et al. (1990) showed that ferrylmyoglobin (ferrylMb), which was formed when deoxymyoglobin (deoxyMb) and metmyoglobin (metMb) were exposed to hydrogen peroxide, was reduced to metMb in the presence of ERT.

Bao et al. (2008) found that *F. velutipes* contains 300 ug/g ERT and can prevent lipid oxidation as well as stabilize the color of beef and fish meats during low-temperature storage. In addition, Bao et al. (2009a) evaluated the antioxidative activity of hydrophilic extract prepared from solid waste medium obtained during *F. velutipes* cultivation with regard to value-added utilization of the waste for recovery of bioactive compounds. The extract contained 341 ug/mL ERT and was used for stabilizing the lipids and the color of the dark meat of two year-old yellowtail, *Seriola quinqueradiata* through feeding prior to harvest (Bao et al., 2009a).

The DPPH radical scavenging activity of the extracts correlated with their ERT content is shown in Figure 2. In this study, the effective content of ERT was 0.3 ug in the extract in which the DPPH radicals were scavenged by 50% of the original.

Changes in the HPO content of the yellowtail dark muscle during chilled storage are shown in Figure 3. The HPO content in the dark muscle of the control yellowtail increased with

et al., 1988), nucleotides, peptides, amino acids (Boldyrev et al., 1987), and phospholipids (Ohshima et al., 1993). These natural antioxidants are usually extracted from fruits,

Mushrooms have been known as a potential source of antioxidants. Numerous studies have shown that certain mushroom extracts (ME) have antioxidative activity *in vitro* and *in vivo* (Mau et al., 2002; Jang et al., 2004; Cheung & Cheung, 2005; Elmastas et al., 2007). Active compounds, including ergothioneine (ERT), certain phenolic compounds, and saccharides, have been found in extracts from different mushroom species (Wasser, 2002; Quang et al.,

Mushrooms are widely cultivated because of their known uses and health benefits. However, accumulation of industrial waste has been an issue with the increasing production of mushrooms worldwide. Although spent medium is still underutilized, it is normally used as compost or raw material for extracting soluble sugars (Makishima et al., 2006). Among the mushroom species that are cultivated, *Flammulina velutipes* has been known as a medicinal mushroom and has also been cultured as an edible mushroom on a

Jang et al. (2004) reported the antioxidative properties of the *F. velutipes* extracts against the oxidation of cod liver oil in oil-in-water emulsions. In addition, water extracts from this mushroom have been reported to inhibit the oxidation of oxymyoglobin (MbO2) isolated from cattle meats (Ashida & Sato, 2005). The activity of phenolic compounds depends on their structure and is relative to the number and location of the hydroxyl (OH) groups involved (Dziedric & Hudson, 1984). Recently, ERT is another potent antioxidant which usually exists in mushrooms. It has been known as a powerful scavenger of hydroxyl

is catalyzed by iron or copper ions (Akanmu et al., 1991). Arduini et al. (1990) showed that ferrylmyoglobin (ferrylMb), which was formed when deoxymyoglobin (deoxyMb) and metmyoglobin (metMb) were exposed to hydrogen peroxide, was reduced to metMb in the

Bao et al. (2008) found that *F. velutipes* contains 300 ug/g ERT and can prevent lipid oxidation as well as stabilize the color of beef and fish meats during low-temperature storage. In addition, Bao et al. (2009a) evaluated the antioxidative activity of hydrophilic extract prepared from solid waste medium obtained during *F. velutipes* cultivation with regard to value-added utilization of the waste for recovery of bioactive compounds. The extract contained 341 ug/mL ERT and was used for stabilizing the lipids and the color of the dark meat of two year-old yellowtail, *Seriola quinqueradiata* through feeding prior to harvest

The DPPH radical scavenging activity of the extracts correlated with their ERT content is shown in Figure 2. In this study, the effective content of ERT was 0.3 ug in the extract in

Changes in the HPO content of the yellowtail dark muscle during chilled storage are shown in Figure 3. The HPO content in the dark muscle of the control yellowtail increased with

which the DPPH radicals were scavenged by 50% of the original.

OH generation from hydrogen peroxide; **.**

OH generation

**3.2 Prevention of fish meat discoloration and lipid oxidation by dietary** 

large scale in Japan and other Asian countries (Wasser, 2002).

OH) and an inhibitor of **.**

vegetables, and other edible materials in nature.

2006; Dubost et al., 2007).

**supplementation with ME** 

radicals (**.**

presence of ERT.

(Bao et al., 2009a).

Fig. 2. DPPH radical scavenging activity of the mushroom (*Flammulina velutipes*) extract. Results are presented in terms of mean ± standard deviation (*n* = 3) (Bao et al., 2009a).

Fig. 3. Changes in the total lipid hydroperoxide level of yellowtail (*Seriola quinqueradiata*) dark muscle during chilled storage. ( ), fed a diet without mushroom extract; ( ), fed with diet containing 1% concentrated mushroom extract; ( ), fed with diet containing 10% mushroom extract. Data are presented in terms of mean ± standard deviation (n = 5). The values with different superscript letters represent significant difference (*P* < 0.05) (Bao et al., 2009a).

prolongation of storage time, whereas HPO accumulation in the ME-supplemented fish was significantly suppressed. This effect is believed to be because of the radical scavenging behavior of ERT (Franzoni et al., 2006). ERT is also known to not only protect organs against lipid peroxidation but also conserve endogenous glutathione and α-tocopherol (Deiana et al., 2004). The same study also showed that feeding yellowtails with the ME remarkably delayed metMb formation in the dark muscle during chilled storage, as shown in Figure 4.

Fig. 4. Changes in the metmyoglobin content of yellowtail (*Seriola quinqueradiata*) dark muscle during chilled storage. ( ), fed a diet without mushroom extract; (■), fed with diet containing 1% concentrated mushroom extract; ( ), fed with diet containing 10% mushroom extract. Data are presented in terms of mean ± standard deviation (n = 5). The values with different superscript letters represent significant difference (*P* <0.05) (Bao et al., 2009a).

Because ERT conserves endogenous antioxidants such as glutathione and α-tocopherol, it enhances the color stability of fish meat during postharvest handling and chilled storage. In addition, discoloration of meats is known to result from interaction between Mb and lipid oxidation (Renerre, 1990). Mb exists in 3 forms in fresh meat: deoxyMb, oxyMb, and metMb. metMB is an undesirable form not only because of its brown color but also because of its catalytic effect during the oxidation of unsaturated lipids (Love & Pearson, 1971). Grunwald & Richards (2006) reported that the reaction between metMb and lipid HPO generates ferrylMb, which can abstract a hydrogen atom from lipid (LH) to form an alkyl radical (L. ). In the presence of oxygen, the alkyl radical then generates a peroxyl radical (LOO. ), which can in turn abstract a hydrogen atom from another unoxidized lipid, resulting in the production of lipid HPO. The lipid HPOs generated are continuously involved in the redox reactions of Mb to generate radicals. Once underway, the alkoxyl radical (LO. ) formed rearranges to generate an epoxyradical (epoxyL. ), which reacts with molecular oxygen to form LOO**.** . The epoxyL. can also react with ferrylMB to form metMB, which continuously plays a role as an intermediate for oxidizing lipids in meat. In this case, the antioxidants that are present in the ME and accumulated in the meat could have acted as chain-breaking antioxidants. The radicals generated in meat from the interaction between Mb and lipid oxidation could be scavenged by the antioxidants, and the ferrylMb produced from this process could be reduced by donating electrons from the thiol groups of the antioxidants (Romero et al., 1992).

The proposed mechanism for ERT-delayed oxidation of lipid and Mb in minced big eye tuna (*Thunnus obesus*) meat to which *F. velutipes* extract has been added is shown in Figure 5.

ESH, ergothioneine; ESSH, dimer of ergothioneine; LO, lipid; LO•, alcoxyl radical, LOOH, lipid hydroperoxide, LOO, Peroxy radical; Mb, myoglobin; Mb(Fe=O)2+, ferryl myoglobin; •Mb(Fe=O)2+, ferryl myoglobin radical; MbO2, oxymyoglobin; Mb•, myoglobin radical; metMb, metmyoglobin.

Fig. 5. Proposed mechanism for ERT-delayed oxidation of lipid and myoglobin in minced big eye tuna *(Thunnus obesus)* meat to which *F. velutipes* extract has been added (Bao et al., 2009b).

Thus, the delay in metMb formation in the dark muscle of yellowtail fed with ME might result from both suppression of lipid oxidation and conservation of endogenous antioxidants. The *in vivo* study of Bao et al. (2009a) was the first model to show that dietary supplementation with ME can be used on an industrial scale for preventing discoloration and lipid oxidation in the dark muscle of yellowtail.
