**4. Melanosis in crustaceans**

164 Aquaculture

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).

which can abstract a hydrogen atom from lipid (LH) to form an alkyl radical (L.

presence of oxygen, the alkyl radical then generates a peroxyl radical (LOO.

to generate radicals. Once underway, the alkoxyl radical (LO.

electrons from the thiol groups of the antioxidants (Romero et al., 1992).

an epoxyradical (epoxyL.

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,

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

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

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.

), which reacts with molecular oxygen to form LOO**.**

). In the

can

), which can in

. The epoxyL.

) formed rearranges to generate

The development of melanosis or blackspot during the postharvest period of crustaceans is a well-known postmortem phenomenon attributed to the polymerization of phenol into an insoluble black pigment, melanin. Phenol polymerization is mainly initiated by the action of an enzymatic complex called polyphenoloxidase (PPO; also known as tyrosinase). Severe blackspot formations can cause tremendous economic losses because of the high value commanded by these aquatic products in the marketplace (Kim et al., 2002). Its prevention has been a challenge to the industry, especially for food scientists.

Intensive studies on PPO in crustaceans could help understand the mechanisms underlying the development of melanosis in crustaceans; the properties of PPO; their substrates and inhibitors; and the physical, biological and chemical factors that affect each of these parameters. Complete understanding of this phenomenon and the mechanisms underlying it may help provide a scientific approach to prevent melanosis and slow its rate, thus extending the shelf life and acceptability of the product.

#### **4.1 PPO in melanosis development**

PPO plays an important physiological role in crustaceans, particularly in the process of sclerotization. The melanization cascade has been reported to be closely associated with the occurrence of factors that stimulate cellular defence by aiding phagocytosis and encapsulation reactions (Cerenius et al., 2008). Thus, melanization is an important immune response in crustaceans, similar to that observed in plants. In plants, the compounds produced as a result of the polymerization of quinones have been reported to exhibit both antibacterial and antifungal activities, whereas in crustaceans, PPO is thought to be involved in wound healing and sclerotization of the cuticle. In live crustaceans, the activation of prophenoloxidase (proPO) to PPO requires proteases and microbial activators such as polysaccharide-binding proteins (García-Carreño et al., 2008). This activation system plays an important role in the primary immune response, cuticle sclerotization, and injury healing in crustaceans (Buda & Shafer 2005; Martínez-Álvarez et al., 2005; José-Pablo et al., 2009). Simple diagram on the induction of the proPO cascade in the invertebrate immunity has been shown in Figure 6.

Fig. 6. Induction of the prophenoloxidase (proPO) cascade in the invertebrate immunity. Pathogen-associated molecular patterns (PAMPs; e.g., peptidoglycans, lipopolysaccharides, and β-1,3-glucans) are bound by host recognition proteins. This initiates a serine proteinase cascade that leads to the conversion of zymogenic proPO into catalytically active phenoloxidase and ultimately results in the generation of cytotoxic products and encapsulation of the pathogen (Cerenius et al., 2008).

Postharvest PPO-catalyzed blackening of the shell in crustaceans adversely affects both quality and the consumers' acceptance of these products. Crustaceans such as lobsters, shrimps, and crabs are extremely vulnerable to enzymatic blackening or melanosis. Although the occurrence of melanosis in these aquatic products does not necessarily mean that they are unfit for human consumption, consumers tend to be selective regarding these

PPO plays an important physiological role in crustaceans, particularly in the process of sclerotization. The melanization cascade has been reported to be closely associated with the occurrence of factors that stimulate cellular defence by aiding phagocytosis and encapsulation reactions (Cerenius et al., 2008). Thus, melanization is an important immune response in crustaceans, similar to that observed in plants. In plants, the compounds produced as a result of the polymerization of quinones have been reported to exhibit both antibacterial and antifungal activities, whereas in crustaceans, PPO is thought to be involved in wound healing and sclerotization of the cuticle. In live crustaceans, the activation of prophenoloxidase (proPO) to PPO requires proteases and microbial activators such as polysaccharide-binding proteins (García-Carreño et al., 2008). This activation system plays an important role in the primary immune response, cuticle sclerotization, and injury healing in crustaceans (Buda & Shafer 2005; Martínez-Álvarez et al., 2005; José-Pablo et al., 2009). Simple diagram on the induction of the proPO cascade in the invertebrate immunity has been shown in Figure 6.

Fig. 6. Induction of the prophenoloxidase (proPO) cascade in the invertebrate immunity. Pathogen-associated molecular patterns (PAMPs; e.g., peptidoglycans, lipopolysaccharides, and β-1,3-glucans) are bound by host recognition proteins. This initiates a serine proteinase

Postharvest PPO-catalyzed blackening of the shell in crustaceans adversely affects both quality and the consumers' acceptance of these products. Crustaceans such as lobsters, shrimps, and crabs are extremely vulnerable to enzymatic blackening or melanosis. Although the occurrence of melanosis in these aquatic products does not necessarily mean that they are unfit for human consumption, consumers tend to be selective regarding these

cascade that leads to the conversion of zymogenic proPO into catalytically active phenoloxidase and ultimately results in the generation of cytotoxic products and

encapsulation of the pathogen (Cerenius et al., 2008).

**4.1 PPO in melanosis development** 

products, mainly because browning in the carapace connotes spoilage; thus, melanosis decreases the product's market value.

Enzymatic browning or melanosis takes place in the presence of oxygen when tyrosinase and their polyphenolic substrates are mixed. Tyrosinase catalyzes 2 fundamental reactions: 1) hydroxylation at the *o*-position adjacent to an existing hydroxyl group of phenolic compounds (monophenolase activity) and 2) oxidation of diphenol to *o*-benzoquinones (diphenolase activity).

The subunits of tyrosinase have been reported to differ with respect to chemical, physical and kinetic properties, which were believed to be responsible for the relative affinities of the enzymes for both mono- and diphenolic substrates (Kim et al., 2002). The aforementioned considerations have led to many studies on the molecular mechanism underlying the monophenolase and diphenolase activity of tyrosinase. Many studies have been performed on the monophenolase activity of tyrosinase, based on 3 forms of the enzyme. The monophenolase cycle is shown in Figure 7, wherein monophenol reacts with the oxy form and binds at the axial position of one of the coppers ions of this oxy form. It has been reported that rearrangement through the trigonal bipyramidal intermediate leads to binding of peroxide and thereby generates a coordinated *o*-diphenol that is oxidized to *o*-quinone, resulting in a deoxy form ready for dioxygen binding. In the diphenolase cycle, both the oxy and met forms react with *o*-diphenol, oxidizing it to *o*-quinone. However, monophenol can compete with *o*-

Fig. 7. Catalytic cycles of the hydroxylation of monophenol and oxidation of *o*-dipehol to *o*quinone by tyrosinase (Kim and Uyama, 2005).

diphenol for binding to the met form site, thereby inhibiting its reduction (Kim & Uyama, 2005). Diphenolases have received much attention because of their high catalytic rate and their association with quinone formation that in turn leads to melanin production.

#### **4.2 Control of melanosis**

The fundamental step in melanosis is the transformation of an *o*-diphenol such as 3,4 dihydroxyphenylalenine (L-DOPA) to the corresponding *o*-quinone, which undergoes further oxidation to form brown or black pigments (Martinez & Whitaker, 1995). *o*-Quinones are known to be powerful electrophiles that can be attacked by water, other polyphenols, amino acids, peptides, and proteins, leading to Michael-type products (Mayer et al., 1990).

Many studies have been performed on controlling or inhibiting the activity of PPO in foods, and various mechanisms and techniques have been proposed and developed over the years to mitigate concerns regarding undesirable products of enzyme activity. This involves elimination of one or more of the important components involved in the enzymatic reaction, such as oxygen, copper, substrate, and the enzyme itself. There are also known compounds that react with the product of enzyme activity, thus inhibiting the formation of colored compounds. Many other techniques are also being applied to prevent melanosis in foods, such as processing methods, different types and kinds of inhibitors, and a molecular approach to control PPO, as presented in the following section.

#### **4.2.1 Processing**

Traditionally, heating is one of the most popular method to destroy microorganism and to inactivate enzymes. The catalytic activity of PPO is inhibited at temperatures ranging from 70ºC to 90ºC (Vamos-Vigyoso, 1981). Lee et al. (1988) reported that blanching green beans at temperatures above 82 ºC inactivated its enzymes, including PPO.

Similar to heat treatment, low-temperature treat can also help control enzyme-catalyzed reactions. At low temperatures, the kinetic energy of the reactant molecules decreases, subsequently leading to a decrease in the mobility and reaction necessary for the formation of enzyme-substrate complexes and their products (Kim et al., 2002). Thus, low-temperature preservation and storage are used during distribution and retailing to control the development of melanosis in food products. Temperatures as low as -18ºC can be used to inactivate enzymes. However, this changes the physical attributes of the products.

Dehydration can also be used to control enzyme catalyzed reactions that cause browning in foods. Generally, water greatly influences enzyme activity because it acts as a solvent or a reactant (Ashie & Simpson, 1996). Water activity can be controlled by physical drying (i.e., dehydration or freeze drying) or by chemical methods (i.e., addition of water-binding agents such as sugars and salts).

Irradiation is another method used for controlling enzymatic browning in foods. It uses ionizing radiation to inactivate microorganisms as well as enzymes. Irradiation is being increasingly recognized as a method for reducing postharvest losses, maintaining food quality, as well as ensuring the hygienic quality of food products.

The rearrangement or destruction of noncovalent bonds such as hydrogen bonds, hydrophobic interactions, and ionic bonds of the tertiary protein structure can lead to enzyme denaturation. This can be achieved by high-pressure treatment that reduces molecular spacing and increases interchain reactions. However, this varies depending the enzymes; the nature of the substrate; and the temperature and duration of high-pressure processing (Cheftel, 1992).

Other processing methods that have been developed and used to control the enzymatic browning of food products included the treatment of food with supercritical carbon dioxide, ultrafiltration, or ultrasonication (Kim et al., 2002). Treatment with supercritical carbon dioxide inactivates the enzyme through carbonic acid production, which causes a significant pH reduction. In contrast, ultrafiltration removes PPO during the process, whereas ultrasonication causes severe shear stresses, thereby promoting enzyme denaturation.

## **4.2.2 Inhibitors**

168 Aquaculture

diphenol for binding to the met form site, thereby inhibiting its reduction (Kim & Uyama, 2005). Diphenolases have received much attention because of their high catalytic rate and

The fundamental step in melanosis is the transformation of an *o*-diphenol such as 3,4 dihydroxyphenylalenine (L-DOPA) to the corresponding *o*-quinone, which undergoes further oxidation to form brown or black pigments (Martinez & Whitaker, 1995). *o*-Quinones are known to be powerful electrophiles that can be attacked by water, other polyphenols, amino acids, peptides, and proteins, leading to Michael-type products (Mayer et al., 1990). Many studies have been performed on controlling or inhibiting the activity of PPO in foods, and various mechanisms and techniques have been proposed and developed over the years to mitigate concerns regarding undesirable products of enzyme activity. This involves elimination of one or more of the important components involved in the enzymatic reaction, such as oxygen, copper, substrate, and the enzyme itself. There are also known compounds that react with the product of enzyme activity, thus inhibiting the formation of colored compounds. Many other techniques are also being applied to prevent melanosis in foods, such as processing methods, different types and kinds of inhibitors, and a molecular

Traditionally, heating is one of the most popular method to destroy microorganism and to inactivate enzymes. The catalytic activity of PPO is inhibited at temperatures ranging from 70ºC to 90ºC (Vamos-Vigyoso, 1981). Lee et al. (1988) reported that blanching green beans at

Similar to heat treatment, low-temperature treat can also help control enzyme-catalyzed reactions. At low temperatures, the kinetic energy of the reactant molecules decreases, subsequently leading to a decrease in the mobility and reaction necessary for the formation of enzyme-substrate complexes and their products (Kim et al., 2002). Thus, low-temperature preservation and storage are used during distribution and retailing to control the development of melanosis in food products. Temperatures as low as -18ºC can be used to

Dehydration can also be used to control enzyme catalyzed reactions that cause browning in foods. Generally, water greatly influences enzyme activity because it acts as a solvent or a reactant (Ashie & Simpson, 1996). Water activity can be controlled by physical drying (i.e., dehydration or freeze drying) or by chemical methods (i.e., addition of water-binding agents

Irradiation is another method used for controlling enzymatic browning in foods. It uses ionizing radiation to inactivate microorganisms as well as enzymes. Irradiation is being increasingly recognized as a method for reducing postharvest losses, maintaining food

The rearrangement or destruction of noncovalent bonds such as hydrogen bonds, hydrophobic interactions, and ionic bonds of the tertiary protein structure can lead to

inactivate enzymes. However, this changes the physical attributes of the products.

their association with quinone formation that in turn leads to melanin production.

approach to control PPO, as presented in the following section.

temperatures above 82 ºC inactivated its enzymes, including PPO.

quality, as well as ensuring the hygienic quality of food products.

**4.2 Control of melanosis** 

**4.2.1 Processing** 

such as sugars and salts).

Melanosis can be inhibited by targeting the enzyme, the substrate, and/or the reaction products involved in the process. Inhibition that targets the enzyme includes metal chelators and compounds that consist of carboxylic acids of the benzoic and cinnamic series. These compounds can be competitive owing to their structural similarity with phenolic substrates. Inhibition that targets the substrate can be performed by removing either oxygen or the phenolic substrate from the reaction. Inhibition targeting the products involves compounds that can react with *o*-quinones to produce a colorless addition product.

These PPO inhibitors can be classified into 6 categories that include: 1) reducing agents such as sulfites, ascorbic acid and its analogs, cysteine, and glutathione; 2) chelating agents such as phosphates, EDTA, and organic acids; 3) acidulants such as citric acid and phosphoric acid; 4) enzyme inhibitors such as aromatic carboxylic acids, aliphatic alcohols, anions, peptides, and substituted resorcinols; 5) treatments with enzymes such as oxygenases, *o*methyltransferase and proteases; and 6) complexing agents such as cyclodexrins (McEvily et al., 1992). To date, there have been many studies on the different compounds used to inhibit melanosis in foods.

#### **4.2.3 Molecular approach**

The nature of PPO and its role in the immune system of plants and crustaceans have been extensively reviewed. One possible approach to reduce the activity of PPO and the resultant enzymatic browning reactions is to characterize and inactivate the PPO coding genes. This can be done by generating antisense RNAs specific for PPO.

Antisense genes have been successfully used for altering plant processes and improving crops. It involves blocking gene expression of the plant enzymes involved in a certain process. The process is based on blocking the flow of information from DNA via RNA to protein by introducing an RNA strand complementary to the sequence of the target mRNA. The antisense approach involves the insertion of a gene or a significant part of it into the cell in a reverse orientation. This approach has been used to increase the shelf life of fruits (Fray & Grierson, 1993), and commercial applications of this technology now include alteration of flower color, viral resistance induction, and fruit ripening. It has been reported that the lack of bruising sensitivity in transgenic potatoes without any side effects opens up the possibility of preventing melanosis in wide varieties of food crops and even to crustaceans, without the use of any physical and chemical treatments. Thus, by using this antisense technology, it is possible to develop fruits, vegetables and crustaceans that are resistant to enzymatic browning.

#### **4.3 Application of ERT-rich edible mushroom (***Flammulina velutipes***) extract for controlling melanosis**

As mentioned earlier, many technologies and techniques have been developed for the inhibition of melanosis in food products, and some more new approaches are under study. However, these alternatives must be evaluated in terms of their impact on the overall food quality, effectiveness, cost, and regulatory status. Because of health concerns, only a number of inhibitors have been accepted for food application, based on the basis of government health regulations.

A relatively new approach involves the design and development of another application of ERT-rich edible mushroom (*F. velutipes*) extract for inhibiting melanosis in commercially important crustaceans.

#### **4.3.1 Dietary supplementation**

The extract from the edible mushroom *F. velutipes* has been found to significantly inhibit mushroom PPO activity; prevent browning in apples; and delay melanosis in shrimps which usually develops during storage (Jang et al., 2002; 2003). These observations suggest that the mushroom extract contains certain compounds contributing to such actions. In section 3, it has been mentioned that ME containing ERT at the level of 3.03 ± 0.07 mg/mL has been reported to show remarkable DPPH radical scavenging activity and suppress lipid oxidation in bigeye tuna meat (Bao et al., 2008). ME have been reported to stabilize the fresh color of tuna meat during ice storage (Bao et al., 2009a). These results strongly suggest that ERT could be one of the major compounds in the ME that effectively inhibited mushroom PPO activity, apple browning, and melanosis in shrimps in the previous studies (Jang et al., 2002; 2003).

The *in vivo* application of L-ERT as an antioxidant in rats (Deiana et al., 2004) and the effects of feeding fish with ERT-rich ME have been studied; however, before this study, no study had been performed to determine the efficacy of extracts that contains this compound, when fed to crustaceans to control melanosis. Therefore, a study was performed on the use of a hydrophilic extract prepared from the fruiting body of *F. velutipes* for controlling melanosis and lipid oxidation in cultured kuruma shrimps (*Marsupenaeus japonicus*) *in vivo*.

In a study performed by Encarnacion et al. (2010), the hot water ME containing 2.05 ± 0.24 mg/mL ERT remarkably inhibited mushroom PPO. The PPO activity is usually found to be higher in the carapace area during postmortem of crustaceans (José-Pablo et al., 2009) and this study showed that the development of melanosis in the carapace area of the shrimps during ice storage was relatively controlled in the ME-fed group than in the control (Fig. 8). The accumulation of ERT from the ME added in the diet during feeding could have contributed to this effect.

In the same study, the PPO activities of hemolymph from shrimps fed a diet containing the ME was significantly lower than that of the control group, and the expression of the proPO genes in the hemocytes of shrimps fed with the ME was relatively lower than that in the

technology, it is possible to develop fruits, vegetables and crustaceans that are resistant to

As mentioned earlier, many technologies and techniques have been developed for the inhibition of melanosis in food products, and some more new approaches are under study. However, these alternatives must be evaluated in terms of their impact on the overall food quality, effectiveness, cost, and regulatory status. Because of health concerns, only a number of inhibitors have been accepted for food application, based on the basis of government

A relatively new approach involves the design and development of another application of ERT-rich edible mushroom (*F. velutipes*) extract for inhibiting melanosis in commercially

The extract from the edible mushroom *F. velutipes* has been found to significantly inhibit mushroom PPO activity; prevent browning in apples; and delay melanosis in shrimps which usually develops during storage (Jang et al., 2002; 2003). These observations suggest that the mushroom extract contains certain compounds contributing to such actions. In section 3, it has been mentioned that ME containing ERT at the level of 3.03 ± 0.07 mg/mL has been reported to show remarkable DPPH radical scavenging activity and suppress lipid oxidation in bigeye tuna meat (Bao et al., 2008). ME have been reported to stabilize the fresh color of tuna meat during ice storage (Bao et al., 2009a). These results strongly suggest that ERT could be one of the major compounds in the ME that effectively inhibited mushroom PPO activity, apple browning, and melanosis in shrimps in the previous studies (Jang et al., 2002;

The *in vivo* application of L-ERT as an antioxidant in rats (Deiana et al., 2004) and the effects of feeding fish with ERT-rich ME have been studied; however, before this study, no study had been performed to determine the efficacy of extracts that contains this compound, when fed to crustaceans to control melanosis. Therefore, a study was performed on the use of a hydrophilic extract prepared from the fruiting body of *F. velutipes* for controlling melanosis

In a study performed by Encarnacion et al. (2010), the hot water ME containing 2.05 ± 0.24 mg/mL ERT remarkably inhibited mushroom PPO. The PPO activity is usually found to be higher in the carapace area during postmortem of crustaceans (José-Pablo et al., 2009) and this study showed that the development of melanosis in the carapace area of the shrimps during ice storage was relatively controlled in the ME-fed group than in the control (Fig. 8). The accumulation of ERT from the ME added in the diet during feeding could have

In the same study, the PPO activities of hemolymph from shrimps fed a diet containing the ME was significantly lower than that of the control group, and the expression of the proPO genes in the hemocytes of shrimps fed with the ME was relatively lower than that in the

and lipid oxidation in cultured kuruma shrimps (*Marsupenaeus japonicus*) *in vivo*.

**4.3 Application of ERT-rich edible mushroom (***Flammulina velutipes***) extract for** 

enzymatic browning.

**controlling melanosis** 

health regulations.

important crustaceans.

contributed to this effect.

2003).

**4.3.1 Dietary supplementation** 

Fig. 8. Development of melanosis (a) and changes in the mean gray values of the carapace area (b) of kuruma shrimps (*Marsupenaeus japonicus*) during ice storage. Results are presented interms of mean±standard deviation (*n* = 3). The values with different superscript letters represent significant difference between groups for different storage periods (*p* < 0.05) (Encarnacion et al., 2010).

Fig. 9. (a) Enzymatic oxidation of catechol and L-DOPA (b) in the hemolymph of kuruma shrimps (*Marsupenaeus japonicus*) after 7 days of feeding. Results are presented in terms of mean ± standard deviation (*n* = 4). The values with different superscript letters represent significant difference (*p* < 0.05) at the end of the reaction period for each assay. (b) Gene expression analysis of prophenoloxidase (proPO) transcripts in the hemocytes of hemolymphs of kuruma shrimp (*M. japonicus*) after 7 days of feeding (representative gel and quantitative analyses of band intensities obtained using the ImageJ software. Elongation factor1-α gene (EF1-α) was used as the standard for computing relative gene expression level for each sample band. Results are presented in terms of mean ± standard deviation (*n* = 3). The values with different superscript letters represent significant difference (*p* < 0.05) (Encarnacion et al., 2010).

hemocytes of the control samples, showing that the ME inhibited PPO activation in the hemolymphs of the supplement-fed shrimps (Fig. 9). Because PPO activity mainly depends on the activation of the proPO system, decreasing the expression of proPO genes in hemocytes consequently reduces the activity of PPO.

The results of a study performed by Amparyup et al. (2009) indicated the significant role of the proPO activating system as one of the major immune responses in shrimps and also reported that inhibiting proPO activation in shrimps could cause death. However, the study did not show any significant differences in mortality between the supplement-fed shrimps the control samples. A study by Leclerc et al. (2006) reported that microbial infections in *Drosophila* did not require the activation of the proPO system for survival thus, raising questions regarding the precise function of phenoloxidase activation and its level of participation in the immune defense system of invertebrates because they use 2 broad but interacting categories of defense responses against pathogens, namely, the cellular and hormonal responses.
