**5. Summary**

178 Aquaculture

Fig. 14. Inhibitory effects of commercial L-ergothioneine (a), mushroom (*F. velutipes*) extract

polyphenoloxidase. Results are presented in terms of mean ± standard deviation (*n=3*). The values with different superscript letters represent significant difference (*p*<0.05) at the end of

Fig. 15. Inhibition of the activation of the proPO system in hemocyte lysate supernatant by commercial L-ergothioneine (L-ERT) and *p*-amidinophenyl methanesulfonyl fluoride hydrochloride (*p*-APMSF). Polyphenoloxidase activity (a) and peptidase activity (b). 7 amino-4-methylcoumarin (AMC) was used as the standard solution in the peptidase activity assay. The final concentration of each inhibitor in the reaction system was 1 mM. Results are

the proPO cascade leading to PPO activation and is itself tightly regulated (Buda & Shafer, 2005). The results of the peptidase activity assay indicated that L-ERT inhibited serine protease activity but that this effect was not as strong as that of *p*-APMSF (Fig. 16b). However, the presence of L-ERT could possibly affect the overall activation of the proPO system because it inhibited PPO activity (Fig.16a). At least 4 mechanisms underlying PPAE regulation have been recognized: gene induction, activation by another protease, requirement for noncatalytic serine protease homologues as cofactors, and inactivation by serine protease inhibitors (Wang & Jiang, 2004). Thus, other proPO activating factors such as

presented in terms of mean ± standard deviation (n = 3). The values with different superscript letters represent significant difference between groups for different storage

(b), and residues of diets used in the feeding trial (c) on the activity of mushroom

the reaction period (Encarnacion et al., 2010).

periods (*p* < 0.05) (Encarnacion et al., 2010).

Mushroom trimmings are cost-effective and scalable source of ME and contain significant amounts of ERT, which has been proven to have antioxidative and melanosis-inhibiting properties. Yellowtail fish *S. quinqueradiata* that were fed a diet, including ME showed significantly reduced myoglobin and lipid oxidation in dark muscles during chilled storage. The results of the feeding trial are also supported by data from similar trials on other aquacultured fish species such as jack mackerel and rainbow trout (data not shown). Immersion of live *M. japonicus* shrimps and *C. japonicus* crabs in an *F. velutipes* ME solution containing a significant amount of ERT effectively inhibited postharvest melanosis in shrimps and crabs, and this result was comparable to those obtained using melanosisinhibiting agents such as HR, AA and SS. *In vitro* experiments with exogenous ERT confirmed that it inhibits PPO activity and proPO gene expression in shrimp HLS. Thus, ME containing ERT is a promising natural alternative to the synthetic melanosis-inhibiting agents used to prevent postharvest melanosis in shrimps and other crustaceans. Furthermore, the use of biowaste as the raw material for extracting and producing antioxidants or of melanosis-inhibiting compounds such as ERT for food and industrial applications could also help reduce the growing problem of agricultural waste.

#### **6. References**


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