BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come
276 Part 2: Biotechnology and EnzymologyTable 14.5.Characteristic of polyphenoloxidase from CrustaceansSourceMW
(kDa)pH
OptimumpH
StabilityTemperature
Optimum (◦C) ReferenceKuruma prawn 160 6.5 3–10 35 Benjakul et al. (2005)
Imperial tiger prawn – 5–8 Basic pH 40–60 Montero et al. (2001)Deepwater pink shrimp 200 4.5 4.5–9.0 15–60 Zamorano et al. (2009)
Clam 76.9 7.0 - 40 Cong et al. (2005)
White shrimp 30 6.5–7.5 8.0 45 Simpson et al. (1987)
Lobster – 6.5 7.5 30 Opoku-Gyamfua et al. (1992)Crab 64.5 6.0 – 40 Liu et al. (2006)2000). The characteristics of PPO from different crustaceans are
summarized in Table 14.5.Activators/InhibitorsOne characteristic of PPO is its ability to exist in an inactive or
latent state. PPO can be released from latency by several agents
or treatments such as incubation with ammonium sulfate, anionic
detergents, carboxymethylcellulose, urea, guanidine salts, short-
time acid or base treatment or a limited proteolysis (Whitaker
1995), and alcohol (methanol) (Adachi et al. 1999).
Several factors have been reported to influence PPO activ-
ity. They include: (a) a proenzyme, (b) a PPO-bound inhibitor,
and (c) a conformation change (Gauillard and Richard-Forget
1997). Chazarra et al. (1997) reported that the presence of SDS
in the assay medium provokes a conformation change in the
latent iceberg lettuce PPO, leading to an increase in PPO ac-
tivity with increasing concentration. Adachi et al. (1999) and
Mazzafera and Robinson (2000) also found that proPPO from
kuruma prawn was activated by SDS. Some ion or salts af-
fect the PPO activity. MnSO 4 and CaCl 2 enhanced PPO activity
(Adachi et al. 1999). Kim et al. (2000) observed that the activity
was increased by addition of 1 mM Mg^2 +,K+,orCu^2 +.How-
ever, the activity of PPO was dependent on the concentrations
of divalent cations (Adachi et al. 1999). Adachi et al. (1999)
reported that the highest activity of PPO from kuruma prawn
was obtained at a concentration of 50 mM for both Mg^2 +and
Ca^2 +. Ascorbic acid, cysteine, glutathione, thiourea, and hexyl-
resorcinol were demonstrated to control enzymatic browning
induced by PPO (Guerrero-Beltran et al. 2005). Benjakul et al.
(2005) reported that phenylthiourea, a copper-chelating agent,
and cysteine exhibited inhibitory effects against PPO from ku-
ruma prawn. Total inhibition of the extract from imperial tiger
prawn was achieved with ascorbic acid and citric acid at pH
3.0 (Montero et al. 2001). Nirmal and Benjakul (2009a) found
that ferulic acid and oxygenated ferulic acid inhibited PPO from
Pacific white shrimp, and ferulic acid was the more effective
inhibitor than oxygenated ferulic acid. Recently, Nirmal and
Benjakul (2009b) reported that catechin showed inhibitory ac-tivity toward PPO of Pacific white shrimp in a dose-dependent
manner.MelanosisMelanosis is a process that is triggered by the oxidation of phe-
nols to quinones induced by PPO or PO (Montero et al. 2001).
PO is responsible for a discoloration in crustacean species such
as lobster, shrimp, and crab. The postmortem dark discoloration
in crustaceans, called melanosis or blackspot, connotes spoilage
(Kim et al. 2000). Melanosis reduces the market value of these
foods and causes large financial losses to the food industry
(Williams et al. 2003).
Melanosis or blackspot appears very frequently during post-
mortem storage of crustacea prior to the onset of spoilage.
Melanosis starts in refrigerated crustaceans within a few hours
of capture, and the susceptibility is often greater in autumn
and winter, coinciding with molting and lower food availability
(Montero et al. 2001). Marine crustaceans form the black
spots on their tail, cephalothoraxes, and podites during stor-
age (Adachi et al. 1999). In chilled prawns and shrimps, the
melanosis reaction begins from the head region and spreads to
the tail (Simpson et al. 1987), and the rate of spread of melanosis
differs among various species. This could be related to differ-
ences in levels of substrate or levels of enzyme concentration or
enzymatic activity in each species (Simpson et al. 1987). Pink
shrimp PPO oxidized DOPA 1.8-fold more rapidly than white
shrimp PPO. Pink shrimp carapace has relatively higher levels of
free Tyr and phenylalanine, the natural substrates of melanosis,
than white shrimp carapace (Rolle et al. 1991). The enzymatic
oxidation to form melanin is associated with the stages of molt-
ing cycle, sex, and occurrence of injury or trauma (Ogawa et al.
1984). To prevent or retard melanosis, several PPO inhibitors
including reducing agents or antioxidants inhibitors such as sul-
fiting agent, cysteine, acidulants, and metal ion chelators have
been used. Because of the susceptibility of certain individu-
als to the residual level of sulfiting agents commonly used for
melanosis prevention in crustaceans, natural additives such as
plant phenols have been used to reduce melanosis formation in