BLBS102-c14 BLBS102-Simpson March 21, 2012 13:17 Trim: 276mm X 219mm Printer Name: Yet to Come
278 Part 2: Biotechnology and Enzymologykidney by heat treatment, ammonium sulfate precipitation, and
a series of chromatographies including Sephacryl S-300 and
DEAE–cellulose. The addition of partially purified TMAOase
from lizardfish kidney to haddock natural actomyosin in the
presence of cofactors (FeCl 2 , ascorbate, and cysteine) acceler-
ated formaldehyde formation throughout the storage either at
4 ◦Cor− 10 ◦C.Prevention of TMAOase ActivityTMAOase inhibition has been focused on preventing loss of
quality in frozen fish, especially in some species such as cod.
The inhibition of the TMAO demethylation can be achieved us-
ing several inhibitors. Most of them are general inhibitors of
redox reactions, being strong oxidants, such as H 2 O 2 , which in-
hibits the reaction (Phillipy and Hultin 1993). The presence
of oxygen or oxidants can promote inhibition of enzymatic
TMAO breakdown in frozen red hake (Racicot et al. 1984).
Iodoacetamide, cyanide, and azide have been shown to inhibit
TMAOase activity (Sotelo and Rehein 2000); while sodium cit-
rate and H 2 O 2 have been found to slow down the rate of DMA
and FA formation in frozen gadoid mince (Racicot et al. 1984).
Treating Alaska pollack fillets with sodium citrate and sodium
pyruvate before freezing resulted in a less texture toughening
(Krueger and Fennema 1989). Furthermore, the addition of 0.5%
alginate could lower the DMA and FA formation in minced
fillet of mackerel stored at− 20 ◦C for 2 months (Ayyad and
Aboel-Niel 1991).
Apart from reducing protein denaturation, cryoprotectants
have also been reported to prevent FA formation. Sotelo and
Mackie (1993) found a protective role of different cryoprotec-
tants (amino acids and sugars) in preventing FA-promoted ag-
gregation of bovine serum albumin during frozen storage. The
binding of FA during frozen storage was dependent on protein
rearrangements in the way that reactive groups become avail-
able. The constraints of cryostabilizers on molecular diffusion
reduced the exposure of these groups (Herrera et al. 1999). Her-
rera et al. (2000) also reported that the addition of maltodextrins
to minced blue whiting muscle inhibited FA production during
storage at− 10 ◦C and− 20 ◦C. Sucrose, however, was effective
only at− 20 ◦C. Herrera et al. (2002) studied the effects of vari-
ous cryostabilizers on inhibiting FA production in frozen-stored
minced blue whiting muscle. Several maltodextrins (dextrose
equivalent 9, 12, 18, and 28) or sucrose minimized the decrease
in protein solubility during storage at− 10 ◦C and− 20 ◦C. These
effects were greater at− 20 ◦C, and DE 18 maltodextrin seemed
to be the most effective treatment at both temperatures. Con-
versely, sucrose was as effective as maltodextrins at− 20 ◦C,
but showed hardly any effect at− 10 ◦C. A high correlation was
found between the effectiveness of cryostabilizers in prevent-
ing protein alterations and their effectiveness in inhibiting FA
production, particularly at− 10 ◦C. In addition, a sigmoidal re-
lationship between protein solubility and FA content was found
at this temperature, which supports the hypothesis of the coop-
erative nature of the effect of FA on protein alterations (Herrera
et al. 2002).Addition of TMAOase inhibitors could be a means to retard
the loss in textural properties of some fish species associated
with FA formation. Leelapongwattana et al. (2008b) reported
that sodium citrate and pyrophosphate could inhibit TMAOase
activity from lizardfish muscle in a concentration dependent
manner, most likely due to their chelating property. Sodium al-
ginate is a hydrocolloid possessing inhibitory activity toward
TMAOase. During the storage of lizardfish mince at− 20 ◦Cfor
24 weeks, the addition of 0.5% sodium alginate and 0.3% py-
rophosphate in combination with 4% sucrose and 4% sorbitol as
the cryoprotectants slowed down TMAOase activities and low-
ered the formation of DMA and FA. The addition of TMAOase
inhibitor (0.3% pyrophosphate and 0.5% alginate) in haddock
mince containing TMAOase retarded FA formation throughout
storage at− 10 ◦C for 6 weeks. On the other hand, antioxidants
(250 mg ascorbic acid/kg and 250 mg tocopherol/kg) induced
FA formation (Leelapongwattana et al. 2008a, 2008b).
Moreover, TMAO has also been found to exert a protective
role over proteins. It has been shown that TMAO stabilizes fish
muscle proteins and some enzymes during the frozen storage
(LeBlanc and LeBlanc 1989).LIPASE
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) constitute a
group of enzymes defined as carboxylesterases that catalyze the
hydrolysis (and synthesis) of long-chain acyl-glycerols at the
lipid–water interface (Cherif and Gargouri 2009). The two main
categories in which lipase-catalyzed reactions may be classified
are given in Figure 14.8.
From Figure 14.8, the last three reactions are often grouped
together into a single term, namely, transesterification.
Lipases are important because of the role they play in the
postmortem quality deterioration of seafood (and other food-
stuffs) during handling, chilled, and frozen storage. Lipases are
finding increasing uses as food and others industrial processing
aids (Aryee et al. 2007). Compared with other hydrolytic en-
zymes (e.g., proteases and carbohydrases), lipases are relative
less well studied and in this regard, lipases from aquatic animals
are even less well-known versus their counterparts from mam-
malian, plant, and microbial sources (Aryee et al. 2007). The
presence of a lipolytic activity and partial characterization of a
lipase in marine organisms have been reported in few studies.
The pH and temperature optima of most lipases lie between 7
and 9 and 35◦C and 60◦C, respectively. Lipases are active over
a very wide temperature range of− 20 ◦Cto65◦C, but the more
usual range is 30–45◦C (Shahidi and Kamil 2001).
The presence of a lipase activity has been described for some
fish and aquatic organisms. Mukundan et al. (1985) described
the purification of a lipase from the hepatopancreas of oil sar-
dine using anion-exchange chromatography and gel filtration.
The glycosylated enzyme (6.1% carbohydrate) showed a MW
of 54–57 kDa (by gel filtration), was marginally activated by ap-
proximately 1 mM Ca^2 +, was completely inhibited by approx-
imately 1 mM F−, and hydrolyzed tributyrin most efficiently
among C2–C18 monoacid triglycerides (TGs). Sardine lipase
exhibited optimum activity against tributyrin at pH 8–8.5 and