Food Biochemistry and Food Processing (2 edition)

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250 Part 2: Biotechnology and Enzymology

during capture is often intensive, the muscular glycogen stores

may be almost depleted even before slaughtering takes place.

The activation of myosin ATPase by spinally mediated reflexes

continues after slaughter, but the extent to which it occurs can be

reduced by employing rested harvest techniques such as anes-

thetization (Jerrett and Holland 1998, Robb et al. 2000); by

destroying the nervous system by use of appropriate methods

such asiki jime,where the brain is destroyed by a spike (Lowe

1993), or by use of still other techniques (Chiba et al. 1991, Berg

et al. 1997).

ATPases hydrolyze the terminal phosphate ester bond, form-

ing ADP. ADP is subsequently degraded by adenylate kinase,

forming AMP. The degradation of ADP is favored by the removal

of AMP by AMP deaminase (EC 3.5.4.6), producing inosine

monophosphate (IMP) and ammonia (NH 3 ). AMP deaminase

is a key regulator of the intracellular adenine nucleotide pool

and as such is a highly regulated enzyme inhibited by inorganic

phosphate, IMP and NH 3 and activated by ATP.

It is widely accepted that IMP contributes to the desirable taste

of fresh seafood (Fluke 1994, Gill 2000, Haard 2002). IMP is

present in small amounts in freshly caught relaxed fish but builds

up during the depletion of ATP (Berg et al. 1997). IMP can be

hydrolyzed to form inosine, yet the rate of hydrolysis in fish is

generally low, following zero-order kinetics, indicating that the

IMP-degrading enzymes are fully substrate saturated (Tomioka

et al. 1987, Gill 2000, Itoh and Kimura 2002). However, a large

species variation in the degradation of IMP has been shown,

and thus IMP may persist during storage for several days or

even weeks (Dingle and Hines 1971, Gill 2000, Haard 2002).

The formation of inosine is often rate limiting for the overall

breakdown of nucleotides and is considered to constitute the last

purely autolytic step in the nucleotide catabolism of chilled fish.

The formation of inosine usually indicates a decline in the prime

quality of seafood (Surette et al. 1988, Gill 2000, Haard 2002).

IMP can be hydrolyzed by various enzymes, 5’-nucleotidase

(EC.3.1.3.5) being regarded as the most important of these in

the case of chilled fish (Gill 2000, Haard 2002). Several forms

of 5’-nucleotidase exist, of which a soluble sarcoplasmic form

has been documented in fish (Itoh and Kimura 2002).

The degradation of inosine continues via hypoxanthine and

its oxidized products xanthine and uric acid. These reactions, all

of them related to spoilage, are catalyzed by both endogenous

and microbial enzymes, the activity of the latter depending upon

the spoilage flora that are present and thus differing much be-

tween species and products. Inosine can be degraded by either

nucleoside phosphorylase (EC 2.4.2.1) or inosine nucleosidase

(EC 3.2.2.2), leading to the formation of different by-products

(see Fig. 13.1). The final two-step oxidation of hypoxanthine is

catalyzed by one and the same enzymes, xanthine oxidase (EC

1.1.3.22). The oxidation requires a reintroduction of oxygen in

the otherwise anaerobic pathway, which often limits the extent

of the xanthine oxidase reaction and causes inosine and hypox-

anthine to accumulate. Hypoxanthine is an indicator of spoilage

and may contribute to bitter taste (Hughes and Jones 1966).

The hydrogen peroxide produced by the oxidation of hypoxan-

thine can be expected to cause oxidation, but the technological

Figure 13.1.The successive reactions of the main pathway for nucleotide degradation in fish. Substances listed below the dotted line are

generally considered to be indicators of spoilage. Piis inorganic phosphate.