Dairy Chemistry And Biochemistry

ENZYMOLOGY OF MILK AND MILK PRODUCTS^329

least part of which is transported to the mammary gland via the blood-

stream. A similar enzyme is found in various animal tissues and several

bacterial species (Farkye, 1993).

Isolation. Numerous methods have been developed for the purification

of XO from milk; since the enzyme is concentrated in the MFGM, in which

it is one of the principal proteins, all methods employ cream as starting

material, use a dissociating agent to liberate XO from membrane lipo-

proteins and some form of chromatography for further purification.

Milk XO has a molecular weight of c.300kDa and consists of two

subunits. The pH optimum is about 8.5 and the enzyme requires flavin

adenine dinucleotide (FAD), Fe, Mo and an acid-labile compound as

co-factors; cows deficient in Mo have low XO activity. The amino acid

composition of XO has been determined by a number of workers; at least

five genetic polymorphic forms have been reported.

Activity in milk. Various processing treatments affect the XO activity of

milk. Activity is increased by about 100% on storage at 4°C for 24 h, by

50-100% on heating at 70°C for 5 min and by 60-90% on homogenization.

These treatments cause the transfer to XO from the fat phase to the aqueous

phase, rendering the enzyme more active. The heat stability of XO is very

dependent on whether it is a component of the fat globules or is dissolved

in the aqueous phase; ageing and homogenization increase susceptibility and

explain the inconsistency of early work in which the history of the sample

was unknown or unrecorded. XO is most heat stable in cream and least in

skim milk. Homogenization of concentrated milk prepared from heated milk

(90.5"C for 15 s) partially reactivates XO, which persists on drying the

concentrate, but no reactivation occurs following more severe heating (93°C

for^15 s); apparently, homogenization releases potentially active, unde-

natured XO from the MFGM. All the major milk proteins can act as either

activators or inhibitors of XO, depending on their concentration, and may

have some significance in the activation, inactivation and reactivation of the

enzyme.

Sign$cance of xanthine oxidase
Lipid oxidation
XO, which can excite stable triplet oxygen (302), is a pro-oxidant. Milk
which undergoes spontaneous rancidity contains about^10 times the normal
level of XO, and spontaneous oxidation can be induced in normal milk by
the addition of XO to about four times normal levels. Heat-denatured or
flavin-free enzyme is ineffective and the susceptibility of unsaturated fatty
acids to oxidation increases with the degree of unsaturation.