For the determination of galactose, the enzymatic method and the so-called our microbioassay method have been compared in various points.
Galactose oxidase method is a kind of colour reaction, using galactose oxidase, substrate, peroxidase, chromogen and spectrophotometer. Microbioassay method is the ordinary cup-plate method, using highly galactose-sensitive mutants of enteric bacteria as the test organisms in order to demonstrate the inhibition zone.
In the oxidase method, the combined galactose is oxidized by the enzyme as well as free galactose with few exceptions. In microbioassay method, free galactose is most active against the mutants. Therefore, if the galactosides are not reactive to the mutants employed, they must previously be hydrolyzed by dilute mineral acids to release the free galactose. Event when the galactosidase-positive mutants are used as the test organisms, the appearance of inhibition zone is greatly interfered in the case of galactosides.
In the enzymatic method, the end point of the titre is 25 γ/2ml ; and 200-500 γ/2ml when deproteinizing or desalting is necessary in the biological samples. In the microbioassay, the end point is 1.25 γ/0.1 ml, and usually there is no need to carry out deproteinizing or desalting. The range of reliable determination for galactose is 25-100 γ/2ml in the enzymatic method, and 1-10 γ/0.1 ml in the microbioassay method. The length of diameter of inhibition zone by galactose is not influenced by the presence of other hexoses, unless their amounts exceed that of galactose by 7-8 times.
The time required for the procedures is 3-4 hours, or some hours more if deproteinizing or desalting is necessary, in the enzymatic method, and about 8 hours in the microbioassay when stocked seed-layer plates are utilized.
In the galactose oxidase method, the result should be read within 6 hours. On the contrary, the inhibition zone remains unchanged for a few days in the microbioassay.
In the oxidase method, lactulose is reactive, but lactose is not. In the microbioassay, lactulose is not reactive, but lactose is reactive when the mutant producing -galactosidase is employed as the test organism.
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