Lxiv. the Reduction of Methylene Blue in Milk

THREE types of reaction which will bring about the reduction of methylene blue added to milk have been known for a considerable time and have been utilised in various ways. (i) Schardinger [1902] has shown that milk contains an enzyme which is able to catalyse the reduction of methylene blue in the presence of an aldehyde. Trommsdorff [1909] has proved that the enzyme is a constituent of the milk itself, and is independent of micro-organisms. Other observers have shown that aldehydes may be replaced by xanthine, and methylene blue by nitrates. Hence the reaction is now recognised as a typical oxidation-reduction in which hydrogen donator and hydrogen acceptor are enabled to interact through the presence of an enzyme or catalyst. (ii) Milk which has been heated for a short time to a temperature over 100° will bring about the decoloration of methylene blue. The mechanism of this reaction has not yet been definitely established. The possibility of enzyme action is of course excluded. Barthel [1925] considers that an oxidationreduction occurs in which citrate acts as hydrogen donator, methylene blue as hydrogen acceptor, and some inorganic constituent of milk as catalyst. Schwartz [1929], however, has brought forward evidence in favour of the theory that hydrolytic products of the milk proteins, probably those containing sulphydryl groups, are the reducing agents. (iii) Milk containing living bacteria will decolorise methylene blue. The reaction in this instance is caused by an enzyme associated with the microorganisms, and most probably is an oxidation-reduction in which the methylene blue acts as a hydrogen acceptor. The corresponding oxidation, which occurs simultaneously, must be a reaction which supplies energy for the growth of the organisms. Clark, Cohen, and Gibbs [1928] have shown that a reaction takes place in the absence of methylene blue, and that the dye, if present only in small quantity, is merely an indicator of reducing potential, analogous to an indicator of hydrogen ion concentration. Milk has therefore no specific function in the reaction, but is merely the source of substances suitable for the supply of energy to bacteria.