The colorimetric determination of lactic acid in silage.

The fundamental reaction which takes place in the process of ensilage is the conversion of a proportion of the soluble carbohydrates present to lactic acid. This reaction is of such importance that when material of high protein content is being ensiled, it is desirable to add molasses to provide a readily available substrate for the lactobacilli which occur along with coliform organisms in the original grass crop. When lactic acid is formed in suitable amount it acts as a preservative for the ensiled material and serves, on account of the lowering of pH which accompanies its formation, to inhibit the activity of coliform bacteria which produce undesirable byproducts. Despite the importance of this reaction it has never been fully investigated, partly on account of the pre-occupation of many investigators with the question of quality in silage, for the determination of which the pH is a reasonable guide, and partly because the methods used for its estimation are tedious and in some cases inaccurate. For example, the method of Foreman (1920) depends upon a difference calculation which involves the experimental error of the other acid determinations, and gives a result which represents the total non-volatile acid content, ofwhich but a part is lactic acid, a fact which has been stressed by Watson & Ferguson (1937) in their study of the method. The method of Karstr6m & Virtanen (1937), modified from the original techniques of Hirsch-Kauffmnann (1924) and Lieb & Zacherl (1932), involves the use of large amounts ofmaterial and complicated procedures for the elimination of proteins and carbohydrates. Miller & Muntz (1938) developed a method, later modified by Barker & Summerson (1941), for the colorimetric estimation of lactic acid which depends upon the fact that lactic acid is oxidized by sulphuric acid to acetaldehyde which gives a purple-red colour with p-hydroxydiphenyl in the presence of metallic ions, of which copper has been found to be the most suitable (Barker & Summerson, 1941). The reaction is similar to that of Mendel & Goldscheider (1925) who, however, used o-dimethoxybenzene as the colour-producing reagent. Davidson (1949) hasusedthe Barker& Summerson technique for the determination of lactic acid in milk and milk products, demonstrating its applicability to amounts of 20-400 mg. lactic acid. In the case of these latter, and blood or minced tissue, steps have to be taken to remove the protein matter involved. Silage, however, may be looked upon as solid matter covered with, or impregnated with, lactic acid to a greater or lesser extent and the amounts of protein extractable by water from silage are relatively small. Of the 5 g. portions of fresh silage which are used in the method of analysis described below, less than 0 4 g. is protein and of this only a part is soluble in water. This small proportion may be removed from the prepared extract at the same time as the soluble carbohydrates. The technique of Barker & Summerson, as used for the determination of lactic acid in silage, is described below.