UBC Theses and Dissertations
An evaluation of the microbiological assay technique for the determination of the amino acids Mowatt, James Graham
The microbiological techniques employed for the determination of amino acids have been examined critically, particularly with respect to the limitations of the method in the assay of natural products. The efficiency of the method in recovering given amounts of an added amino acid when subjected to the conditions of hydrolysis in the presence of purified protein with and without added carbohydrate was investigated. A study of the methods employed for hydrolysis in relation to the results obtained by the microbiological assay technique for these amino acids was also undertaken. The technique was applied to the assaying of a natural product. The microbiological method of Stokes and Gunness was found particularly advantageous in the case of lysine, leucine, isoleucine, valine, methionine, threonine and tryptophane released hydrolytically from casein. Good recovery results were also obtained in the case of arginine, histidine and phenylalanine but in the presence of added carbohydrate the results for these amino acids were considerably lower. Irregular results were obtained in the case of tyrosine. The use of titanous chloride in the acid digestion mixture permitted the intact release of tryptophane during acid hydrolysis. The amount of tryptophane released was shown to be dependent upon the concentration of acid, temperature and time of hydrolysis. The rate of hydrolysis in the presence of titanous chloride was shown to be lower than that brought about by acid in its absence. In the case of a tryptophane solution evidence was obtained indicating that this amino acid is not racemized when treated with five normal sodium hydroxide at 120˚C. The extent to which tryptophane from the alkaline hydrolysis of a protein undergoes racemization is a matter which warrants further investigation. Except in the case of histidine excellent results were obtained for the recovery of added amino acids from a dehydrated sample of meat. The use of 2 ml. and 3 ml. portions of basal medium in place of the 5 ml. quantities usually employed gave irregular results.
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