Amino acids microbiological methods

Gravimetric, photometric, chromatographic, enzymatic, and microbiological methods for the determination of amino acids are reviewed and discussed. Marked advances have been made during the present decade in methods applicable to the determination of amino acids, and with the development of new analytical methods it should soon be possible to determine all the amino acids of biological importance with a degree of accuracy sufficient for practical as well as many theoretical purposes. 

The percentages of amino acids in silk fibroin which Poison et al. (224) found by direct visual and indirect photometric analysis of ninhydrin paper-partition chromatograms are shown in Table VII. The percentages obtained for alanine, glycine, and serine appear to be reasonably accurate, inasmuch as they agree closely with those found by other methods. It would be of interest to determine alanine by the microbiological method reported recently by Sauberlich and Baumann (238), in view of the widely different values found for this amino acid by the described ninhydrin-chromatographic procedure and the selec-... 

In more recent times chemically defined basal media have been elaborated, on which the growth of various lactic acid bacteria is luxuriant and acid production is near-optimal. The proportions of the nutrients in the basal media have been determined which induce maximum sensitivity of the organisms for the test substance and minimize the stimulatory or inhibitory action of other nutrilites introduced with the test sample. Assay conditions have been provided which permit the attainment of satisfactory precision and accuracy in the determination of amino acids. Experimental techniques have been provided which facilitate the microbiological determination of amino acids. On the whole, microbiological procedures now available for the determination of all the amino acids except hydroxy-proline are convenient, reasonably accurate, and applicable to the assay of purified proteins, food, blood, urine, plant products, and other types of biological materials. On the other hand, it is improbable that any microbiological procedure approaches perfection and it is to be expected that old methods will be improved and new ones proposed by the many investigators interested in this problem. 

Table XII. Microbiological Assay Methods First Used to Determine Amino Acids...

The improved methods introduced in the years 1940 to 1945 created new possibilities in this field. The Van Slyke method for quantitative determination of amino acids, based on the measurement of the volume of carbon dioxide evolved in the course of the reaction between amino acids and ninhydrin (V2), was much more reliable than the older methods. At the same time the microbiological methods designed for amino acid determinations (D3, S6) made possible the detection of very small concentrations of these compounds. The application of these... 

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

With respect to amino acid excretion Woodson and co-workersl3 and othersl4,15 have found by microbiological methods large variations. Stein 16 obtained evidence of wide variations in the amino acid excretion of cystinurics. Further data are also available with respect to creatine and creatinine excretion which bear out our conclusion regarding individuality of excretion patterns. 17,18,19... 

By far, the predominant methods for determination of amino acids in foods are based on HPLC. However, alternative methods for amino acid analysis do exist. Many of the earliest determinations for certain amino acids were based on microbiological tests (and other bioassays), but these are no longer widely employed. Cost and analysis time are obvious factors in the demise of these type of methods. Also, these types of methods are very prone to biased results and high variance. 

Pentz et al. (P5) estimate taurine with fluorodinitrobenzene in urine passed through Dowex 50 H+ columns, but there are doubts as to whether this procedure is really specific for taurine (B38). Dent et al. have compared results obtained for the estimation of sulfur-containing amino acids in urine of cystinuric patients, by polarographic and microbiological methods (D18, D19). Hier (H12) and Schreier and Pliickthun (S10, Sll) have published data on amino acid excretion as determined microbiologically. Enzymatic methods have been used with success in the case of histidine in urine with specific decarboxylase preparations (S23). 

Comparison with values from the literature obtained by microbiological methods has been discussed by Stein (S32). The main objection to microbiological methods lies in the fact that amino acid derivatives or combined amino acids may be as readily available to the microorganisms used in these methods as are the parent amino acids from which they... 

In early infancy the situation is known to be quite different from that among older children. Simon noticed in 1911 that the newborn infant excreted 10 % of its total nitrogen output in the form of amino nitrogen, whereas in adults it drops to 2 % (S15). This was confirmed in later years (B4, Cll, G5) and extended to the study of individual amino acid excretion by microbiological methods (S10, Sll), by paper chromatography (S20, S21), and finally by ion exchange column chromatography (D28, D30) at the time Moore and Stein s first method of 1951 was still the only one available. 

Amino acids are monomeric units of polypeptides and proteins. They are widely used in the food and chemical industries as flavor enhancers, seasonings and sweeteners e.g. for the improvement of bread quality, also in the production of drugs, cosmetics, synthetic leather and surfactants, in medicine for infusions and as therapeutic agents. Amino acids are produced by chemical synthesis or extraction from protein hydrolyzate. They may be also produced by microbiological methods. 

Richard Block s Amino Acid Handbook is a compendium of analytical procedures and analytical results. Among the analytical procedures a chapter is devoted to the microbiological methods, and it is from a study of microbiological methods for the determination of amino acids that the studies here summarized have developed. More detailed accounts of various phases of the work have been published (1-15). 

The modern ion-exchange methods for amino acid analysis are po ibly more accurate, and certainly more reliable than microbiological procedures with careful use the experimental variations are small and the methods are sufficiently reliable to show up small differences in amino acid composition where comparative surveys are carried out in the same laboratory under identical conditions. Gk)tte et al. (1961) used the method of Moore and Stein... 

Barton-Wright, E. C. Practical Methods for the Microbiological Assay of the Vitamin B Complex and Essential Amino Acids. Ashe Laboratories Ltd. 1946. 

Microbiological Methods in Amino Acid Analysis. Annals New York... 

Methionine-enriched protein was produced also from an enzymatically prehydrolyzed milk protein (SR) by the EPM process, and the methionine contents of SR and the EPM product were determined by a microbiological method. The methionine content in the EPM product was more than twice as high as in the substrate. These protein fractions were separated by thin-layer chromatography [83]. The separated peptides and peptide mixtures were eluted and their molar amino acid contents were determined. The ratio of polar and apolar amino acids in the peptides was found to be influenced basically by transpeptidation taking place in the EPM reaction. The analysis of the peptides of these products showed that methionine was incorporated mainly in peptides with a relatively high ratio of apolar amino acids. 

The amino acid produced in the fermentation broth can be quantified by employing different colorimetric assays (e.g., ninhydrin, 8-hydroxy quinoline), microbiological assays, or more specifically by HPLC methods using pre- or postcolumn derivatization with OPA, dansyl chloride, etc. 

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