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An evaluation of the microbiological assay technique for the determination of the amino acids Mowatt, James Graham 1948

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AN .VALUATION ,0F, THE MICROBIOLOGICAL ASSAY TECHNIQUE JOE THE DETERMINATION OF THE AMINO ACIDS - ty -James Graham Mowatt A Thesis Submitted In Partial Fulfilment of the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE in the DEPARTMENT OF DAIRYING THE UNIVERSITY OF BRITISH COLUMBIA May, ISkS, ABSTRACT The microbiological techniques employed for the deter-mination of amino acids have "been examined critically, particularly with respect to the limitations of the method in the assay of natural pro-ducts. 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 carbo-hydrate 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 arid phenylalanine but in the presence of added carbohydrste 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 raceraization 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 m l - portions of basal medium in place of the 5 ml. quantities usually employed gave irregular results. TABLE OF CONTENTS Introduction Part I. The Recovery of Amino Acids from Protein Hydrolysates in the Presence and Absence of Carbohydrate Historical Outline Experimental Discussion Summary Part II. The Liberation of Tryptophane During Acid Hydrolysis in the Presence of Titanous Chloride. Historicp.1 Outline Experimental Discussion Summary Part III.The Application of the Microbiological Method to tho Determination of Amino Acids in a Natural Product. Experimental 'Summary Bibliography INTRODUCTION Advancement in our knowledge of the needs of the animal body has led to the discovery of the importance of the individual amino acids in nutrition. Certain of these ."building blocks of protein are essential and must be supplied for animal metabolism while others axe synthesised by the anabolic processes and are therefore termed non-essential. With the increasing emphasis being placed on the amino acids present in protein, investigators have been concerned with the development of new analytical techniques to supplement and replace the rather inaccurate, costly, time-consuming, and cumbersome methods of analysis generally employed. Most of these methods f a i l to provide sensitive, specific, fast, and readily reproducible techniques easily adapted to routine analysis. Microbiological techniques for the assay of amino acids in natural products have been developed as the result of studies on the growth requirements of various microorganisms, particularly the fastidious l a c t i c acid bacteria. (62,63.6U,65) The establishment of the precise vitamin and amino acid requirements of these micro-organisms for growth, has led to the development of assay techniques, for both the vitamins and the amino acids, i n which growth response as determined either by turbidity or acid production, i s , under -2-controlled conditions, a quantitative measure of the particular sub-stance under investigation. It i s the purpose of this study to examine c r i t i c a l l y certain microbiological methods for the determination of the amino acids as tools for research in the f i e l d of protein chemistry and nutrition. In order to obtain more detailed information with respect to the limitations of the method In the assay of natural products, i t was considered essential to determine f i r s t the efficiency of the method i n recovering given amounts of an added amino acid when sub-jected to conditions of hydrolysis i n the presence of purified casein and varying amounts of carbohydrate material. A study of the methods employed for hydrolysis in relation to the results obtained by the microbiological assay technique for certain of the amino acids was also undertaken. It was f e l t that in this way a more desirable type of hydrolytic procedure might be evolved. The application of the microbiological assay technique to the determination of amino acids i n a natural product was inves-tigated. For the purposes of this study a dehydrated meat sample was prepared. PART I THE RECOVERY or AMINO'ACIDS EROM PROTEIN HYDROLYSATES I N THE PRESENCE AFD ABSENCE OP CARBOHYDRATE.. - 3 -HISTORICAL OUTLINE In order to obtain a more complete picture of the investigations concerning the amino acid requirements of the l a c t i c acid bacteria i t is of advantage f i r s t to review b r i e f l y the work that has been done regarding the vitamin and other requirements of these microorganisms. The f i r s t comprehensive studies on the growth requirement s of the l a c t i c acid bacteria were made by S. Orla-Jensen (62 and 63) who established the fact that these microorganisms require a complex medium including riboflavin and certain amino acids for their rapid growth and acid production (6U and 65) Based on the early observations of Orla-Jensen, Snell and his co-workers (85,86,88,89,90.91) along with other Investigators, carried out studies on the vitamin requirements of the l a c t i c acid bacteria* These investigations led to the development of micro-biological methods for the determination of the vitamins i n natural products. Snell, et a l . established proof of the need of such factors as pantothenic acid (89 and $0), nicotinic acid (89 and 90) and riboflavin (86) by certain members of the l a c t i c acid group of baeertia. An early paper by Snell, Tatum and Peterson, in 1937i (88) described the use of a peptone medium in which the riboflavin had been destroyed by photolysis i n the presence of a l k a l i . Later Snell, Strong, and Peterson reported (90) the use of a casein hydrolysate wlth added tryptophane and cystine as the nitrogen source for their growth medium. Through such studies on the growth factor requirements of the l a c t i c acid bacteria, a number of microbiological procedures using these microorganisms have been developed. The method devised by Snell and Strong (87). i a 1 9 ^ . for the growth factor riboflavin was the f i r s t technique employing a bacterial culture (#) to be described i n the literature. This technique, involving the use of -Lactobacillus easel, is now accepted by the Association of O f f i c i a l Agricultural Chemists ( l ) , as the o f f i c i a l microbiological procedure for the quantitative determination of riboflavin. This publication was followed by a paper by Pennington, et a l . (66), which outlined a method for the microbiological deter-mination of pantothenic acid. This procedure was c r i t i c a l l y examined and reported upon by Heal and Strong, i n 19^3 (6l)» Lactobacillus arabinosus was used by Snell and Wright (92), i n I9U1, to determine nicotinic acid microbiologically. In the same year,.Mitchell and Snell, (58), working at the University of Texas, reported a microbiological method for the deter-mination of " f o l i c acid" using Streptococcus faecalis. # The f i r s t microbiological assay technique was: SCHOPFER, W.H. (1935) PHYCOMYCES BLAKESLEEANUS FOR B (THIAMINE) ASSAY ARCH. MIKROBIOL. 6: 139. - 5 -Lewis (50), i n 19^2, published a procedure for the deter-mination of p-amino benzoic acid using Lactobacillus arabinosus. This microorganism was also used by Wright and Skeggs (107) for the microbiological assay of biotin. The growth factor thiamine was determined microbiologically in I9UU, by Sarett and Cheldelin (72) using the assay organism Lactobacillus fermentum 36. The purine requirements of the l a c t i c acid bacteria have been studied by a number of investigators. Most of these organisms require adenine, guanine or xanthine, but the ease with which each compound i s u t i l i z e d varies among the different members of the l a c t i c acid group. Moller (59) reported adenine stimulatory to Lactobacillus plantarum. Lactobacillus arabinosus was also found by Snell and Mitchell (SU) to respond to this purine . These investigators further reported the stimulation of Lactobacillus arabinosus and Leuconostoc mesenteroides by guanine, the increased growth of the latter organism in the presence of xanthine, and the stimulatory effect of u r a c i l and thymine on Lactobacillus arabinosus and Streptococcus faecalis respectively. The growth promoting effect of thymine for Streptococcus faecal i s can be demonstrated only i n the absence of the growth factor f o l i c acid. Prior to the determination of the complete vitamin require-ments of the Lactobacilli, work began on their amino acid requirements. As early as 1939• Mdller (59) grew Lactobacillus plantarum -6-on a medium containing pure amino acids instead of a protein hydro-lypate. This investigator reported aspartic acid, glutamic acid, leucine, tryptophane, and valine essential to the growth of this microorganism while other amino acids, although unessential, were found to improve growth. Wood, Geiger and Werkman (102) suggested the use of Lactobacillus mannitopoeus and Lactobacillus lycopersici for the determination of threonine. These microorganisms were shown to require a number of amino acids. In 19H2, Pollack and Lindner (6?) claimed glutamine and glutamic acid to be of equal growth promoting activity for Lactobacillus arabinosus. These investigators used a synthetic medium composed of twenty amino acids, salts, dextrose, sodium acetate and vitamins. Hutchings and Peterson (46), in I9U3, working with Lactobacillus arabinosus and Lactobacillus casei, showed the latter test organism to be inhibited by histidine and isoleucine. Lysine and alanine were found to be stimulatory for Lactobacillus easel exhibiting a more or less additive effect. The amino acid requirements of Lactobacillus arabinosus were investigated by Kuiken, et a l . (4g). ^rom this work methods for the microbiological determination of valine, leucine and isoleucine were evolved. (4$). -a this publication (H9) a hitherto unknown growth promoting substance in tomato juice extract was reported as being required by this organism. A description of the - 7 -preparation of an active eluate of this substance was given TABLE I. THE AMINO ACID RETIREMENTS OF  LACTOBACILLUS ARABINOSUS 17-5 (^9). ESSENTIAL ACCESSORY NO EFFECT Glutamic acid Alanine Norvaline Tryptophane Arginine Norleucine Threonine Aspartic acid ocAmino isohutyric acid Valine Histidine ocAmino n. butyric acid Leucine Proline Glycine Isoleucine Serine Hydroxyproline Cystine Methionine Alanine Lysine Tyrosine Phenylalanine Shankman (78) reported the amino acids leucine, isoleucine, valine, cystine, methionine, tryptophane, glutamic acid and threonine essential to the growth of Lactobacillus arabinosus. This Investigator noted two ranges of stimulation for arginine with an intermediate range of inhibition. Shankman and Dunn (79) determined seven amino acids including axginine, glutamic acid, leucine, phenylalanine, tryptophane, tyrosine, and valine using Lactobacillus easel. Of these amino acids, glutamic acid, leucine, tryptophane, and valine were also assayed microbiologlcally with Lactobacillus arabinosus 17-5. In 194U, Dunn, Camien, Rockland, Shankman and Goldberg (10) published a microbiological method for the determination of glutamic acid i n protein hydrolysates using Lactobacillus arabinosus. The amino acid requirements of Leuconostoc mesenteroides P-60 were studied by Dunn, et a l . (lU) and a technique for the microbiological assay (l4 and 15) of lysine i n proteins was reported by these workers. The use of a tryptophane-free, charcoal-treated caBein hydrolysate with added cystine and growth factors was reported by Greene and Black (25) for use i n the detection of tryptophane. Hegsted (32) concluded that "the single omission of axginine, cystine, glutamic acid, isoleucine, methionine, phenylalanine, tryptophane, tyrosine or valine from an adequate medium containing nineteen amino acids prevents the growth of Lactobacillus arabinosus." A mixture of these ten amino acids however was found inadequate for the growth of this test organism, Hegsted reported the further addition of aspartic acid, glutamic acid, threonine, and lysine to these ten amino acids to be necessary for the growth of Lactobacillus arabinosus. Leucine valine, and phenylalanine were determined microbiologically by this investigator. (32) - 9 -McMahan and Snell (57) determined valine and. arginine using Lactobacillus casei. They also used Lactobacillus arabinosus for the determination of valine. Owing to the small amounts of acid produced by test organisms such as Streptococcus lactis R and Leuconostoc mesenteroides,- they suggested the use of turbi dime trie determinations as a measure of growth response. Schweigert, Mclntire, Elvehjem and Strong (75). reported the direct determination of valine and leucine in fresh animal tissues using Lactobacillus arabinosus as the test organism. They claimed hydrolysis was possible without the•preliminary removal of fat, water, and water-soluble constituents of the tissues. The availability of d, 1, and dl isomers of the amino acids to the Lactobacilli was reported upon by Stokes and Gunness in lS^ 1 (°3) . They found the d and 1 forms of aspartic acid to have equal growth promoting power for Lactobacillus delbruckii. This is an exceptional case where the d or unnatural isomer can substitute for the 1 or natural form in the growth of the Lactobacilli. In I9U5, Dann, Schott, Ffankl and Rockland (IS) determined the apparent free tryptophane of the blood using a microbiological method. Lactobacillus arabinosus was claimed by Hac, Snell and Williams (31) to be more active to glutamine than to 1 ('+') glutamic acid. Increased activity to glutamic acid could be obtained by increasing the size of the inoculum, by lengthening the incubation time, by lowering the initial pH, or by adding ammonium salts to the ' -10-medium. Ia the light of these observations Hac, et al.claimed that the amino acid was changed to the amine before being used by the test organism. Hac and Snell (30) reported a procedure for the deter-mination of aspartic acid using Leuconostoc mesenteroides. They stated the assay organism was able to u t i l i z e aspartic acid directly without prior change to the corresponding amine. Further investigations of the specificity-of the leucine, isoleucine, and valine requirements of Lactobacillus arabinosus 17-5 were made by Hegsted (33) 19^» In the same year a microbiological method for the determination of 1 (+) glutamic acid using Lactobacillus arabinosus 17-5 with a glutamic acid free casein hydrolysate was described by Lewis and Olcott (51). The removal of glutamic acid from the casein hydrolysate was accomplished by the conversion of this amino acid to pyrrolidonecarboxylic acid and extraction with ethyl acetate. These workers were concerned with the specificity of 1 ( - ) . glutamic acid for Lactobacillus arabinosus, the racemization of 1 (-) glutamic acid and the period of hydrolysis required to liberate this amino acid from various protein sources. Lyman, Kuiken, Blotter and Hale (52) used Lactobacillus arabinosus in the determination of glutamic acid. The investigations of these workers agree with the findings of others (Pollack and Lindner, I9U2 t Hac, Snell and Williams I9U5). Kuiken, et a l . -11-claimed, however, that glutamine will not form if only a small amount of glutamic acid is present. Gunness and co-workers (28), in 19^ +5. reported a microbiological procedure for the determination of aspartic acid and serine. These workers outlined an entirely synthetic basal medium for the technique using Lactobacillus delbruckii. Tryptophane was determined microbiologically by Wooley and Sebrell (106) in enzymatic hydrolyses of proteins using Lactobacillus arabinosus and Eberthella typhosa as test organisms. The growth response of Lactobacillus erabinosus was measured titrimetrically after a three day incubation period at 37° C; the response of Eberthella typhosa was determined turbidimetrically after a period of sixteen hours incubation. These investigators claimed enzymatic hydrolysis to give more consistent results than sodium hydroxide because of incomplete racemization of tryptophane by the latter method. At this time Stokes, Gunness, Dwyer and Casvrell (96) published a uniform assay procedure for the ten essential amino acids, histidine, arginine, lysine, leucine, isoleucine, valine, methionine, threonine, tryptophane and phenylalanine. Streptococcus faecalis and Lactobacillus delbruckii were used as the test organisms. This technique was particularly adapted to routine analysis and the determination of amino acids in such proteins as casein, gelatin, blood meal, alfalfa meal, peas, tankage, using an entirely synthetic medium was described. -12-Dunn, Shankman, and Camien ( l l ) reported microbiological procedures for the determination of phenylalanine using Lactobacillus casei and Leuconostoc mesenteroides. Studies (20) on the amino acid requirements of Lactobacillus fermenti 36 by Dunn, et a l . resulted in the developement (2l) of a procedure for the determination of histidine using this microorganism. Dunn and Rockland (17) reported the microbiological determination of histidine i n casein hydrolysates using Leuconostoc mesenteroides P-60. They suggested the value 3.00$ histidine in casein as an average of a number of assays. The amino acid requirements for Streptococcus faecalis and the use of that organism for the determination of threonine was discussed by Greenhut, Schweigert and Slvehjem (26). Leucine, isoleucine, threonine, glutamic acid, asparagine, lysine, valine, methionine, arginine, histidine, tryptophane and cystine were reported as being essential while alanine, tyrosine,, phenylalanine, and glycine were found to be stimulatory. Gunness, Dwyer and Stokes extended the uniform assay (95) for the ten essential amino acids to include tyrosine (28). Alkaline hydrolysates of protein were assayed-microbiologically for this amino acid using Lactobacillus delbruckii. Dunn, et a l . (12) described the microbiological deter-mination of methionine using Lactobacillus fermenti 36. This microorganism was found to respond equally to d (+) methionine or -13-1 (-) methionine. These investigators also reported (13) a microbiological assay procedure for threonine i n protein hydro-lysates using this assay organism. Hiesen, Schweigert and Elvehjem (69) compared Lacto-bacillus arabinosus 17-5. Streptococcus faecalis, and Leuconostoc mesenteroides P-60 for the microbiological determination of methionine i n protein and foodstuffs. These workers concluded that methionine values, as determined by the three test organisms, agreed at normal assay levels. Comparable results on the microbiological determination of methionine using the two test organisms Streptococcus faecalis and Leuconostoc mesenteroides F-60 and a colorimetric chemical procedure were observed by Lyman, Moseley, Butler, Wood and Hale (53) • These investigators applied Leuconostoc mesenteroides P-60 to the determination of methionine in a dehydrated meat sample (54). Methionine was determined microbiologically by Horn,, Jones, Blum ( 4 l ), i n 1946. using Lactobacillus arabinosus 17-5* Procedures for the determination of both threonine (42) and valine (43) were also reported by these investigators i n the following year. Lactobacillus arabinosus 17-5 and Streptococcus faecalis were both recommended as test organisms for valine, however these workers used the latter mainly i n their investigations of the amino acid content of proteins and foodstuffs, Horn, Jones and Blum (44) have recently reported, 1948, a method for the microbiological determination of histidine i n -lH-proteins. This technique outlined the procedure for using either Leuconostoc mesenteroides P-60 or Streptococcus faecalis as test organism. Henderson and Snell have recently brought together the present vast number of microbiological methods for the deter-mination of amino acids in a publication .(35) describing a standard procedure for the assay of fourteen amino acids by a number of widely used test organisms. Arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophane, tyrosine and valine can be deter-mined by Lactobacillus arabinosus 17-5. Streptococcus faecalis, and Leuconostoc mesenteroides P-60. The composition of the basal medium is such that it permits the use of Lactobacillus casei, Lactobacillus delbruckii L.D. 5 . . Lactobacillus delbruckii L.D. 3» and Lactobacillus - fermenti 36 also for microbiological assay work. The value of such an inclusive technique for routine analysis.is readily realized when a review of the literature reveals so many different micro-biological procedures. Using the technique developed by Henderson and Snell (35)» Henderson, et al. (36) reported a micro-microbiological technique for the determination of fourteen amino acids. Precision "approaching that of the macro-method" was reported by these workers using total volumes per tube of 0.2 ml. It.is claimedthat by this technique the determination of fourteen amino acids using as little as 20-kO mgm. of protein sample can be carried out. -15-Although this review of the literature has "been concerned only with the study of the lactic acid "bacteria as test organisms, certain other unicellular organisms, requiring one or more amino acids, have "been used for the quantitative determination of these substances in protein hydrolysates. Rengnery (68) reported a "leucineless mutant strain" of Neurospora crassa that responded specifically to leucine and the keto acid analogue of leucine. This leucineless strain was developed "by the treatment of Neurospora crassa with ultra violet light. Ryan and Brand (71) described a technique for the deter-mination of leucine in protein hydrolysates and foodstuffs using the mutant strain of Neurospora crassa developed "by Rengnery (6S). Similarly induced mutations of Neurospora developing lysine (9) , isoleucine and valine (7) requiring characteristics and the use of- these mutants in bioassays were reported "by Doermann(9) and Bonner (7). A mutation induced "by X-ray in E. coli developing threonine and leucine- requiring characteristics was reported "by Simmonds, Tatum, and Eruton (Si). Wooley and Sebrell (106) reported the microbiological determination of tryptophane using turbidimetric determination of the growth response of Eberthella typhosa after sixteen hours incubation. Since a great deal of work has been done on the amino -16-acid requirements of the la c t i c acid bacteria and the subsequent development of microbiological methods of their determination, two charts have been prepared to present, i n more condensed form, the existing knowledge, f i r s t l y , as to the amino acids essential to certain of the assay organisms and secondly, as to the technique recorded i n the literature at the present time. THE REQUIREMENT -17-TABLE 2. OP VARIOUS TEST ORGANISMS. Str. faecalis R CO © o tH Q) 4» • a O Q) ^  Xi « rH a o a •s CM • to 03 (0 o n •H r — <D a a! o^. VD at •H •3 • u 4> a) Alanine X 0 c;' # 0 ' Arginine X X X # X X Aspartic Acid X X f # X Cystine # X X X X *. Glutamic Acid X X X X X Glycine X X 0 0 0 ' Histidine 0 X 0 # # X Isoleucine X X X X f X Leucine X X X X X X Lysine. X X # X # X Methionine 0 X X # # X Phenylalanine 0 X X # X 0 Proline 0 X 0 0 0 0 Serine X X 0 0 X Threonine - X X # # * X Tryptophane X X X X X X Tyrosine * X X # X # Valine 0 X X X X X X— essential : #— stimulatory : 0— non-essential. -18-TABLE 3 . METHODS FOR THE MICROBIOLOGICAL ASSAY OF THE AMINO ACIDS. AMINO ACIDS REFERENCES ARGININE 35, 36 • Lactobacillus arabinosus Streptococcus faecalis 3 , 29, 37, 35, 57, 36, 79 96 ASPARTIC ACID 35, 36 Lactobacillus delbruckii Leuconostoc mesenteroides 9^ 30 GLUTAMIC ACID 35. 36 Lactobacillus arabinosus Lactobacillus easel Streptococcus faecalis 5 . 37, 64 10, 64, 31. 7? 51, 52. 78 HISTIDINE 35, 36 Lactobacillus fermenti 36 Leuconostoc mesenteroides P-60 Streptococcus faecalis 11, 16, 96, 21 17. 5 29. 44 ISOLEUCINE 35, 36 Lactobacillus arabinosus Lactobacillus casei Streptococcus faecalis 5 , 5 , 5 , 25, 79 96 •37, ^9, 75, 77, 79-LEUCINE 35, 36 1 Lactobacillus arabinosus Lactobacillus casei Streptococcus faecalis 5 , 5 , 5 , 25, 79 96 37, ^9. 75, 77, 79 LYSINE 35. 36 Leuconostoc mesenteroides P-60 Streptococcus faecalis 15, 5 , 29 96 -19-TABLE 3 CONTINUED AMINO ACIDS REFERENCES METHIONINE 35, 36 Lactobacillus arabinosus Lactobacillus fermenti 36 Leuconostoc mesenteroides Streptococcus faecalis 12, 69 12, 45 12, 53, 4, 53, 69 6?. 96 PHENYLALANINE 35, 36 Lactobacillus arabinosus Lactobacillus casei Lactobacillus delbruckii Leuconostoc mesenteroides 25,40, 69 19. 79 96 19. to, 69 PROLINE 35, 36 THREONINE 35, ?6 Lactobacillus arabinosus Streptococcus faecalis 37 4, 26, 27, 40, 42, 96 TRYPTOPHANE 35/ 36 Lactobacillus arabinosus Lactobacillus casei Streptococcus faecalis 18, 24, 25, 79 4, 96 7^. 76, 79. 106 TYROSINE 35, 36 Lactobacillus casei Lactobacillus delbruckii Leuconostoc mesenteroides Streptococcus faecalis 36, 79 28 40 4 VALINE 35. 36 Lactobacillus arabinosus Lactobacillus casei Streptococcus faecalis 25. 37, 57, 4, 29, U3, U3. 49, 57,. 75. 79 96 . As investigations of the growth requirements of the lactic acid bacteria proceeded and techniques were developed for the microbiological assay of the vitamins and amino acids, it became apparent that certain minerals, fatty acids and other substances exerted an influence on the growth response of these micro-organisms which might seriously limit the applicability of these procedures. Wooley, as early as I3HI, (103) and later in 19UU (lOH) reported the stimulatory effect of streptogenin on Lactobacillus casei, some hemolytic Streptococci and certain other microorganisms.. Since streptogenin was destroyed by strong acid and alkali, this worker believed it to be a peptide or peptide-like structure (105).' Eoberts and Snell (70) in describing the use of tryptic digest of casein as a constituent of the basal medium to be used in the microbiological determination of the vitamins, reported, that this enzymatic hydrolysate contained unknown substances capable of inducing greater growth of Lactobacillus casei. In the light of Wooley's work (105), it would appear that this increased growth promoting effect was possibly due to the presence of streptogenin in the tryptic digest of casein. Since variations in the composition of the basal medium can cause differences in the qualitative as well as the quantitative response of microorganisms, Lyman, Moseley, V/ood, Butler and Hale (55) in 1947. studied the effect of some chemical factors which influenced the amino acid requirements of the lactic acid bacteria. These -21-investigators reported the effect of pyridoxamine and.carbon dioxide on the amino acid requirements of certain assay organisms. Lactobacillus delbruckii have been previously reported (90 not to require alanine, lysine, and threonine in the presence of pyridoxamine.- Investigation of the alanine, lysine and threonine requirements of Lactobacillus prabinosus (55) in the presence of this growth factor showed a similar condition to exist. Phenylalanine, tyrosine and arginine were found not essential for the growth of Lactobacillus arabinosus in the presence of carbon dioxide. Lyman, et al. reported that evidence was obtained which indicated that one pathway of the amino acid synthesis may be the reversal of the amino acid decarboxylase reaction. The requirements of this organism for valine, leucine, isoleucine, glutamic acid, cystine and tryptophane were found unchanged both in the presence and absence of pyridoxamine or carbon dioxide. These investigators further reported Streptococcus faecalis R able to synthesize the amino acid lysine in the presence of succinate-acetate buffers, whereas, in the presence of an acetate buffer alone no such phenomenon was observed. Pyridoxamine and carbon dioxide were found to exert no . effect on the growth requirements of Streptococcus faecalis for the amino acids valine, leucine, isoleucine, arginine, histidine, -22-txyptophane, threonine, methionine and glutamic acid. MacLeod and Snell (56) reported the inhibition of lactic acid "bacteria "by large amounts of citrate. These workers stated that citrate inhibition could be prevented by the addition of further Mri*"*an& Mg"*+ions, the amount of the former required being decreased as the amount of the latter added is increased, Bivalent calcium ions were reported to form complex ions with citrate and thus when added decreased the amount of Mn*"*required to overcome citrate inhibition. Oleic acid was reported as a growth stimulant to Lacto-bacillus casei by Williams and Pieger (101) in 1946. The following year W.L.Williams, H.P, Broquist and S. Snoll (100) reported the effect of oleic acid and related compounds • as growth factors for lactic acid bacteria. The even numbered saturated fatty acids from to Cjgwere found completely inactive. These investigators observed further that most lactic acid bacteria did not require oleic acid in the presence of biotin. Since the stimulation occurred only over a small range the effect of oleic acid during the assay of natural products appears of remote significance when a number of assay levels axe used. As the mode of action of the factors essential to the growth of lactic acid bacteria become more apparent and as the ways in which non-essential outsido factors effect these micro-organisms are established, even more sound and rigidly controlled methods for the microbiological analysis of amino acids in protein -23-can be brought forth. A great number of results, as established by microbiological methods have been published concerning the amino acid content of various purified proteins and natural products. The amino acid content of purified casein as determined by a number of investigators using different test organisms, as well as figures for this protein as established by certain chemical procedures are presented i n Table U to provide a basis of comparison for the experimental phase of this study. -2k-TABLE U THE AMINO ACID CONTENT OF CASEIN • MICROBIOLOGICAL VALUES CHEMICAL VALUES (96) (37) (6) (80) ARGININE 3.6 3 . 6 * * K5 3.8 HISTIDINE 2.6 2.6*** . 3.0 1.83 ISOLEUCINE 6.1 6 . 0 * 6.3 — LEUCINE 9.1 9 . 9 * 9.7 9.7 LYSINE 7.7 7.6*** 7.6 6.3 METHIONINE 2.6 — — PHENYLALANINE 5.9**** 5 . 3 * 5.0 3.9 THREONINE k.i i * . l * 3.8 — TRYPTOPHANE 1.07 — 1.2 22. TYROSINE 6 . 5 * * — — _ VALINE 6.25 7.1* 6.5 6.7 Employing L. arabinosus# L. casei ## L. delbruckii* -*** Leuc. mesenteroidesJt)hf Str. faecalis -25-EXPEEIMMTAL The microbiological technique employed in this study was that of Stokes and Gunness (96), a uniform method for determining the ten essential amino acids arginine, histidine, lysine, leucine, isoleucine, valine, methionine, threonine, tryptophane and phenyl-alanine. The procedure for tyrosine, reported by these workers in a later publication (28) was also. used. Before describing the experimental plan followed in establishing the accuracy and validity of .their technique it is first necessary to outline the various steps involved in the pre-paration of the microbiological assay, the preparation of the basal medium, hydrolysing the sample, making the dilutions, inoc-ulating the assay, titrating the acidity produced, and calculating the results. It is the purpose of the basal medium to furnish in double the final concentration of all the required amino acids, with the exception of the one to be determined, all the vitamin, purine, mineral and carbohydrate substances in addition to certain accessory factors stimulatory to the growth of the microorganism. The basal medium is prepared as outlined in Table 5 with the omission of the amino acid to be determined. It is convenient when making up basal media for more than one amino acid assay at a time that the amino acids: to be assayed be kept in separate solutions thus facilitating the preparation. - 2 6 -TABLE 5 CONSTITUTION OP THE BASAL MEDIUM Glucose 5 gm. GROWTH FACTORS Na acetate (anhyd.) 3 gm. SOLUTION I. INDIVIDUAL SOLUTIONS Pantothenic acid 100 1 dl-Leucine' 100 mgm. Thiamine H CI 100 i dl-Isoleucine . 100 mgm. Nicotinic acid 100 * dl-Valine 100 mgm. p-Amino benzoic acid 20 X dl-Methionine 100 mgm. Pyridoxamine . 200 $ dl-Tryptophane 200 mgm. 1 (-) Tyrosine 100 mgm. SOLUTION 2 dl-Phenylalanine 100 mgm. Riboflavin 100 i dl-Threonine 100 mgm. 1 (+•) Lysine 50 mgm. • SOLUTION 3 0.1 t \ 1 (+) Arginine 100 mgm. Biotin 1 (+) Histidine 100 mgm. 1 ('-) Cystine 100 mgm. SOLUTION 1+ 1 (-)• Proline 100 mgm. Folic acid 1 . 0 * SOLUTION I. PURINE BASES dl-Glutaraic acid 100 mgm. Adenine 5 mgm. dl-Aspartic acid 100 mgm. Guanine 5 mgm. j Uracil 5 mgm. j SOLUTION 2. dl-Alanine 100 mgm. SALTS A dl-Serine 100 mgm. Kst HPo* 250 mgm. ; dl-Norleucine 100 mgm. KH^Po* 250 mgm. Glycine 100 mgm. SALTS B | MgSo^ .JHy.o 100 ragm. 1 NaCl 5 mgm. FeSo^ . 7H3O 5 mgm. MnSoq.•kE%o 5 mgm. Adjusted to pH 6*8 - 7*0 and made up to 250 ml. in a volumetric flask. -27-All of the amino acids are soluble in either distilled water, dilute acid or alkali, dl-leucine, dl-tryptophane, dl-phenylalanine, dl-glutamic acid, dl-aspartic acid, dl-alanine, dl-serine, dl-norleucine and glycine are best dissolved in dis-tilled water at a concentration of 10 mgm. per ml. dl-isoleucine, dl-valine, dl-methionine, dl-threonine, 1 (+) lysine, 1 (+) arginine, 1 1+) histidine, and 1 (—) proline are readily prepared in distilled water at concentrations of 20 mgm. per ml. Tyrosine and cystine are soluble in 0.2 E KaOH and 0.2 H HC1 respectively and are made up to the concentration of 10 mgm. per ml. All amino acid solutions for the basal medium are stored under toluene at 5° C, It was found advantageous to keep as many of the growth factors as possible in one solution. Pantothenic acid, thiamine, nicotinic acid, p-amino benzoic acid and pyridoxamine, being of similar solubilities are kept in one solution of such concentration that 1 ml. of a 1:10 dilution of this solution provides the required number of micrograms of the respective factors for 250 ml. of basal medium (Table 5)-Owing to the greater insolubility of riboflavin and folic acid, (#) these growth factors are made up separately. Riboflavin is dissolved in 0,02 N acetic acid and made up to a concentration of 0.1 mgm. per ml. Folic acid is prepared in the (#) The author is indebted to Dr. T.H.Jukes, Lederie Laboratories Inc. for the Folic acid used in this study. -28-form of an aqueous "suspension" containing 10 micrograms per ml. When used for the preparation of media, this suspension is thoroughly shaken prior to use. For the preparation of the "biotin solution used in this study, the contents of a vial, containing 25 micograms in 1 ml,, are diluted to 250 ml. with distilled water containing 1 ml, of the phosphate salt solution A. It was found that "biotin in a concentration of 0.1 microgram per ml. is unstable in distilled water and must be stabilized with phosphate salts. All growth factor solutions must be renewed at 30 day intervals and are stored under toluene at 5° C. The purine bases (Table 5) are prepared by dissolving 0.870 gm. of adenine sulphate, 0.620 gm. of quanine hydrochloride and 0,500 gm. of uracil in 200 ml. of distilled water containing 10 ml. of concentrated hydrochloric acid. Five ml. of this solution are required for 250 ml. of basal medium. This solution is also stored in the refrigerator under toluene. Salt solutions A and 5 are prepared in the proportions indicated in Table 5 in such concentrations that 5 ml. of each provides the required amount for a 250 ml. volume of the basal medium. These growth factors, purine bases, amino acids and salt solutions are added to a volumetric flask by means of chemically cleaned pipettes. The glucose and sodium acetate -29-required are added i n crystalline form. After adjustment to a pH of 6*8 - 7*0. the medium i s made up to volume and dispensed i n 5 ml. quantities in test tubes. In the determination of amino acids i n protein hydrolysates the method employed for hydrolysis of the proteins i s of paramount importance. In Stokes' microbiological procedure (28 and 96) a method of acid hydrolysis suitable for the determination of 9 amino acids is described. In the case of tryptophane and tyrosine, the degree of microbial response to alkaline hydrolysates,under controlled conditions,is a quantitative measure of the concentration of these two amino acids. One gram of protein is hydrolysed with 10 ml. of 10$ H CI (#) in a sealed ampule of 20 ml. capacity. The hydrolytic reaction i s maintained at 15 lbs. pressure per sq. in. (120* C) for a period of ten hours. At the end of this time the hydrolysate is immediately brought to pH 6*8 - 7*0 with 10 N sodium hydroxide solution and f i l t e r e d . Any residue or precipitate i s washed and the hydrolysates are made up to volume in chemically clean 100 ml. volumetric flasks. (#) Concentrated H CI is made to 10$ by volume, with d i s t i l l e d water. - 3 0 -Alkaline hydrolysates for tryptophane and tyrosine determinations are prepared by digesting one gram of protein material in similar sealed ampules to the acid hydrolysis using 5 N NaOH for a period.of 10 hours* at 15 lbs. pressure per sq.. in. Neutralization of the hydrolysates with 12 N hydrochloric acid yields a copious gelatinous material believed to be s i l i c a dissolved from the glass tube. This substance i s best removed by centrifugation. The residue is washed once with d i s t i l l e d water and the hydrolysate and washings are made up to a 100 ml. volume. A l l assay values obtained from alkaline hydrolysates for tryptophane and tyrosine are multiplied by 2 as complete racemization is assumed under these conditions of hydrolysis (Historical Part I, Historical Part 2, Discussion Part 2) The next step i n the preparation of a microbiological assay for an amino acid involves setting up the reference curve and dilution of the unknown substance to obtain suitable response curves w i t h both the standard and unknown solutions. The standard amino acid solutions for the reference curve are prepared i n concentrations of 1 mgm. per ml. and are kept under toluene in the refrigerator. It i s important to renew these solutions at 30 day intervals. The concentration of the f i n a l dilution i s such that 5 nJ-. of the solution furnished the greatest extreme of the range. For most protein hydrolysates, prepared as outlined above, a 1 : 12*5 dilution i s usually sufficient to give a suitable response curve for the specific unknown amino acid. The addition of increasing amounts of the unknown dilution up to 5 ml, quantities to 5 ml. of -31-the basal medium provides a response curve that is referred to the standard for purposes of calculation. A l l tubes are made to 10 ml. volume with d i s t i l l e d water and s t e r i l i z e d at 15 lbs. pressure for 15 minutes. The two assay microorganisms used i n this study are Streptococcus faecalis (A.T.C. 9790) and Lactobacillus delbruckii L.D.5 (A.T.C. 9595). These microorganisms are transfered daily i n the following broth medium: Glucose 1 gm. Bacto peptone 0.5 gm. Na acetate (anhyd.) 0.6 gm. SALTS, A KJJH Po* 50 mgm. K H X Po + 50 mgm. SALTS B MgSo4 • 7Hxo 20 mgm. NaCl 1 mgm. PeSo^. • 7%o 1 mgm, MnSo^  • UHjO 1 mgm. Made up to 100 ml. with d i s t i l l e d water and adjusted to pH 6*8 - 7»0. The washed c e l l suspension used for inoculating the assay tubes, i s obtained by centrifuging a 20 - 24 hour culture of the test organism grown i n 8 ml. of the broth medium, washing the cells .once with s t e r i l e d i s t i l l e d water and i n the case of Lactobacillus delbruckii suspending the cells i n 20 ml. of ste r i l e d i s t i l l e d water and i n the case of Streptococcus faecalis in 100 ml. of st e r i l e water. The assay tubes are inoculated with one drop of the suspension from a one ml. pipette and are incubated at 37° c » -32-Streptococcus faecalis attains maximum acid production in UO hours at this temperature while Lactobacillus delbruckii i s incubated for three days before active acid production ceases. Bacterial response is guaged titrimetrically using 6.05 N NaOH and Brom Thymal Blue as indicator. Stock transfers of these microorganisms are made in duplicate at 30 day intervals on freshly prepared broth to which 1.5$ agar has been added. These cultures are kept in the r e f r i g -erator and fresh broth inoculations are made from them every 15 to 20 days. An example of the technique employed in the calculation of results i s given below. THE CALCULATION OF LYSINE IN A CASEIN HYDROLYSATE ML. OF I: 12.5 DLL. ML. OF ACID PROD. MICROGRAMS OF LYSINE FROM STANDARD CURVE. 0.5 ml . 3.5 ml . 52 0 .5 ml. 3.5 ml. 52 1.0 ml . k.S ml . 53 1.0 ml . U.8 ml. 52 1.5 ml. 6.2 ml. 52 1.5 ml . 6.H ml. 5»+ 52-*52+53f 52-h52t51* * 12.5x100= 0.0635 gm. Percent Lysine= 6.35$ -33-Before applying Stokes' microbiological technique to specific investigation i t is essential that the limits of accuracy and the degree of reproducability of the procedure be established. Microbiological methods for the determination of amino acids are best appraised by their agreement with values.obtained using existing chemical methods and microbiological procedures involving other organisms, by obtaining comparable results upon repeated assay of protein hydrolysates at more than, one dilution level, and by the recovery of known amounts of added amino acids after hydrolysis with a purified protein. Since certain of the values obtained by s t r i c t l y chemical methods are under suspicion, i t would appear that other procedures are of greater value for the purposes of analyzing and evaluating the dependability of the microbiological technique. In order to study the recovery of added amino acids using Stokes' microbiological technique, a series of hydrolysea were planned using purified casein with and without added amino acids. These investigations were carried out i n duplicate at more than one dilution l e v e l . The effect of the presence of aldehyde radicles during hydrolysis, such as w i l l be present in natural tissues, on values obtained by the microbiological method was demonstrated by the addition of xylose to the material subjected to hydrolysis. A member of the pentose group was selected because these sugars are known to be the most reactive i n humin and melanin formation - 3 ^ processes which involve the condensation of certain amino acids with aldehydes. Nine hydrolysis tubes were prepared, each containing 1 gm. of dried casein for the purposes of investigating the recovery of added histidine from acid hydrolysates in the presence of varying amounts of xylose. These 9 tubes were divided into three series of 3 tubes each and to one series of 3 tubes was added 0.25 gffl. of xylose, to the second set was added 1.0 gm. of xylose, while the third series was left free of any carbohydrate material. To 1 tube of each series was added 10$ of the reported amount of histidine present in 1 gm. of casein (80) (i.e. 10$ of 0.0183 gm. or 0.00183 gm.). To a second tube from each series was added 20$ of the histidine claimed present in casein (i.e. 20$ of 0.0183 gm» or 0.00366 gm.). No histidine was added to the remaining tube in each set. All nine tubes were sealed and hydrolysed at 15 lbs. pressure (120* ) for a ten hour period with 10$ hydrochloric acid. TABLE 6 THE STUDY OP HISTIDINE RECOVERY. CASEIN 1 XYLOSE HISTIDINE ' PRESENT HISTIDINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. E .0183 gm. .0183 gm. .0183 gm. .00183 gm. .OO366 gm. .0183 gm. .OI9I gm. .0209 gm. 1.81 $ 1.90 # 2.18 % 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .0183 gm. .0183 gm. .0183 gm. .00183 gm. .OO366 gm. .0183 gm. .0191 gm. .0209 gm. 1.51 $ 1.59 % 1.66 $ 1 gm. 1 gm. 1 gm. 1.0 gm. 1.0 gm. 1.0 gm. .0183 gm. .0183 gm. .0183 gm. .00183 gm. . O O 3 6 6 gm. .0183 gm. .0191 gm. .0209 gm. 1.35 # 1.53 $ -36-To study the recovery of added arginine from acid hydrolyses of casein in the presence of varying amounts of xylose a plan of hydrolysis identical to that in the Table 6 was followed using similar conditions of digestion. Arginine was added in the amounts of 10$ and 20$ of the reported arginine content of casein (SO) (i.e. 3 .8$) . An outline of this plan of experimentation appears in Table 7. TABLE 7 THE STUDY OF ARGININE RECOVERY. CASEIN XYLOSE ARGININE PRESENT ARGININE ADDED THEORETICAL . RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. — ,038 gm. .038 gm. .038 gm. .0038 gm. .0076 gm. .038 gm. .01+2 gm. . ,0U6" gm. 3.50 $ 3.87 $ U.12 £ 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .038 gm. .038 gm. .OJS gm. .0036 gm. .0076 gm. .038 gm. ,0U2 gm. ,0U6 gm. 3.38 $ 3.62 % 3.75 $ 1 gm. 1 gm. 1- gm. 1.0 gm. . 1.0 gm.. 1.0 gm. .038 gm. .038 gm. .038 gm. .0038 gm. .0076 gm. .038 gm. .0k2 gm. .OhS gm. 2.62 $ 3.00 $ 2.98 $ - 3 8 -To study the recovery of added lysine from the hydrolysates in the presence of varying concentrations of xylose the plan of hydrolysis outlined in Table 8 was followed. Lysine was reported in the literature (80) to be the lysine in one gram of casein would then be 0.0063 gm. and 0.0116 gm. respectively. Xylose v/as added to the extent of 0.1 gm. , 0.25 gm. and 1.0 gm. per gram of casein. Hydrolysis was carried out for a ten hour period at 15 lbs. pressure with 10 ml. of 10$ hydrochloric acid. Ten percent and 20$ of TABLE 8 THE STUDY OF LYSINE RECOVERY. CASEIN XYLOSE LYSINE PRESENT LYSINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. .063 gm. .063 gm. .063 gm. .0063 gm. .0126 gm. .063 gm. .0693 gm. .0756 gm. 6.35 $ 6.95 % 7.60 $ 1 gm. 1 gm. 1 gm. .10 gm. .10 gm. .10 gm. .063 gm.• .063 gm. .063 gm. .0063 gm. .0126 gm. .063 gm. .0693 gm. .0756 gm. 6.35 Sf 7.00 $ 1 gm. 1 gm.1 1 gm. .25 gm. .25 gm. .25 gm. .063 gm. .063 gm. .063 gm. .0063 gm. .0126 gm. .063 gm. .0693 gm. .0756 gm. 6.25 $ 7.75 $ 6.37 $ 1 gm. 1.0 gm. .063 gm. .063'. gm. 5.75$ -40-To study the recovery of added leucine from acid hydrolysates in the presence of varying concentrations of xylose the plan of hydrolysis outlined in Table 9 was followed. Acid hydrolysis was carried out with ten ml. of 10$ hydrochloric acid for 10 hours at 15 lbs. pressure. The leucine content of casein was reported by Sherman (SO)' to be 9.9$.0n the basis of this figure 10$ and 20$ of the leucine present in 1 gm. of purified casein then would be 0.0099 gm. and 0.019S gm. respectively. TABLE 9 THE STUDY OF LEUCINE RECOVERY CASEIN XYLOSE LEUCINE PRESENT LEUCINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. —— .099 gm. .099 gm. .099 gm. .0099 gm. .0198 gm. .099 gm. .1089 gm. .1188. gm. 9.93 i ' 10.10 $> 11.50 % 1 gm. 1 gm. 1 gm. »10 gm. .10 gm. .10 gm. .099 gm. .O99 gm. .099 gm. ,0099 gm.-.0198 gm. .099 gm. .1089 gm. .1188 gm. 9.87 * 10.50 $ 11.1*0 % 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. • .099 gm. .099 gm. .099* gm. .0099 gm. .0198 gm. .099 gm. .1089 gm. .1188 gm. 10.10 j> 10.70 $ 6.50 $ -U2-To study the recovery of added isoleucine from acid hydrolysates prepared in the presence of varying concentrations of xylose the plan of hydrolysis outlined in Table 10 was followed. The theoretical value for isoleucine was taken as 5.65$. Ten percent and 20$ added isoleucine would then be 0.005S gm. and 0.0112 gm. respectively. The conditions of hydrolysis followed were the same as for the previously mentioned amino acids. TABLE 10 THE STUDY OP ISOLEUCINE RECOVERY CASEIN XYLOSE ISOLEUCINE PRESENT ISOLEUCINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm.' 1 gm. 1 gm. .056 gm. .056 gm. .056 gm. .0056 gm. .0112 gm. .056 gm. .0616 gm. .0672 gm. 4.81 $ 5.17 $ 5.6S $ 1 gm. 1 gm. 1 gm. . 10 gm. .10 gm. .10 gm. .056 gm. .056 gm. .056 gm. .0056 gm. .0112 gm. .056 gm. .06l6- gm. .0672 gm. 4.81 $ 5.06 $ 5.13 $ ' 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .056 gm. .056 gm. .056 gm. .0056 gm. .0112 gm. .056 gm. .06l6 gm. .0672 gm. 4.60 $ 4.87 $ 5.08 $ • . 1 gm. 1.0 gm. .056 gm. .056 gm. 4.30 $ Second wash recovery (1.0 gm. xylose) 0.31$ 4.30$ 4.6l$ -44-To study the recovery of added valine from'casein hydrolysates in the presence of xylose the scheme of hydrolysis outlined in Table 11 was followed. The theoretical value for valine was given as 6.7$ (80) Ten percent and 20$ of this figure would then "be 0.0067 gm. and 0.0134 gm. respectively. Hydrolysis was carried out with 10 ml. of 10$ hydro-chloric acid for io hours- at 15 lbs. pressure. TABLE 11 THE STUDY OP VALINE RECOVERY CASEIN XYLOSE VALINE PRESENT VALINE ADDED THEORETICAL RECOVERY ' CALCULATED VALUE 1 gm. 1 gm. 1 gm. .067 gm. .067 gm. .067 gm. .0067 gm. .0134 gm. .067 gm. .0737 gm. ,080U gm. 6.68 # 7.00 £ 8.05 % 1 gm. 1 gm. 1 gm. .10 gm. .10 gm. .10 gm. .067 gm. .067 gm. .067 gm. .0067 gm. .013U gm. .067 gm. .0737 gm. .080U gm. 6.62 j> 6.9.1)- & 7.9S i 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .067 gm. .067 gm. .067 gm. .0067 gm. .013U gm. . .067 gm. .0737 gm. .OSOU gm. 6,75 i 7.00 $ 7.S7 $ To study the recovery of added methionine from acid hydrolysates of purified casein in the presence of added xylose, the scheme of hydrolysis outlined in Table 12 was followed. The theoretical value for methionine in casein was taken as 2.60$ (SO). Ten percent and 20$ of this value for 1 gm. of the protein then would be 0.0026 gm. and 0.0052 gm, respectively. The hydrolysis was carried out at 15 lbs, pressure for 10 hours using 10 ml, of 10$ hydrochloric acid. TABLE 12 THE STUDY OF. METHIQNIEB RECOVERY CASEIN XYLOSE METHIONINE. PRESENT METHIONINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. .026 gm. .026 gm. .026 gm. .0026 gm. .0052 gm. , ,026 gm. .0286 gm, .0312 gm. 2.50 1» 2.50$ 2.60 f, 1 gm. 1 gm. .1 gm. .10 gm. .10 gm. .10 gm. .026 gm. .026 gm. .026 gm. .0026 gm. .0052 gm. .026 gm. .0286 gm. .0312 gm. 2.50 % 2.75 $ ' 3.12 % 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .026 gm. .026 gm, .026 gm. .0026 gm. .0052 gm. .026 gm. .0286 gm. .0312 gm. 3.00 £ 2.76 $ 3.18 <$> 1 gm. 1.0 gm. .026 gm. .026 gm. 2.60 £ -48-To study the recovery of added, threonine from acid hydrolysates of purified casein i n the presence of added xylose the plan of hydrolysis was followed as outlined in Table 13. The theoretical value for threonine in casein was taken as 4.2$ (80). Ten percent and 20$ of the threonine content of 1 gm. of purified casein then would be 0.0042 gm. and, 0.0084 gm. respectively. The hydrolysis was carried out for 10 hours at 15 lbs. pressure using 10 ml. of 10$ hydrochloric acid. TABLE 13 THE. STUDY, OP .THREONINE.RECOVERY CASEIN XYLOSE THREONINE " PRESENT THREONINE ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. .042 gm. .042 gm. .042 gm. ,00U2 gm. .0084 gm. .042 gm. .0462 gm. .0504 gm. 3.75 £ 4.06 i 1 gm. I gm. 1 gm. .10 gm. .10 gm. .10 gm. .042 gm. .042 gm. .042 gm. .0042 gm. .00S4 gm. .042 gm. .0462 gm. .0504 gm. 3.50 $ 3.62 £ 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .042 gm. .042 gm. .042 gm. .0042 gm. .0084 gm. .042 gm. .0462 gm. .0504 gm. ' 3 . 6 2 $ 4.00 j> 1 gm. 1.0 gm. .042 gm. .042 gm. 3.13 * -50-To study the recovery of added amounts of phenylalanine i n the acid hydrolysis of purified casein in the presence of added xylose the plan of hydrolysis as outlined in Table lk was followed. The theoretical value of phenylalanine i n casein was taken as 3»9$ (80). Ten percent and 20$ of the phenylalanine content of 1 gm. of purified casein would then be 0.0039 gm. and 0.0078 gm. respectively. The hydrolysis was carried out for 10 hours at 15 lbs. pressure using 10 ml. of 10$ hydrochloric acid. TABLE l4 THE STUDY, OF, PHEHYLALAHIME RECOVERY CASEIN XYLOSE PHENYLALANINE PRESENT • PHENYLALANINE • ADDED THEORETICAL RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. .039 gm. .039 gm. .039 gm. .0039 gm. .007S gm. .039 gm. .0429 gm. .0468 gm. 5.00 $ 5.^ 7 $ 5.63 $ 1 gm. 1 gm. 1 gm. .10 gm., .10 gm. .10 gm. .039 gm. .039 gm. .039 gm. .0039 gm. .0078 gm. .039 gm. .0429 gm. .0468 gm. .^39 * 4.87 $ 5.00 $ 1 gm. 1 gm. 1 gm. .25 gm. .25 gm. , .25 gm. i .039 gm. .039 gm. • .039 gm. .0039 gm. .0078 gm. .039 gm. .0429 gm. .0468 gm. 4.12 % -^37 $ 4.87 $ 1 gm. 1.0 gm. j .039 gm. .O39 gm. 4.25 £ -52-The recovery of added amounts of tryptophane during the alkaline hydrolysis of purified casein in the presence of added xylose was followed as outlined in Table 15. The theoretical value for tryptophane was taken as 2.2$ (80). Ten percent and 20$ of the tryptophane content of 1 gm. of casein would then he 0.0022 gm. and 0.00U4 gm. respectively. Calculation of the amino acid content of tryptophane from an alkaline hydrolysate as determined microbiologically involved multiplying the figure received by 2 since complete racemization of the substance was assumed (96). Hydrolysis was carried out for 10 hours at 15 lbs. pressure using 10 ml. of 5 N NaOH per gm. of casein. TABLE 15 THE. STUDY, Off TRYPTOPHANES .RECOVERY CASEIN XYLOSE TRYPTOPHANE PRESENT TRYPTOPHANE ADDED THEORETICAL • RECOVERY CALCULATED VALUE 1 gm. 1 gm. 1 gm. .022 gm. .022 gm. .022 gm. .0022 gm. .0044 gm. .022 gm. .0242 gm. .0264 gm. 2.00 i» 2.37 * 2.63 * 1 gm. 1 gm. 1 gm. .10 gm. .10 gm. .10 gm. .022 gm. .022 gm. .022 gm. .0022 gm. .0044 gm. .022 gm. .0242 gm. .0264 gm. • 2.01 $ 2.3S $ 2.60 $ 1 gp. 1 gm. 1 gm. .25 gm. .25 gm. .25 gm. .022' gm. .022 gm. .022 gm. .0022 gm. .0044 gm. .022 gm. .0242 gm. .0264 gm. 2.00 $ 2.45 $ 2.81 $ 1 gm. 1.0 gm. .022 gm. .022 gm. 2.10 # -54-To study the recovery of added tyrosine during the alkaline hydrolysis of purified casein i n the presence of varying amounts of xylose the plan of expermintation as outlined i n Table l6 was followed. The theoretical value for tyrosine was taken as 6.5$ (SO). Ten percent and 20$ of the tyrosine content of 1 gm. of casein would be 0,0065 gm and 0.0130 gm. respectively. Calculation of the tyrosine content of an alkaline hydrolysate as determined microbiologically involved multiplying the value received by. 2 since complete raceraization of the sub-stance was assumed (96). Hydrolysis was carried out for 10 hours at 15 lbs. pressure using 10 ml. of 5 N NaOH per gm. of casein. TABLE 16 THE STUDY OP TYROSINE RECOVERY CASEIN XYLOSE TYROSINE TYROSINE THEORETICAL CALCULATED • PRESENT ADDED RECOVERY VALUE 1 gm. .065 gm. .065 gm. 5.50 f> 1 gm. .065 gm. • .0065 gm. .0715 gm. 5.88 £ 1 gm. .065 gm. .0130 gm. .0780 gm. 6.25 £ 1 gm. .10 gm. .065 gm. .065 gm. 5.00 £ 1 gm. .10 gm. .065 gm. .0065 gm. .0715 gm. 5.25 £ 1 gm. .10 gm. .065 gm. .0130 gm. .0780 gm. 7.00 £ . 1 gm. .25 gm. .065 gm. • ™" ™ "* .065 gm. 5.25 £ 1 gm. .25 gm. .065 gm. .0065 gm. .0715 gm. 5.^5 £ 1 gm. .25 gm. .065 gm. .0130 gm. .0780 gm. 6.12 £ 1 gm. 1.0 gm. .065 gm. .065 gm. 3.50 £ Added tyrosine would appear to have "been racemized during alkaline hydrolysis. -56-DISCUSSION In considering the experimental evidence hearing out the validity and accuracy of the technique employed "by Stokes, it would appear that the method is of value in quantitative deter-minations of the 11 amino acids studied. Acid digestion of a purified protein in the presence of xylose and the subsequent analysis of these hydrolysates showed the influence of the carbohydrate material on the amino acids released by hydrolytic cleavage of the casein molecule. The values obtained under these conditions were indicative of the destruction of amino acids likely to be encountered during the hydrolysis of impure proteins or natural products containing carbohydrate material. In first considering the data for histidine, the reference value for this amino acid, taken as l.S3$ (SO), compared favorably with the figure established for casein hydrolysed in the absence of xylose, (i.e. 1.81$). Hydrolyses of casein prepared in the absence of xylose have good results with and without added histidine, the recovery being approximately 100$ (Table 6). The presence of 0.25 gm. of xylose during hydrolysis resulted in an approximate 20$ decrease in the values obtained by this method. Recovery of added histidine, hydrolysed with casein in the presence of 0.25 gm* of xylose, was found possible with this technique. Similarly hydrolysates prepared i n the presence of l'.O gm. of xylose exhibited a 25$ decrease i n histidine content over the figures obtained for acid digestions with no xylose .present. It i s d i f f i c u l t to postulate the nature of the loss of histidine i n this case, whether i t be chemical or physical. However, the fact that extra washings of the black, amorphous humin residue gave qualitative indication of the presence of histidine and suggested that some of the loss, at least, was a physical one. It seemed possible also that a certain amount of histidine was rendered .inactive or destroyed during acid hydrolysis of casein in the presence of carbohydrate materials. The microbiological value established for the percentage of arginine i n a purified casein hydrolysate, determined using Streptococcus faecalis, was 3*5$ as compared to the reference value of 3*3$ (80). Stokes, et a l . (96) found arginine present!; in casein/ in the proportion of 3*6$. Directly proportional increases were observed, in the values obtained for this amino acid i n casein hydrolysates prepared with added arginine, both in the presence and absence of xylose. The hydrolysates prepared in the presence of carbohydrate material gave consistently lower figures for arginine, however (Table 7). These observations suggested p a r t i a l destruction of arginine during acid hydrolysis of casein i n the presence of xylose. -58-Extremely good agreement between the values for the acid hydrolysates of lysine prepared both i n the presence and absence of xylose with the reference value of 6.3$ (80) were obtained. The f u l l recovery of. lysine was well illustrated by this plan of hydrolysis (Table 8). On the basis of these observations, i t would appear that the procedure outlined by Stokes (96) for the microbiological determination of lysine using Streptococcus faecalis i s of particular value. Values of 9.93$ were obtained for leucine, using this technique, as compared with the reference value of 9*9$. Here again the presence of xylose during the hydrolysis of casein did not influence the values obtained from acid digestions so prepared (Table 9). The recovery of added leucine was also accomplished under these conditions of hydrolysis. The r e l i a b i l i t y of this method for the microbiological determination of leucine was then established. Similar results were obtained with isoleucine. Xylose did not influence the values obtained from proteins hydrolysed i n i t s presence (Table 10) Good recovery of added isoleucine was noted in hydrolysates prepared with and without added carbohydrate material. It was further interesting to note the added recovery of isoleucine from the insoluble amorphous humin material by i n -creased washing. When 1 gm. of purified casein was hydrolysed with 1 gm. of the pentose a value of 4.30$ was obtained, while extra washings were found to contain 0.31$ isoleucine bringing the total -59-value'to 4.6l$. The value of 4.81$ was obtained by this method on casein hydrolysed without added xylose or isoleucine. The recovery of isoleucine by additional washing indicated any loss of this amino acid, as a result of acid hydrolysis in the . presence of carbohydrate material, to be caused by the adsorption of the amino acid on the humin residue. The figure obtained for valine of 6.68$ was found to be in good agreement with the reference value of 6.7$ (80). No loss of valine (Table 11) occured as a result of the hydrolysis of casein with xylose, lurthermore, good recovery results were observed when known amounts of this amino acid were added to casein prior to hydrolysis. In the light of these results it would appear that the method of hydrolysis employed was not detrimental to the values obtained using Stokes' microbiological method (96) of analysis for the amino acid valine. Methionine did not present difficulties in being recovered quantitatively from acid hydrolysates of casein prepared in the presence of added xylose (Table 12). This amino acid was found, using Stokes' technique, to exist in casein in the con-centration of 2.5$ as compared to the reference value of 2.6$ (80). On the basis of these observations, it was concluded that the validity of this method for methionine had been established. Although 3.75$ threonine was found, by this microbiological technique, to exist in casein hydrolysates as compared to 4.2$ -6o-threonine reported in the literature (SO), no appreciable loss of this amino acid occured when the protein was hydrolysed in the presence of xylose. The riason for lower values using the technique for threonine was not clear. It is safe to say, however, that no significant loss of threonine occured when this amino acid was exposed to carbohydrate material during the hydrolytic procedure. The microbiological determination of phenylalanine using Lactobacillus delbruckii yielded the value 5»00$, The reference value (SO) was taken as 3.9$ while Stokes (96) and Block (6) report phenylalanine to be present in casein in the proportions of 5.9$ and 5.0$ respectively. A loss of phenylalanine was observed when the acid hydrolysis of casein was carried out in the presence of carbohydrate material (Table lk). The recovery of added phenylalanine from hydrolysates prepared both in the presence and absence of xylose was satisfactory. These results indicated this m ethod to be fairly reliable although the figures obtained for lysine, leucine, isoleucine, methionine and valine appear better and gave greater confidence with these techniques. Assay values obtained for tryptophane in alkali hydrolysates of casein with added xylose indicated that the presence of this carbohydrate did not cause destruction of this amino acid. Added increments of tryptophane were also recovered when the various samples were measured quantitatively using the microbiological technique (Table 15). -61-The results obtained by the microbiological determination of tyrosine using Lactobacillus delbruckii indicated alkaline hydrolysis of casein in the presence of added xylose caused irregular values. Figures obtained on alkaline hydrolysates pre-pared in the presence of xylose were found to be lower than values from alkaline hydrolysates of casein without added xylose. However the extent to which the results were lowered did not correspond directly to the amount of carbohydrate material present (Table 16). Since no humin formation took place under alkaline con-ditions of hydrolysis, it might be postulated that xylose participated in certain condensation reactions yielding non-specific inhibitory or stimulatory products that effect the growth of Lactobacillus delbruckii when cultured with tyrosine as the limiting growth re-quirement. : The reference figure for tyrosine was taken as 6.5$ (80) whereas a value of 5.5$ was found using this technique. One half of the added 1-tyrosine was recovered from the alkaline hydrolysates suggesting complete racemization of the added amino acid under these conditions. It was claimed by Gunnes, et al. (28) that tyrosine was racemized under these conditions of hydrolysis, thus necessitating multiplication of the calculated value by 2 in order to express the figure for this amino acid in terms of the 1 or natural form. The value of the microbiological procedure reported by Stokes, et al. (28 and 96) n a s been established by the determination -62-of amino acids i n hydrolyses of casein and by the recovery of added amino acids after they have been subjected to the conditions of hydrolysis i n the presence of purified casein and added carbohydrate material. Particularly good results were obtained in the deter-mination and recovery of the amino acids lysine, leucine, isoleucine, valine, methionine, threonine and tryptophane. Figures obtained i n the microbiological determination of histidine, arginine, phenylalanine and tyrosine indicated a slight loss of these amino acids when hydrolysis was carried out i n the presence of xylose. Partial loss of amino acids when hydrolysed in the presence of carbohydrate material has been previously reported in the case of arginine (99), histidine (99), phenylalanine (47), and tyrosine (8). The medium through which these losses might have occured was not reported. In the presence of abnormal amounts of xylose much more humin was formed than would be encountered i n the hydrolysis of natural products. Qualitative determinations of arginine, histidine, and phenylalanine washings of the humin residue i n their respective hydrolysates revealed appreciable amounts of the amino acids to be adsorbed on the black charcoal-like precipitate. Some of the loss at least was the result of the apparent adsorptive nature of the humin residue. A hydrolytic procedure for the liberation of tyrosine - 6 3 -from protein material much similar to the one recommended "by-Stokes, et a l . (28),was reported "by Brand and Kassel (8) to cause partial destruction of this amino acid. These investigators showed an approximate 4.00$ increase i n values obtained for tyrosine when released from protein by a sodium hydroxide — stannous chloride mixture. In the proceeding plan of experimentation i t was found that figures established for tyrosine liberated from the casein molecule using NaOH alone were lower when hydrolysis was conducted in the presence of added xylose. Since this loss of tyrosine occured despite the fact that humin and melanin formation did not take place under these conditions, i t would appear that an un-known mechanism causing the partial destruction or inactivation of tyrosine functioned during the digestion of casein with a l k a l i i n the presence of added xylose. The possibility of con-densation products forming involving the carbohydrate in the presence of a l k a l i which influenced the growth and acid production of Lactobacillus delbruckii also seemed possible. Stokes' outline of procedure was found extremely specific for each of the eleven amino acids described. The fact that no growth occured in the control tubes was singularly significant proof of the specificity of this method. The lack of growth in the control tubes was directly dependent upon two factors: namely, the actual composition of the synthetic medium and the purity of the constituents of the basal medium. -64-It was evident in the literature that different combinations of amino acids in a synthetic medium gave rise to the specific requirement of different amino acids by the assay organism. This is illustrated by the work of Stokes and Gunness (96) where they report the specific requirement of 9 amino acids by Streptococcus faecalis, whereas Henderson and Snell (35) increased the number of required amino acids for this microorganism by chang-ing the constitution of the basal medium. In considering the composition of the synthetic basal medium used in the present study (96) it was evident the constituents were present in such numbers and concentrations to supply all the growth requirements of the test organism in addition to certain amino acids and other factors not essential for but often stimul-atory to growth and acid production. Proline, hydroxyproline,. norleucine and glycine as well as thiamine, p-amino benzoic acid , guanine and uracil were not required by Streptococcus faecalis and Lactobacillus delbruckii but a basal medium prepared omitting these factors might have been unduly sensitive to non-specific stimul-atory or inhibitory substances introduced by the unknown hydrolysate ( The addition of an excess of purines to the basal medium is also justified by the fact that dips observed in the standard curves,particularly those of lysine, were eliminated by further additions of adenine, guanine or uracil. -65-Th e growth and amino acid requirements of Streptococcus faecalis was not influenced "by the presence of pyridoxamine. The Lactobacilli, in the presence of vitamin E^ ,were found not to require lysine and threonine thus making Streptococcus faecalis a better test organism for the microbiological determination of these amino acids. (96). In most cases the specificity of amino acid requirements of Streptococcus faecalis were not influenced bv chemically and physiologically related organic compounds. OrnitMne and citrulline would not replace arginine for this microorganism (96) nor would choline plus homocystine influence the methionine require^ ments of Streptococcus faecalis. Indole and anthranillic acid, which replaced tryptophane for Lactobacillus arsbinosus, were fount1 Inactive to Streptococcus faecalis (96). Streptococcus- faecalis, however, exhibited the ability to synthesize phenylalanine after 16 to 20 hours in the absence of this amino acid. Since the phenylalanine requirement of Lactobacillus arabinosus was dependent upon the composition of the basal medium ( 7 7 ) , the choice of Lactobacillus delbruckii for the microbiological deter-mination of this amino acid was a wise one. The purity of the amino acid crystals used in the preparation of basal medium has presented difficulties in the past (34). This author found the dl-isoleucine obtained from one supply house to be contaminated with leucine. Investigation of the purity of 7 samples of leucine v/ss -66-undertaken by Hegsted and Wardell ( 3 4 ) . These workers found two samples pure while the others contained approximately 1 to 20$ isoleucine. % On the basis of such observations it would appear that in studying the growth requirements of a microorganism, an amino acid might easily be classified as stimulatory or non-essential to growth and acid production, when it actually was essential or stimulatory to the test organism. -67-SUMMABY 1. The uniform microbiological method for the deter-mination of eleven amino acids as proposed by Stokes and Gunness has been critically examined and the re-liability of the technique established. 2. This procedure was found particularly advantageous for the microbiological determination of lysine, leucine, isoleucine, valine, methionine, threonine and trypto-phane in purified casein. 3. Good recovery results were also obtained in the micro-biological determination of histidine, arginine and phenylalanine, although lower values were observed with these amino acids in the presence of added car-bohydrate material. It was established that this loss was in large measure caused by physical adsorption of these amino acids on the insoluble humin residue formed under these conditions. k. The response of Lactobacillus delbruckii to alkaline hydrolysates for tyrosine prepared in the presence of added xylose was observed to be irregular. The reason for this phenomenon was not clear. THE LIBERATION OF TRYPTOPHANE DURING ACID HYDROLYSIS IN THE PRESENCE OF TITANOUS CHLORIDE. -68-HISTORICAL OUTLINE Since studies on the hydrolytic procedures for the release of amino acids from protein material are as old as the field of protein chemistry itself, and since excellent reviews of this topic are available (73). n o attempt is made to give a complete bibliography on this aspect of the problem. Hydrolytic procedures are generally classified in three main groups, those involving the use of either acids, alkalis or enzymes. Acid hydrolysis is usually carried out with varying concentrations of sulfuric or hydrochloric acid. Hydrochloric acid is probably a better hydrolytic agent when the preparation of hydrolysates for microbiological work is being carried out as sub-sequent neutralization with sodium hydroxide yields sodium chloride which does not materially influence the growth and acid production of the commonly employed test organisms. Sulfuric acid, on the other hand, gives rise to undesirable complications in that sulphate must be removed from the hydrolysate before microbiological analysis can be carried out. No advantage has been found in using hydrobromic, hydriotic, hydrofluoric, formic, acetic or phosphoric acid other than their application to the release of specific amino acids from protein material. Acid hydrolysis causes the liberation of all the amino acids with the exception of tryptophane and tyrosine which are destroyed partially or in whole during the normal hydrolytic period. Alkaline hydrolysis is carried out using various con-centrations of "barium hydroxide or sodium hydroxide. The use of sodium hydroxide for the alkaline digestion of protein material is generally considered the more convenient of the two methods. Alkaline hydrolysis is of advantage in the liberation of tryptophane and tyrosine as a racemic mixture from protein. • This type of hydrolysis has, however, several serious limitations. There is complete destruction of serine and cystine in the presence of alkali while arginine is converted to ornithine. Racemization of most amino acids is observed when proteins are subjected to alkaline hydrolysis. Furthermore, alkali dissolves silica from the glass tubes .when subjected to the heat of hydrolysis which when neutralized forms a highly absorptive colloidal silica gel precipitate. The use of enzymatic preparations for protein hydrolysis have been reported by several workers. A digestion method utilizing pancreatin in the liberption of tryptophane for microbiological analysis was described by Green and Black (25). Wooley and Sebrell (106) applied the proteolytic activity of an enzyme preparation consisting of trypsin, pepsin and erepsin to the liberation of tryptophane from protein material. These workers claimed higher and more consistent results were obtained using -70-enzymatic hydrolyses than were observed with sodium hydroxide digestions. The enzymatic methods, however, have several serious disadvantages. These reactions are slow and do not go to com-pletion. A further disadvantage is seen in the fact that after enzymatic hydrolysis there is present in protein the enzyme preparation as well as its products of the resulting digestion. The actual liberation of tryptophane from acid hydrolysates has received little investigation by workers in the field of protein chemistry and nutrition. Investigators have been more concerned with the reactions involved in the destruction of this amino acid when subjected to acid hydrolysis. Tryptophane is claimed to be involved, under acid conditions of hydrolysis, in a condensation with aldehyde or aldehyde-like substances as,released, along with the tryptophane, during hydrolytic cleavage (22). This interaction of the indole nucleus with three molecules of aldehyde is known as humin formation. The first aldehyde molecule would appear to form on condensation with tryptophane a substituted indolidene-methane molecule. The second molecule of aldehyde forms a compound of the rosindole type while the third molecule of aldehyde condens.es with the rosindal type of compound to eliminate water and form the insoluble humin. The hydrolysis of protein using acid without destruction of tryptophane is not easily accomplished. Hlasiwetz and Haberman - 7 1 -as early as 1871. reported the use of stannous chloride in pre-, venting humin formation in hydrolysates of proteins (39 and kO). The stannite was subsequently removed through precipitation with hydrogen sulphide. Sullivan and Hess (97) reported the use oftitanous chloride in stopping the acid destruction of cystine. Hydrolysis in the presence of this compound was believed by these workers to •^e faster than under ordinary conditions. Colorless or slightly yellow hydrolysate solutions were observed by these investigators. They stated that insoluble humin and soluble melanin formation was arrested in the presence of titanous chloride. Subsequent removal of this compound was achieved by the dropwise addition of 5 N NaOH to the hydrolysate until pH 6.0 had been reached. The fact that titanous chloride stops humin formation suggested the possible use of this compound in the liberation of tryptophane from protein material by acid hydrolysis for the purpose of microbiological assay. -72-EXPERIMENTAL After considering the advantages and disadvantages of various methods of hydrolysis, it was decided to investigate the conditions surrounding the liberation of tryptophane from protein by acid in the presence of a reducing substance. Hlasiwetz and Haberman (38 and .39) reported the use of stannous chloride for the liberation of tryptophane during hydrolysis while Sullivan and Hess (97) suggested the use of titanous chloride in this capacity. Since the removal of stannous chloride involved the use of hydrogen sulphide and consequently give rise to highly adsorptive precitpitates, whereas the removal of titanous chloride was more readily achieved through precipitation by neutralization, the latter mentioned compound was selected for this study. Stokes' microbiological method for the determination of tryptophane (96), using Streptococcus faecalis as the test organism, was employed. The methods for the quantitative measure of phenylalanine (96) and tyrosine (28) reported by this investigator were also used. Various methods of hydrolysis were followed including that described by Sullivan and Hess (97) and. by Stokes, et al. (96). The procedure employed by Gunness, et al. (28) for alkaline hydrolysis was also used. -73-The extent to which titanous chloride, under varying conditions of hydrolysis, was effective in preventing amino acid destruction was determined by estimation of the tryptophane content of the various hydrolysates. Tryptophane has been shown to be the most sensitive of the amino acids to destruction by acid (73). 1.33 of titanous chloride was added, prior to digestion, to acid hydrolysates as a 15$ weight/volume solution with hydrochloric acid. At the end of the hydrolysis period the titanous chloride was precipitated from the hydrolysates- by the dropwiso addition of 5 N NaOH. The precipitate, an insoluble blue-gray hydroxide of titanium was removed at pH 6.0 or less. The precipitate was centrifuged and washed once with 10-20 ml. of distilled water. The hydrogan ion concentration of the hydrolysate with the" washings was adjusted to 6.8 - 7-0 and made to 100 ml. in a volumetric flask. If centrifugation did not removo the precipitate completely or i f further titanous hydroxide came down after the hydrogen ion concentration was adjusted to 6.8 - 7.0, the hydrolysate plus weshings was.carefully filtered before being made up to volume. Hydrolysates prepared in the presence of titanous. chloride as described above were colorless and, on standing, took on s. yellowish tint. .All hydrolysates were stored under toluene and away from light to await assay. In certain casps a determination of the oxtent of hydrolysis was made using Sorensen's Formol Titration. -74-This procedure was carried out "by neutralizing to phenolphthaiein using 0.2 N NaOtf a 20 ml. quantity of hydrolysate prepared as previously described. To this solution was added 10 ml. of a freshly prepared formaldehyde solution, made by neutralizing, with 0.2 N NaOH., 100 ml. of 40$ formol containing 2 ml. of a 0.5$ phenolphthaiein solution. The mixture of 20 ml. of hydrolysate and 10 ml. of formaldehyde solution was titrated to a faint pink using 0,2 N NaOH. Back titration to a clear solution was carried out using 0.2 N HC1. A' blank titration was conducted on water to serve as a control. One ml. of 0.2 N NaOH was equivalant to 2.8 mgm. of amino nitrogen. CALCULATION:t Amount of 0.2 N NaOH required= 4.0 ml. Amino nitrogen = 4.0X2.8*100 s 56 mgm. 20 A general study was first carried out on the ability TiCl^ to prevent the destruction of tryptophane liberated from casein by hot mineral acid. Pour test tubes were prepared for hydrolysis with 1 gm. of dried casein in each. The 4 tubes were divided into 2 groups of 2 tubes each and to one'group was added 1.33 ml. of TiClg . To one tube from each group was added 0.002 gm. of 1-tryptophane, the other tube from each group being left free of added tryptophane. All tubes 'were sealed and hydrolysed at 120° C for 10 hours in the presence of 10 ml. of 10$ by volume HC1. -75-An outline of the plan of experimentation, along with the values established for tryptophane are given in Table 17. TABLE 17. THE LIBERATION OF TRYPTOPHANE FROM ACID HYDROLY- SATES PREPABED IN THE PRESENCE OF TiClg. CASEIN TiCl, in ml. TRYPTOPHANE • ADDED. CALCULATED VALUE. 1 gm. nil 1 gm. 1.33 0.8 $ 1 gm. 0.002 gm. nil 1 gm. 1.33 0.002 gm. 0.89 $ The control value for tryptophane determined from an alkaline hydrolysate (5 N NaOH at 120* C for 10 hours ) was 1.5$. Since the degree of racemization' of tryptophane as a result of acid hydrolysis is not known, figures obtained throughout this study for this"amino acid in acid hydrolysates are expressed as determined microbiologically without multiplication by 2. In the case of alkaline digestion the figure obtained is multiplied by 2 to allow for complete racemization of the amino acid. CONCLUSIONS Tryptophane was completely destroyed when TiCl^ was not present during acid hydrolysis at 120° for 10 hours using 10$, by volume, of HC1. The presence of this reducing substance accounted for 0.87$ tryptophane in the hydrolysate. To study the effect of added xylose on the determination of tryptophane from acid hydrolyses,in the presence and absence of added titanous chloride,the following study was made. Nine hydrolyses were prepared with 1 gm. of dried casein weighed accurately into each tube. To one tube was added 0.01 gm. of xylose and 1.33 ml. of titanous chloride. To the second tube was added 0.05 gm. of xylose alSng with 1.33 ml. of TiClj . The remaining 7 tubes were divided into 2 groups of 3 and 4 tubes respectively. To the 3 tubes of the first group, each containing 1 gm. of dried, purified casein, and 0.1 gm. of xylose was added 1.33 ml., 2.0 ml., and 5.0 ml. of TiCl 3 respectively. The 4 tubes of the second group were further divided into two groups of 2 tubes each. To both of the tubes of one group vras added 0.002 gm. of 1-tryptophane, the remaining 2 tubes being left free of added amino acid. To one tube from each group was added 1.33 ml. of TiCl 3. All 9 tubes were sealed after the addition of 10 ml. of 10$ HC1 and digested for a period of 10 hours at 120° C. Since this plan of experimentation (Table IS) and the -77-previously described outline of hydrolysis (Table 17) were carried out concurrently, the same alkaline hydrolysate value was used as a basis of comparison for each experiment . The results for the determination of tryptophane under these conditions are recorded in Table IS. TABLE 18 THE LIBERATION OP TRYPTOPHANE FROM ACID  HYDROLYSATES PREPARED IN THE PRESENCE OF  TiCl 3 AND XYLOSE. CASEIN XYLOSE TiCl 3 in ml. TRYPTOPHANE ADDED CALCULATED VALUE 1 gm. .01 gm. 1.33 0.45 $ 1 gm. .05 gm. 1.33 nil 1 gm. .10 gm. 1.33 nil 1 gm. .10 gm. 2.00 nil 1 gm. .10 gm. 5.00 0.25 $ 1 gm. .10 gm. — nil 1 gm. .10 gm. — .002 gm. nil 1 gm. .10 gm. 1.33 .002 gm. 0.25 £ 1 gm. .10 gm. 1.33 nil The control value for tryptophane determined from an alkaline hydrolysate (5 N NaOH at 120° C. for 10 hours ) was 1.5$. CONCLUSIONS The presence of 0.05 gm. or more of xylose during acid hydrolysis caused' complete destruction of tryptophane released in the presence of TiCl^ . . Attempted recovery of added tryptophane when subjected to acid hydrolysis in the presence of TiCl3 and xylose was also unsuccessful. •To investigate further the possibility of TiCLj preventing the destruction of tryptophane during acid hydrolysis of casein in the presence of xylose, the plan of experimentation outlined in Table 19 was followed. In this study 3 hydrolytic procedures were used involving 10 ml. of 10$, by volume, of HCl for 10 hours at .120° C. 10 ml. of 10$, by weight, of HCl for 10 hours at 120* C and 5 ml. of 20$, by weight, of HCl for 5 hours at 120° C. TABLE 19 THE LIBERATION OF TRYPTOPHANE BY THREE METHODS  OF ACID HYDROLYSIS I? THE PRESENCE OF TIC13 • AND XYLOSE. CASEIN XYLOSE TiCl^ in ml. 10$ VOL. HCl. 10$ V/T. HCl. 20$ ¥T. HCl. 1 gm. 1.33 0.75$ 0.32$ 0.32$ 1 gm. .05 gin. 1.33 nil nil nil 1 gm. .05 gm. 2.00 O.g $ nil nil • 1 gm. .05 gm. 3.00 nil nil nil -79-CONCLUSIONS It would appear that serious limitations were imposed upon the protective action of TiClj by the presence of added xylose. It is thus clear that under such conditions the use of TiClg was of little value in the protective liberation of tryptophane. To investigate further the possibility of titanous chloride preventing the destruction of tryptophane during acid hydrolysis, using the 3 previously described hydrolytic methods, a brief study of the influence of the time of hydrolysis on assay values for tryptophane was followed. An outline of the conditions for the 12 individual hydrolyses with the results obtained are given in Table 20. TABLE 20 THE LIBERATION OF TRYPTOPHANE BY THREE METHODS OF  ACID HYDROLYSIS IN THE PRESENCE OF TiCl x AT JIVE  AND TEN HOUR PERIODS. CASEIN TiCl 3 in ml. TIME . 10$ VOL. HCl. 10$ WE. HCl. 20$ WT. HCl. 1 gm. 1.00 5 hrs. • 0.45 $ 0.45 $ 1 gm. 1.33 5 hrs. 0.53 $ 0.75 $ 1 gm. 1.00 10 hrs. 0.55 $ 0.21 $ nil 1 gm. 1.33 10 hrs. 0.75 $ • 0.53 $ 0.23 $ -80-C0NCLUSI0HS Ten percent by volume of - HCl and 20$ by weight of HCl presented 2 individual types of hydrolysis. The lower concentration of acid appeared to cause a slower, more gradual liberation of tryptophane without excessive destruction of this amino acid on prolonged heating. The 20$, by weight HCl, on the other hand, caused quick liberation of tryptophane with subsequent destruction of. this amino acid in the presence of TiCl^ at 120° C. Ten percent by weight HCl appeared too concentrated to permit safe liberation of tryptophane during the conditions of prolonged heating required by this concentration of acid to release tryptophane. As a result, the use of this acid solution for the liberation of tryptophane in the presence of TiC]jwas not found suitable. Subsequent studies were then confined to the first two methods of hydrolysis described above, one involving the use of 10 ml. of 10$ by volume of HCl at 120° C for 10 hours, the other using 5 ml. of 20$ by weight HCl. at 120* C for 5 hours. To study the influence of titanous chloride on the values obtained for the microbiological determination of the amino acids tryptophane, tyrosine and phenylalanine after acid hydrolysis of casein, an experiment including recovery of the three amino acids under the following conditions of digestion was outlined. Two -Si-concentrations of acid were used, namely 10$ by volume and 20$ by weight of hydrochloric acid. For the purposes of hydrolysis lh tubes were prepared with 1 gm. of driod casein in each. The lU tubes were divided into three groups, two of six tubes each and a third group containing 2 tubes. The first group of 6 tubes was divided iato 2 Bets' of 3» To each tube of the first set of 3 tubes was added a different amino acid. To one tube was added 0.002 gm. of tryptophane , to the second 0.0065 gm. of 1- tyrosine, while to the third tube was added 0.0052 gm. of 1-phenylalanine 1.33 m?-. of TiCl^was then placed in each of the three tubes. Increasing amounts of TiClgsolution were added to the second set of 3 tubes included in the first group of 6. 1.33 raL» 1.66 ml. and 2.00 ml. quantities respectively of the reducing agent were added to one of three tubes each. The first group of 6 hydrolysis tubes prepared as des-cribed above were hydrolysed for 5 hours at 120 C. in the presence of 5 ml. of 20$ by weight HCl. The second group of 6 hydrolysis tubes were prepared . identically to the set described above. Hydrolysis of this set was carried out over a 10 hour period at 120 C. in the presence of 10 ml. of 10$ by volume hydrochloric acid. The last group of 2 tubes v:ere prepared to serve as controls. To one tube containing 1 gm. of dried casein was added 10 ml. of 10$ by volume hydrochloric acid. Ten ml. of 5 N NaOH -82-was added to the second tube and "both these samples were hydrolysed for 10 hours at 120° C. The results obtained in this study along with an outline of the experiment are presented in Table 21. TABLE 21 THE RECOVERY OF ADDED TRYPTOPHANE, TYROSINE, AND. PHENYIALANINE, . FROM CASEIN HYDROLYSATES PREPARED IN THE PRESENCE OF TiCl, USING TWO ACID CONCENTRATIONS CASEIN ACID TIME AMINO ACID TiClj TRYPTOPHANE TYROSINE PHENYLALINE IN HR, ADDED in ml. 1 gm. 20$ Wt. 5 Trypt. 1.33 0.60$ 4.27$ 3.98$ .002 gm. 3.98$ 1 gm. 20$ Wt. . 5 Tyrosine 1.33 0.50$ 5.50$ .0065 gm. 4.50$ 1 gm. 20$ Wt. 5 Phenylal. 1.33 0.32$ 4.87$ .0052 gm. 0.30$ 4.86$ 1 gm. 20$ Wt. 5 1.33 3.98$' 1 gm. 20$ Wt. 5 1.66 0.94$ 5.00$ 3.99$ 1 gm. 20$ Wt. 5 2.00 0.95$ 5.00$ 3.95$ 1 gm. 10$ Vol. 10 Trypt. 1.33 1.35$ 4.86$ 3.98$ .002 gm. 0.95$ 5.25$ 1 gm. 10$ Vol. 10 Tyrosine 1.33 3.98$ .0065 gm. 1.00$ 4.87$ 4.60$ 1 gm. 10$ Vol. 10 Phenylal. 1.33 .0052 gm. 3.98$ 1 gm. 10$ Vol. 10 1.33 0.50$ 4.87$ 1 gm. 10$ Vol. 10 1.66 0.65$ 5.25$ 3.98$ 1 gm. 10$ Vol. 10 2.00 0.65$ 5.25$ 3.98$ 1 gm. 10$ Vol. 10 a i l 4.88$ 3.98$ 1 gm. 5N NaOH. 10 — 1.30$ 5.50$ — -gU-CONCLUSIONS The figures for tryptophane released in the presence of added amino acids and TiClgwere notably higher than those obtained under similar digestion conditions with no added tryptophane or tyrosine. This would suggest that tryptophane is best liberated from a native protein in the presence of added tryptophane, tyrosine or phenylalanine. No adequate explanation of this finding can be advanced. The microbiological assay of these.hydrolysates were repeated in toto and gave identical results to those reported in Table 21. Time did not permit the complete repetition of this experiment in order to obtain verification of these findings. It would appear that tyrosine was destroyed to the extent of 11! $ when liberated by either method of acid hydrolysis as compared to alkaline digestion. The presence of TiCl^indicated no marked increase in the values obtained as a result of acid hydrolysis. Corresponding figures obtained using both acid solutions were similar.* Values obtained for phenylalanine during acid hydrolysis in the presence and absence of TiCl^showed no significant differences. Corresponding values for phenylalanine prepared using both acid concentrations were found to be similar. To study the progressive liberation of tryptophane at 120° C. by 20$ HCl, hydrolysis tubes containing 1.33 ml., 1.66 ml. and no TiCl-were removed.at 2, 3i »^ a n < * 5 hour intervals for assay - 8 5 -by the microbiological technique. The experimental methods with the results obtained are presented in Table 22. TABLE 22. THE PBOG-RESSIVE LIBERATION OF TRYPTOPHANE BY 20$ HCl.  AT 120° C. IN THE PRESENCE OF TiCla CASEIN ACID TEMPERATURE TiCl s in ml. 2 HOURS 3 HOURS U HOURS 5 HOURS 1 gm. 20$ 120° C. .30$ .10$ .06$ nil 1 gm. 20$ 120° C. 1.33 .59$ .50$ .56$ .36$ 1 gm. 20$ 120° C. 1 1.66 .80$ .50$ .50$ .33$ CONCLUSIONS Progressive destruction of tryptophane in the absence of TiClj at 120* C. is illustrated in Table 22. Increased TiClj appeared to be of value only during the early stages of hydrolysis as values obtained using 1.66 ml. of this substance approximated those found using 1.33 ml. after 3 hours. The gradual destruction of tryptophane in the presence of TiClj suggested the study of the liberation of this amino acid under similar conditions of hydrolysis at lower temperatures (i.e. at 110* C., 100° C., 95° C., and 90° C.). - 8 6 -. The progressive liberation of tryptophane at 110*C. using 20$ HCl. both in the presence and absence of TiClg by the deter-mination of this amino acid at 2, 3» *+» 5 and 6 hour periods. The plan of experimentation along with the. results for both the tryptophane content and amino nitrogen content of•the hydrolysates are outlined in Table 23. TABLE 23 THE PROGRESSIVE LIBERATION OF TRYPTOPHANE BY 20$ HCl. ' at 110° C IN THE PRESENCE OF TiClj ' CASEIN TIME TEMPERATURE TiCl s in ml. ACID TRYPTOPHANE VALUE AMINO Nj in mgm. 1 gm. lgm. 1 gm. 1 gm. 1 gm. 2 3 4 5 6 110° c. 110° c. 110° c 110° c 110 c 1.33 1.33 1.33 1.33 1.33 20$ 20$ 20$ 20$ l.< 0.76$ o.59# 0.36$ 0.67$ 54.6 61.6 70.0 77.0 72.8 1 gm. 1 gm. 1 gm. 1 gm. 1 gm. 2 3 4 5 6 n o * c n o " c 110° c 110° c 110° c 20$ 20$ 20$ 20$ 20$ 0.43$ 0.25$ 0.19$ 0.13$ 0.07$ 78.4 84.0 88.6 84.0 91.0 CONCLUSIONS The maximum value for tryptophane released at 110° C. with 20$ HCl. in the' presence of TiCl"3 was found at 2 hours. The values then gradually decreased from hour to hour at this temperature with added TiCl3 suggesting the gradual destruction of tryptophane even in the presence of this reducing agent. -87-The degree of hydrolysis of casein digested in the presence of TiCLj was found to he lower than that obtained in the absence of this substance. This is contrary to the postulate of Sullivan and Hess (97), based on a study of the rate of liberation of cystine from proteins during hydrolysis, that TiClg increased the rate of digestion of proteins. An investigation of the tryptophane content of casein at 2, 3» 4, 5, 6, 7 and 10 hour periods during acid digestion at 100° C. in the presence and absense of TiCl3 was carried out. The values obtained for tryptophane at the various stages of hydrolysis along with the amino nitrogen content of all hydrolysates are recorded in Table 24 with an outline of the experi-ment. • -88-TABLE 24 THE PROGRESSIVE LIBERATION Off TRYPTOPHANE BY 20$ HCl.  AT 100" C IN THE PRESENCE OF TiClT ' CASEIN TIME IN HR. TEMPERATURE TiCl 3 in ml. ACID TRYPTOPHANE VALUE AMINO Nz in mgm. 1 gm. 2 100° c . 1.33 20$ 0.75$ 46.2 1 gm. 3 100° c . 1.33 20$ 0.79$ 53.2 1 gm. k 100°c. 1.33 20$ 0.87$ 60.2 1 gm. 5 100° c . 1.33 20$ 1.00* 61.6 1 gm. 6 100o0.. . 1.33 20$ 0.75^  68.6 1 gm. 7 100°c. 1.33 20$ 0.79$ 71.4 1 gm. 10 100°c. 1.33 20$ 0.57$ 74.2 1 gm. 1 gm. 1 gm. 1 gm. 1 gm. 1 gm. 1 gm. 2 I 5 6 7 10 • 100°c. :100°C. 100° C. 100° C. 100aC. 100°C. 100°C. . — 20$ 20$ 20$ 20$ 20$ 20$ 20$ 0.33$ 0.35$ 0.25$ . 0.19$ 0.15$ 61.6 65.5 68.6 64.2 78.4 81.2 89.6 CONCLUSIONS The release of tryptophane from acid hydrolysates in the presence of TiClj at 100° C. reached a maximum at 5 hours after which time there was gradual destruction or loss of this amino acid. In the absence of TiClj there was almost complete loss of tryptophane. The presence of TiCl3 did not speed up the liberation of amino nitrogen as was claimed by Sullivan and Hess. On the contrary, liberation of amino nitrogen was slower in the presence of this substance, confirming the results reported in Table 23. -29-Investigation of the progressive liberation and gradual destruction of tryptophane at 95° C., "both in the presence and absence of TiClj was carried out by the determination of this amino acid in hydrolysates removed at 2, 4, 5. 6, 7. 8. 9 a n * 1° hour intervals during the digestion. • A plan of this experiment, along with the figures for tryptophane and amino nitrogen content of the hydrolysates, is presented in Table 25. TABLE 25 THE PROGRESSIVE LIBERATION OF TRYPTOPHANE BY 20$ HCl. -AT 9 5 v C. IN THE PRESENCE OF TiCl a CASEIN TIME IN HR. TEMPERATURE TiCl 3 in ml. ACID TRYPTOPHANE VALUE AMINO Nz • in mgm. 1 1 1 1 1 1 1 1 gm. gm. gm. gm. gm. gm. gm. gin. 2 4 5 6 7 8 9 10 95* c . 95" c . 95° c . 9 5 a c . 95" c . 95° c . 95° a . 95° c . 1.33 1.33 1.33 1.33 1.33 1.33 1.33 1.33 20$ 20$ 20$ 20$ 20$ 20$ 20$ 0.87$ 0.82$ 0.76$ 0.74$ 0.7*+$ 0.50$ 0.36$ 0.50$ 30.8 42.0 47.6 53.2 52.4 63.0 70.0 72.8 gm. gm. gm. gm. gm. gm. gm. gm. 2 4 5 6 7 8 9 10 95° c . 95" C. 95* c . 95" c . 95° c . 95" c . 95" c . 95* C. 20$ 20$ 20$ 20$ 20$ 20$ 20$ 20$ 0.75$ 0.62$ 0.55$ 0.46$ 0.45$ 0.30$ 0.29$ 0.21$ 42.0 60.2 63.0 70.0 74.2 •82.6 81.2 84.0 CONCLUSIONS At 95° C. in the absence of TiClj the' destruction of - 9 0 -tryptophane was not as great as at a higher temperature. Pro-longed heating at 9 5 * C» even in the presence of TiCl-^ , caused the progressive gradual destruction of tryptophane. As was found previously, TiClj did not increase the rate of amino nitrogen released, hut on the contrary, would appear to inhibit the rate of hydrolysis. Finally, in studying 2 0 $ by weight HCl. as a hydrolytic agent, an investigation of the tryptophane content and the degree of hydrolysis was carried out on digests of casein prepared at 9 0 ° C. at intervals of 2, k, 5t 6, 7. 8, 9 10 hour periods. The values indicating the progressive liberation and destruction of tryptophane are outlined in Table 26 along with the plan of experimentation. -91-TABLE 26 THE PROGRESSIVE LIBERATION OF TRYPTOPHANE BY 20$ HCl.  AT 90" C. IN THE PRESENCE OP T i c T ^ " CASEIN TIME IN HR. TEMPERATURE TiCl 3 in ml. ACID TRYPTOPHANE VALUE AMINO N«in mgm. gm. gm. gm. gm. gm. gm. gm. gm. 2 4 5 6 7 8 9 10 90° c . 900 c . 900 c . 900 c . 90° c . 900 c . 900 c . 900 c . 1.33 1.33 1.33 1.33 1.33 1.33 1.33 1.33 20$ 20$ 20$ 20$ 20$ 20$ 20$ 0.73$ 0.79$ 0.86$ 0.89$ 0.68$ 0.74$ 0.73$ 0.58$ 22.4 4o.6 43.4 51.8 47.6 57.^  57.4 77.0 gm. gm. gm. gm. gm. gm. gm. gm. 2\ 4-•5 6 7 8 9 10 90° c . 900 c . 900 c . 90* c . 90" c . 90* c . 900 c . 900 c . 20$ 20$ 20$ 20$ 20$ 20$ 20$ 0.81$ 0.65$ 0.55$ 0.42$ 0.46$ 0.49$ 0.39$ 0.42J& 36.4 54.6 60.2 64.4 65.8 74.2 75.6 78.4 CONCLUSIONS At 90° C. the destruction of tryptophane during acid hydrolysis in the absence of TiCljwas not as great as that of higher temperatures. In the presence of TiCl3 the values of tryptophane increased to a maximum at 6 hours and, after that time, exhibited a gradual decline. The amino nitrogen coritent of the digests prepared at defined intervals with added TiClj showed lower values than the corresponding hydrolysates prepared in the absence of this sub-stance further substantiating the findings of preious experiments (Tables 23, 24 and 25). -92-In the light of the fact .that more tryptophane,was liberated in the absence of TiClj at 2 hours, as indicated by the amino nitrogen content of the hydrolysates, and since the destruction of this amino acid during the early stages of digestion at 90° C was slight, it was easily understood why the value obtained for tryptophane was greater, at that time, in the absence of TiCLj than in the pre-sence of this substance. The liberation of tryptophane by 10$ by volume HCl. was carried out at 120° C. in the presence and absence of TiCl3 . An examination of the tryptophane content of casein hydrolysates was made at 5. 6, 7 and. 10 hour periods in order to study the progressive liberation of this amino acid under these conditions. An outline of the plan of experimentation along with the results obtained in the microbiological assay of the various digests for tryptophane are given in Table 27.• TABLE 27 THE PROGRESSIVE LIBERATION OF TRYPTOPHANE BY 10$ HCl. AT 120° C. IN THE PRESENCE OP INCREASING AMOUNTS OP T i c i a . CASEIN ACID TEMPERATURE TiCL^  in ml. • 5 HOURS 6 HOURS 7 HOURS 10 HOURS 1 gm. 10$ 120° C. .15$ .12$ .08$ nil 1 gm. 10$ 120° C. 1-33 .39$ 1.05$ .76$ . ^ 3 $ 1 gm. 10$ 120° C. 1.66 .33$ .73$ .66$ .43$ CONCLUSIONS No advantage is -to be attained in using increased TiCl^ when hydrolysis is carried out at 120* C. with 10$ HCl. After 5 hours in the presence^ of TiCl^ there is a gradual, destruction of tryptophane. '.-The value of 1.05$ obtained at 6 hours would appear to be out of. line with those of 5 and 7 hours. No adequate explanation of this figure can at present be advanced.. To study the fate of pure tryptophane in the presence of varying amounts of xylose and titanous chloride, the recovery of known amounts of this amino acid was attempted after the pure solutions had been subjected to concentrations of acid and tem-peratures comparable to those of hydrolysis. Bight tubes were prepared with 0.025 gm. of pure 1-trypto-phane in each one. This set of 8 was divided into 3 groups, 2 of three tubes each and one of 2 tubes. To the first group of 3 tubes was added 1.33 ml. 1.66 ml. and 2.0 ml. of TiClj respectively. To each tube in this group was also added 10 ml. of 10$ by volume HCl. The tubes were sealed and heated to 120° C. for 10 hours. The second set of 3 tubes was prepared with a similar addition of T1C13 to each one. To this group 5 ml. of 20$ HCl. was added and the tubes were subjected to 120* C. h.eat for 5 hours. -94-To one of the 2 remaining tubes was added 10 ml. of 10$ by volume HCl. while to the other 10 ml. of 5N NaOH was added. These 2 tubes without TiClj , were heated to 120° C. for a 10 hour period. The results of this investigation are reported in Table 2S. TABLE 28 THE MICROBIOLOGICAL RECOVERY OF PURE TRYPTOPHANE WHEN  SUBJECTED TO CONDITIONS OF HYDROLYSIS IN THE PRESENCE OF TiClj. TRYPTOPHANE ADDED TIME IN HR. TiCl 3 in ml. ACID TRYPTOPHANE RECOVERED .25 mgm. 10 1.33 10$ 25.2 mgm. 25 mgm. 10 1.66 10$ 25.1 mgm. 25 mgm. 10 2.00 10$ 25.0 mgm. 25 mgm. 10 — 10$ nil 25 mgm. 5 1.33 20$ 27.5 mgm. 25 mgm. 5 1.66 20$ 25.0 mgm. 25 mgm. 5 2.00 20$ 25.1 mgm. 25 mgm. 10 — 5N NaOH 27.0 mgm. (#) (#) The figure for pure tryptophane subjected to conditions of alkaline hydrolysis was not multiplied by 2. CONCLUSIONS In preliminary experiments it wa's shown that the presence of xylose in pure tryptophane solutions, even with added H C I 3 , caused the destruction of this amino acid under normal conditions of -95-hydrolysis. In the absence of added xylose it was observed that there was no significant destruction of tryptophane when subjected to the conditions of acid hydrolysis in the presence of TiCl_. Attempts o to racemise a pure solution of 1-tryptophane.by subjecting it to 10 hours exposure with 5N NaOH at 120° C. were entirely unsuccessful. This would suggest that radicles not found in pure 1-tryptophane solutions, that are present in the casein molecule, must be responsible for the change in structure of this amino acid as it is liberated from proteins during alkaline hydrolysis. The question as to whether or not tryptophane is completely racemized during the alkaline hydrolysis of proteins is thus raised by the results -. obtained lm this study. It is possible also that, contrary to popular conception, tryptophane may undergo considerable destruction during the alkaline hydrolysis of the proteins. DISCUSSION In reviewing the results obtained during the experimental, phase of this study several important observations were made regard-ing different conditions of acid hydrolysis with and without added TiCly , in the presence of different amounts of xylose, and under varying conditions of hydrolysis. Early experiments in which the value of TiCl^ in prevent-ing the loss of tryptophane during acid breakdown of 1 gm. of casein at 120° C. was studied, showed 0.8$ of this amino acid to be present in acid hydrolysates after 10 hours digestion with 10 ml. of 10$ by volume HCl (Table 17). No tryptophane was detected in acid hydrolysates prepared under similar conditions in the absence of TiClj . Added tryptophane (0.002 gm.) was recovered to the extent of U'5$ in presence of this reducing agent while complete loss of added tryptophane as well as the tryptophane present in casein was observed in the absence of TiCl^. It would appear from this preliminary investigation that this amino acid is completely des-troyed during normal conditions of acid hydrolysis as revealed by its recovery employing a microbiological method for its determination. Since 4-5$ recovery of 1-tryptophane was realized it would appear not unreasonable to postulate that this amino acid is racemized when subjected to these conditions of acid hydrolysis in the presence of casein and added TiCl^ . The evidence from the literature indicates that proteins racemize under acid conditions of hydrolysis, but it is -97-ordinarily thought to "be a limited amount. In studying the influence of added xylose on amino acid destruction, in an endeavor to simulate conditions likely to occur in natural products, it was found that the presence of this car-bohydrate material lowered the results obtained when casein was subjected to acid hydrolysis with TiClg . No tryptophane was detected in hydrolysates prepared using normal amounts of added TiClg in the presence of 0.05 gm. or more of xylose. When casein was digested in the presence of 0.01 gm. of xylose and 1.33 ml. of TiCl^ , 0.4-5$ tryptophane was. found using the microbiological technique. An increased amount of TiClj (5.0 ml.) was responsible for the detection of 0.25$ tryptophane prepared in the presence of 0.10 gm. of carbohydrate material. The recovery of added 1-tryptophane from casein hydroly-sates in the presence of 0.10 gm. of xylose and 1.33 ml. of TiCl^ was not successful (Table 18). It would appear that the presence of carbohydrate material in the form of xylose imposes severe limitations on the "protective" action of this reducing agent. A comparison of 3 hydrolytic procedures, involving the use of different acid concentrations, was carried out in the presence of 0.05 gm. of xylose and increasing amounts of TiCl^ (Table 19). The use of 10 ml. of 10$ by volume HCl at 120° C. for a period of 10 hours yielded 0.75$ tryptophane in the absence of xylose -98-while in the presence of this carbohydrate material under similar digestion conditions the loss of tryptophane was complete. The more concentrated HCl solutions, 10$ by weight and 20$ by weight, were responsible for 0.32$ values obtained for casein when this protein was hydrolysed in the presence of 1.33 ml. of TiCl-j . When xylose was present the latter 2 methods were of no value in the liberation of tryptophane from casein. Increasing increments of TiCl^did not stop the destruction of tryptophane in the presence of 0.05 gm. of xylose. By studying the progressive liberation of tryptophane during acid hydrolysis with added TiCL^ better understanding of the destruction of this amino acid under different method.s of hydrolysis was obtained. It was observed in the plan of experimentation outlined in Table 20 that 10$ by volume HCl and 20$ by weight HCl. were best for the liberation of tryptophane. The concentration of 10$ by weight HCl. was not great enough to hydrolyse the casein as quickly as 20$ HCl solution, but on the other hand its acid strength wp.s great enough to cause serious destruction of tryptophane. The weakest acid solution used appeared to cause a more gentle release of tryptophane over a longer period of time. The most concentrated acid solution (20$, by weight, HCl) appeared to liberate the amino acid quickly, suggesting the possibility of employing a short hydrolytic period advantageously in the liberation, of tryptophane when TiCl-x is used. -99-Sinoe the use of 10$ by volume HCl. and 20$, by weight, HCl. solutions appeared to provide 2 contrasting pathways of casein hydrolysis in the presence of TiClj ,.subsequent studies were carried out using these 2 acid concentrations. The recovery of tryptophane, tyrosine and phenylalanine, as the amino acids claimed to be involved in humin formation, was attempted using the two methods of acid hydrolysis selected from the previous experiment. Increased increments of TiCl^ over the pre-viously added 1,33 m L were found to give significant results (Table 21). The values obtained for tryptophane using 10$ by volume HCl. at 10 hours with TiCl^ were higher than the results established by the microbiological technique on casein samples subjected to hydrolysis with 20$ HCl. in the presence of TiClg for a 5 hour period. Tryptophane was detected to the extent of 1.35$ in an acid hydrolysis of casein with 0.002 gm. of added 1-tryptophane. The reason for this high value is not understood when an acid hydrolysis of casein with no added tryptophane prepared in the presence of TiClj yields only 0.50$ tryptophane. The figures obtained for tryptophane when tyrosine and phenylalanine were added prior to acid hydrolysis with 1.33 ml» of TiCI3 were 0.95$ and 1.00$ respectively, which is considerably higher than the value stated above for a hydrolysate prepared in the absence of added amino acids. -100-Repetition of the microbiological assay of these hydrolysates yielded identical values. Time, however, did not permit the repeat-ing of this plan of hydrolysis. The reason for these results is not clear at this time. Increased amounts of TiCl^ , 1.66 ml. and 2.00 ml., as compared to 1.33 ml. normally used caused a slight increase in the values found as a result of the acid digestion of casein. In the case of 20$ HCl, lower results for tryptophane were obtained with the normal amount (1.33 ml.) of TiClj . When 1.66 ml. and 2.00 ml. of this reducing substance was added to casein sample, the values obtained were 3 times as great as those found using only 1.33 ml. of TiClj(i. e. 0.94$ and 0.95$ as compared to 0.30$). It would appear that a greater amount of TiCljis necessary during the hydrolysis period of 5 hours at 120° C. with 20$ HCl. Slightly higher results were observed for tryptophane in the acid hydrolysates prepared with added tyrosine and 1.33 ml. of TiClj . The extent of the increased value found in this digestion over the acid hydrolysate prepared with TiClj in the absence of added tyrosine was 0.50$ as compared to 0.30$. The reason for this increase as described for both 10$ and 20$ HCl. solutions, when casein is hydrolysed in the presence of these added amino acids, is not clear. It would appear that the presence in solution of the free amino acids, either tryptophane, tyrosine or phenylalanine,as hydrolytic cleavage begins, has a -101-desirable effect on the preservation of hound tryptophane as it is liberated from casein. The "control" value for tryptophane as established by the determination of dl-tryptophane in an alkaline hydrolysis with multiplication of the result by 2 was found to be 1.35$. Acid hydrolysed proteins prepared either in the presence or absence of TiCl3 yielded approximately 11$ less tyrosine than the alkaline control. There appeared to be little difference in the values obtained for tyrosine in acid hydrolysates prepared both with the 10$ and 20$ acid solutions. (Table 21). Recovery of added tyrosine was observed with both acid concentrations. Here again increased TiClj gave slightly higher results. Higher values for tyrosine were not found in acid hydrolysates with TiCl^ prepared with added tryptophane and phenyl-alanine as was observed with tryptophane in acid hydrolysates with added tyrosine and phenylalanine. Phenylalanine was released by acid to an equal degree both in the presence and absence of TiClj . No increase in values, as established by the microbiological technique, were observed when increasing increments of TiClj were made available for the hydrolytic procedure. Both 10$ and 20$ acid solutions released the same amount of phenylalanine when casein was subjected to hydrolysis in their ' presence. There was also noted a normal recovery of added phenyl-alanine from digestions carried out with either concentrations of -102-acid. A more detailed study, at defined intervals, of the progressive liberation of tryptophane gave rise to several significant observations. It was observed when casein was hydrolysed with 20$ HCl. at 120* C. with no added TiCL^ that destruction of tryptophane was complete by 5 hours (Table 22). It is also apparent from the data recorded on this table that there was a gradual destruction of tryptophane even in the presence of TiClj . There appeared to be little advantage in using increased amounts of TiCl 5 during the acid hydrolysis of casein under these conditions. Since heat was believed to have an influence on the des-truction of tryptophane during these conditions of hydrolysis, investigations of digestion at lower temperature were undertaken. Studies of the progressive liberation of tryptophane at definite periods during the acid hydrolysis procedure carried out at o 110 C. revealed the maximum amount of this amino acid to be present at 2 hours (Table 23). A value of 1.00$ tryptophane was.observed when casein was hydrolysed in the presence of TiCl^ and 5 m L of 20$ HCl. After 2 hours a gradual hour by hour decrease of tryptophane occurred in the hydrolysates prepared both in the presence and absence of TiCl3 at 110° C. . Investigation of the degree of hydrolysis as indicated by the amount of amino nitrogen in these hydrolysates (Table 23) -103-revealed that TiCl3 did not accelerate digestion as was believed to be- the case by Sullivan and Hess (97). The opposite was found to be true; that is, hydrolysates prepared in- the absence of TiClg had more amino nitrogen present as detected by the formol titration, than did digestions made in the presence of this reducing substance. Further investigations carried out on acid hydrolysates prepared in the presence and absence of TiCl 3 using 20$ HCl. revealed that even lower temperatures of digestion were possible for the liberation of tryptophane from casein. Acid digests of casein prepared at 100° C. in the presence of TiCljj showed a gradual increase of tryptophane present from 0.75$ at 2 hours to 1.00$ at 5 hours. The values for tryptophane decreased slowly after 5 hours in the presence of TiCl^ to 0.5$ at 10 hours (Table 24). In the absence of TiCl3 acid hydrolysis at 100° C slowly destroyed the tryptophane present (Table 24). The value of 0.39$ at 2 hours drops to 0.15$ at 10 hours in the presence of 5 ml. of HCl. Study of the amino nitrogen content of this series of hydrolysates further verifies the fact that the liberation of amino acids is faster in the absence of TiCl3than in-its presence. The liberation of tryptophane at 95° C. (Table 25) and 90* C. (Table 26) in the presence of 1.33 ml. of TiClj using 20$ HCl. was not found to be as successful as with 110° C. and 100° C. The highest value found for tryptophane when the -io4-hydrolysis was conducted at 95° C in the presence of TiCl s was that of 0.87$ at 2 hours. At 90° C (Table 25) the (0.g9$) tryptophane content of the various hydrolysates prepared with TiCl-^, occurred at 6 hours. After this time there was a gradual decrease of tryptophane to 6.58$ at 10 hours. In both cases the hydrolysates prepared in the absence of TiCl showed a gradual and almost complete destruction of tryptophane as the digestion of casein proceeded. At these lover temperatures the destruction of tryptophane in the presence of 20^ HCl. was neither as rapid nor quite as complete as it was with higher temperatures, o At 95 C. the value for the 2 hour hydrolysate prepared with 5 ml. of 20$ HCl. in the presence of TiCl 5 was 0.27$ as com-pared to 0.75$ tryptophane found in the corresponding 2 hour, hydrolysate prepared in the absence of this reducing substance. The reason for the closeness of these two figures is readily understood when the amino nitrogen content of these two hydrolysates are compared. The amino nitrogen content of the hydroly-sate prepared under the conditions outlined above in the presence of TiClaj was 30.2 mgm. while the corresponding hydrolysate prepared in the absence of TiClj was found to contain H2.0 mgm. of amino nitrogen. This indicated the latter hydrolysate to be more completely broken down, and since the temperature was not high enough to cause complete immediate breakdown of tryptophane in the presence of 20$ HCl, this value (i.e. 0.75$) would be relatively high as compared -105-to the corresponding figure for tryptophane released under similar conditions in the presence of TiCl3. At 90° C the 2 hour hydrolysate prepared, using 5 ml. of 20$ HCl, in the absence of TiCL^ was higher than the corresponding hydrolysate prepared in the presence of this substance. Since more complete digestion of casein was attained in the absence of TiClj and since the temperature of hydrolysis (90° C) was sufficiently low not to permit immediate destruction of tryptophane this observed phenomenon appeared to be in good order. • Determination of tryptophane in subsequent hydrolysis prepared at 90° C. in the presence of 20$ HCl. and TiCl 3 revealed an increase from 0.75$ at 2 hours to a maximum of 0.89$ at 6 hours, followed by a gradual decrease in values to 0.58$ at 10 hours. Corresponding hydrolysates (90° C.) prepared in the absence of TiCLj decreased from the highest value (0.81$) found at 2 hours, to the lowest figure of 0.42$ found at 10 hours. Destruction of tryptophane liberated from casein hydrolysateB was found complete at 10 hours in the presence of 10 ml. of 10$ by volume, HCl. at 120° C, (Table 27) The addition of 1.33 ml. of titanous chloride appeared to be of more value as compared.to 1.66 ml. of the reducing substances in arresting the destruction of tryptophane during the early stages of acid hydrolysis under these conditions (Table 27) Investigations of the action of acid and alkali solutions on pure 1-tryptophane under normal conditions of hydrolysis -106-revealed a number of important facts (Table 28!). In every case where xylose was present with pure 1-tryptophane either with or without added TiCl^ the amino acid was completely destroyed when subjected to the conditions of acid hydrolysis. During acid hydrolysis with TiCl^ and in the absence of xylose, no destruction of tryptophane was observed, this amino acid being recovered quantitatively from a pure solution. It was also found that no racemization or destruction of tryptophane occurred when a pure solution of 1-tryptophane was . heated to 120° C for 10 hours in the presence of 5^  3SJa0H. The racemization of tryptophane then must require other radicles either present in or arising from the hydrolysis of casein. This would also suggest that racemization of tryptophane may occur prior to the hydrolytic cleavage when the protein is first exposed to alkali. Whether or not this is the case has not as yet been demonstrated. It can be stated from the foregoing discussion that the hydrolytic cleavage of casein by acid to yield tryptophane is an action influenced by many factors. When studying the release of tryptophane from casein it would appear that the greater part of this amino acid is split off in the early stages of hydrolysis only to be destroyed during pro-longed heating in the presence of HCl. In the presence of TiCl^ the loss of tryptophane during the early phase of the hydrolytic period is prevented in most cases. However, prolonged heating of acid hydrolysates, even in the presence of TiC]« caused partial loss -ic-7-of tryptophane. It is not known whether the gradual loss of this amino acid in hydrolysates on prolonged heating prepared in the presence of TiClj is caused "by destruction of the tryptophane or "by race-mization. Kacemization of an amino acid appears as destruction whan microbiological assay methods are used for quantitative measure hecause, with very few exceptions, the test organisms respond only to the 1 or naturally occuring form. It is generally stated that the amino acids exist in protein as the 1 or natural form, although it is probable that small amounts of the d forms are present in the unhydrolysed state. Thus any racemization of an amino acid observed as a product of hydrolysis is usually considered as being caused by the conditions of digestion unless the presence of the racemic mixture is definitely shown to be due to its liberation as such from the natural state (98). Amino acids in peptide linkages are claimed more readily racemized than free amino acid solutions, although the latter are maintained to be extenisvely racemized in the presence of hot alkali. Proteolytic enzymes in a medium that does not itself effect racemization have not been demonstrated to bring about this change. In this study, for the purposes of calculation, destruction was assumed, and not racemization under acid conditions of hydrolysis. Two acid concentrations were selected as providing -102-individual approaches to the problem of tryptophane liberation from casein by acid digestion. The more concentrated acid solution, 20$, by weight, HCl, caused a faster digestion of casein with rapid liberation of tryptophane while 10$, by volume, HCl. provided a more gentle release of this amino acid as the result of a slower hydrolytic reaction. Temperature was found to share inverse relationship with the period of hydrolysis. Data in this study further substantiated the work of Greenburg (23) and others (60) on the kinetics of hydrolytic action. Decreased temperature necessitated an increase in the length of time required to liberate amino nitrogen in general and in particular the amino acid tryptophane. However a prolonged heating period at most temperatures contributed to the destruction of this amino acid. In the presence of higher concentrations of acid (i.e. 20$ by weight HCl.) increased amounts of titanous chloride were found to be advantageous during the early stages of hydrolysis. -iog-SUMMARY. 1. Under normal conditions of acid hydrolysis the amino acid tryptophane is completely destroyed in the absence of a • reducing substance such as titanous chloride. 2. In the presence of titanous chloride in the digestion mixture permits the intact release of tryptophane during acid hydrolysis, the amount of this amino acid released being dependent upon the concentration of the acid solution, the temperature, and duration of the hydrolytic procedure. 3. Hydrochloric acid in concentrations of 10$ by volume and 20$ by weight provides two contrasting pathways of hydrolysis. More detailed studies on the hydrolytic action employing these acid solutions were carried out. 4. Lowering the temperature used during acid hydrolysis with 20$ HCl. to 110° C. resulted in the highest yield of tryptophane being obtained after two hours hydrolysis. Further exposure to acid at this temperature resulted in destruction of- tryptophane. At 100° C. the maximum release of tryptophane was observed at 5 hours. -110-5. In the presence of 20$ hydrochloric acid higher tryptophane values were observed during the early stages of hydrolysis when increased amounts of titanous chloride were added to the hydrolytic mixture. This would suggest the protective action of titanous chloride to'be one function-ing during the early stages of hydrolysis or during the initial release of tryptophane. 6. Titanous chloride was found almost completely ineffective in stopping the destruction of tryptophane both in pure solution and as liberated from casein when xylose was present. 7. The liberation of phenylalanine and tyrosine^ is not appreciably affected by the presence of titanous chloride during acid hydrolysis. g. Contrary to the postulate of Sullivan and Hess, the rate of hydrolysis was not found to increase in the presence of titanous chloride. The normal rate of hydrolysis is decreased in the presence of this reducing substance. 9. Pure solutions of 1-tryptophane were not racemized when treated with 5N NaOH. at 120° C. This finding is contrary to the accepted concept with respect to the racemizing action of alkali on amino acids resulting from the alkaline hydrolysis of proteins. PART III. THE APPLICATION OP THE MICROBIOLOGICAL METHOD TO THE DETERMINATION OP AMINO ACIDS IN A NATURAL PRODUCT. -112-EXFERIMEHTAL In order to study the applicability of the microbiological technique of Stokes, et al. (96) (Historical Part I) to natural pro-ducts, a study of the-amino acid content of a dried meat sample was undertaken. Dehydration of the meat sample to a constant dry weight, provided a sound basis for comparison as well as giving a uniform product for analysis. A rump roast, comparatively free from grissle, was cut into pieces of approximately one cubic centimetre and ground by means of a hand grinder. Care was taken to include all the juices while pieces of fat and sinew were removed. This ground sample was dehydrated in a vacuum dryer at 50° C. with a vacuum of 27"± 1. The resulting product was a leathery, slightly brown fibrous substance which was then ground again to a fine granular substance in a large mortar and dried to constant weight in vaccuo at 50° C. The sample prepared as outlined above was kept in a dark brown ground glass stoppered bottle in a desiccator. Acid hydrolysates of the powdered meat sample were examined microbiologically to establish the concentration of the nine amino acids, histidine, arginine, lysine, valine, methionine, leucine, isoleucine, phenylalanine, and threonine in this natural product. -113-i An alkaline hydrolysate was prepared for the microbio-logical determination of tryptophane and tyrosine. The alkaline hydrolysate was also assayed for the other amino acids mentioned above in order to establish the destruction of certain of these substances during this type of hydrolytic cleavage. The values comparing the eleven amino acids mentioned above as found in acid and alkaline hydrolysates of a powdered meat sample axe presented in Table 29 • TABLE 29. AMINO ACID CONTENT OF DRIED POWDERED MEAT. ACID HYDROLYSATE VALUE ALKALINE HYDROLYSATE VALUE Histidine 3.25$ 0.24$ Arginine '5.25$ nil. Lysine 6.06$ 2.63$ Valine 3.87$ 1.56$ Methionine 2.50$ 1.37$ Leucine 6.75$ 4.oo$ Isoleucine 4.06$ 1.13$ Phenylalanine 2.87$ 1.50$ Threonine 3.21$ nil Tyrosine 2.50$ Tryptophane — 1.09$ -114-CONCLUSIONS The figures for histidine, arginine, lysine, valine, methionine, leucine, isoleucine, phenylalanine and threonine, as determined microbiologically, are recorded in Table 29. The alkaline hydrolysate values for tryptophane and tyrosine, also given in Table 29, re-present the percentage of these-amino acids in 1 gm. of the constant dry weight of the prepared meat sample. Microbiological determination of the first mentioned group of nine amino acids in the alkaline hydrolysate revealed extensive loss to complete destruction of these amino acids. Histidine, arginine and threonine were found completely destroyed while lysine, isoleucine and phenylalanine were found almost completely lost when released under normal conditions of alkaline hydrolysis. The remaining amino a,cids, namely valine, leucine and methionine showed serious destruction as a result of alkaline hydrolysis although their loss was not as extensive as that exhibited in the case of the others (Table 29). In order to determine the validity of the results obtained in a nptural product, such as meat, a study of the recovery of amino acids added to the dried meat sample prior to hydrolysis was • carried out. The plan of experimentation as recorded in Table 30 was followed. Since the hydrolysates with added amino acids were prepared at the same time as the ordinary digests for the determination of -115-these substances and as no reliable information pertaining to the amino acid content of such a hydrolysis, under alkaline conditions. TABLE 30. RECOVERY OF AMINO ACIDS ADDED TO DRIED MEAT  PRIOR TO HYDROLYSIS IN MGM. PER. GM. OF SAMPLE. AMINO ACID CONTENT in mgm. ADDED^  in mgm. TOTAL in mgm. FOUND in mgm. PERCENTAGE RECOVERY Histidine 32.5 7.5 40.0 34.0 85$ Arginine 52.5 1.76 5^.3 60.0 110$ Lysine 60.6 7.6 68.2 69.3 101$ Valine 3S.7 2.0 40.7 39.3 96$ Methionine 25.0 . 2.47 27.5 27.5 100$ . Leucine 67.5 6.6 i K l 75.2 101$ Isoleucine 4o.6 6.6 47.2 45.0 95$ Phenylalanine 28.7 1.6 30.3 32.5 106$ Threonine .32.1 2.0 3^.1 3^.0 100$ Tyrosine • 25.0 2.2 27.2 30.0 110$ Tryptophane 10.9 1.25 dl. 12.1 12.0 160$ Alkaline hydrolysis.*" In terms of 1-isomer-Jfjr CONCLUSIONS Extremely good recovery of added amino acids was observed in the case of lysine, valine, methionine, leucine, isoleucine, -116-phenylalanine, threonine and tryptophane. Fairly good recovery of added arginine and tyrosine was also noted (Table 30) Histidine, however, was found somewhat difficult to recover when subjected to hydrolytic conditions in the presence of the dehydrated but otherwise intact meat sample. This observed value along with the results outlined under Experimental, Part I, would suggest this amino acid to be susceptible to slight des-truction during the normal conditions of acid hydrolysis of natural products or during digestion of purified protein in the presence of added carbohydrate material. The results as .a whole show'that the microbiological method applied to the determination of amino acids in a natural product, such as meat, give valid results. In attempting to conserve costly and difficultly available amino acids, investigations of the degree of accuracy in using smaller amounts of basal medium were carried out. Microbiological determinations of acid and alkaline hydrolysates of the dehydrated meat sample, described previously as prepared without added amino acids, were carried out using 2 ml. and 3 ml. quantities of basal medium respectively. The assay values using 2 ml. and 3 ml. quantities of basal medium were obtained by calculation from similarly prepared standard curves using these amounts of basal medium. Results obtained using the normal amount of basal medium, that of 5 ml.., -ii7-(Table 29), provided the basis of.comparison for values found using 2 ml. and 3 ml. quantities. (Table 31) TABLE 31. A COMPARISON OF 5 ML., 3 ML., AND 2 ML.,  QUANTITIES OF BASAL MEDIUM FOR THE SEsH OF AMINO ACIDS IN DRIED MEAT. AMINO ACID VALUE FOUND USING 5 ML. BASAL. VALUE FOUND USING 3 ML. BASAL. VALUE FOUND USING 2 ML. BASAL. Histidine 3.25$ 2.87$ 2.60$ Arginine 5.25$ M5$ 5.25$ Lysine 6.06$ 5.68$ 5*12$ Valine , 3.87$ 3.69$ 3.69$ Methionine 2.50$ 2.56$ 2.25$ Leucine 6.75$ 7.20$ 7.20$ Isoleucine U.o6$ 3.13$ 3.13$ Phenylalanine 2.87$ ' 2.87$ 2.93$ Threonine 3.21$ 3.18$ 3.16$ Tyrosine 2.50$ 2.50$ 2.12$ . Tryptophane 1.10$ 1.10$ 1.10$ CONCLUSIONS When 3 ml. of basal medium was used, the amino acids methionine, phenylalanine, threonine, tyrosine and tryptophane were determined with reasonable accuracy. The remaining amino acids were not detected with sufficient accuracy to deem adviseable the -112-use of 3 ml. basal medium in place of 5 ml. volumes. Microbiological determinations of amino acids carried out with 2 ml. of basal medium were found sufficiently accurate in the case of arginine, phenylalanine, threonine and tryptophane. The determination of the remaining amino acids using 2 ml. basal medium did not appear accurate enough for microbiological assay work. -119-SUMMABY 1. The amino acid content and recovery of a'dded amino acids from a dehydrated hut otherv/ise intact meat sample has been studied. 2. Low recovery values obtained for histidine suggest partial destruction of this amino acid during the acid hydrolysis of this natural product. 3. Study of the effect of alkali as a hydrolytic agent for the liberation of amino acids revealed extensive to complete destruction of the nine amino acids investi-gated. 4. The use of 2 ml. and 3 portions of basal medium in place of the conventional 5 Quantities re-vealed irregular results with certain of the eleven amino acids studied. 10 0 10 20 30 4o 50 MICROGRAMS OP HISTIDINE FIGURE 1 REFERENCE CURVES FOR HISTIDINE ESTABLISHED USING 5 ML., 3 ML., and 2 ML. QUANTITIES OF BASAL MEDIUM. « * « I 0 20 Uo 60 SO 100 MICROGRAMS OP ARGININE FIGURE 2 REFERENCE CURVES FOR ARGININE ESTABLISHED USING 5 ML. , 3 ML. , AND 2 ML. QUANTITIES OF BASAL MEDIUM 10 o Uo so 120 160 • 200 MICROGRAMS OF LYSINE FIGURE 3 REFERENCE CURVES FOR LYSINE ESTABLISHED USING 5 HL., 3 KL. . AND 2 ML. QUANTITIES OF BASAL MEDIUM 11 0 -I ; 1 1 = 1 1 1 0 - 20 UO 60 . SO 100 MICROGRAMS OF VALINE^ . FIGURE k REFERENCE CURVES FOR VALINE ESTABLISHED USING 5 ML., 3ML., AND 2 ML. QUANTITIES OF BASAL MEDIUM 11 0 20 40 60 SO 100 MICROGRAMS OF METHIONINE FIGURE 5 REFERENCE CURVES FOR METHIONINE ESTABLISHED USING 5 ML., 3 ML., AND 2 ML., QUANTITIES OF BASAL MEDIUM 10 0 !• 1 1_ 1 i 0 20 40 60 SO 100 MICROGRAMS Oj? LEUCINE FIGURE 6 REFERENCE CURVES FOR LEUCINE ESTABLISHED USING 5 ML., 3 HL.. AND 2 ML. QUANTITIES OF BASAL MEDIUM MICROGRAMS OP ISOLEUCINE FIGURE 7. REFERENCE CURVES FOR ISOLEUCINE ESTABLISHED USING 5 ML., 3 ML., AND 2 ML. QUANTITIES OF BASAL MEDIUM 11 10 -0 I i i 1 I 0 20 U0 60 ' 80 100 MICROGRAMS OF PHENYLALANINE FIGURE 8 REFERENCE CURVES FOR PHENYLALANINE ESTABLISHED USING 5 ML., 3 ML., AND 2 ML. QUANTITIES OF BASAL MEDIUM 11 o 20 ho 6o go IOO • MICROGRAMS OF THREONINE FIGURE 9 REFERENCE CURVES FOR THREONINE ESTABLISHED USING 3 ML., 3 ML., AND 2 ML. QUANTITIES OF BASAL MEDIUM I • • . 0 20 40 60 SO 100 MICROGRAMS OP TYROS1KB FIGURE 10 REFERENCE CURVES FOR TYROSINE ESTABLISHED USING 5 Ml., 3 ML., AND 2 ML. QUANTITIES . OF BASAL MEDIUM 11 10 . 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ACKNOWLEDGMENT I wish to express my deepest thanks and sincere gratitude to Dr. B.A. Eagles whose kind-ness and unselfish guidance has been a source of inspiration and encouragement throughout the course of this study. It is with gratitude also that I thank Dr. J.J.E. Campbell and Miss Nora Neilson for their helpful advice and technical assistance. To Swift Canadian Company, limited, I am much indebted for the Eellowship which it was my privilege to enjoy during the carrying out of this work. 

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