u AV .53 . I u 2-H. mum.“ M» '13 n m a m.- .. \I....m— [NJ-I'm S “‘03:— J “6‘ 0‘0. ...4.<. . “w. .9» V“ .3 .,. {.2 m ,IN. . Wk“ WW“ (I...- kmm .... . . Y. "Aw .C o . “‘3...“ Mod. . . C a... ‘ k 3 n .s M” at . a. . .. r. f. t . a.-. n. .. pr at In”!- Ifr e .,3:gig...#52:::_é:__:_;,_:: H " LIBR A R Y Michigan State Univesity _ PLEM? NARY SUflAix EMU“ L IN BACK OF BOOK THE AMINO ACIDS OF IRAQI DATES By Nawal .AléRawi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1965 ACKNOWLEDGMENTS I wish to express my sincere thanks to Dr. Pericles Markakis for his valuable guidance and helpful suggestions throughout this research. .Also, my thanks are extended to Dr. Clifford Bedford and Dr. Olaf Mickelson for their advice and help. I also wish to express my deep gratitude to Miss Doris Bauer for her help in the analysis. Special thanks are due to the Iraqi Date Organization for providing the samples of dates and assisting with a financial grant. This thesis, I would like to dedicate to my mother and my husband for their moral support in my effort to advance my education. ii WWW w Hi at ii H mm om , Page MODWT ION O O O O O O O O O O O O O O O O O O 0 O O O O O l REVEW OF WTIM O 0 O O O O O O O O O O O O I O O O 0 0 Dates (Phoenix dectylifera) . . . . . . . . . . . . . Browning and Darkening of Dates . . . . . . . . . . . Classification of Amino.Acids . . . . . . . . . Qualitative and Quantitative Determination of Amino Acids . . . . . . . . . . . . . . . . . . . 1- Chemical methods . . . . . . . . . . . . . . . 2- Physicochemical methods . . . . . . . . . . . a- Titrimetry b- Isotope dilution c- Spectrophotometry d- Optical rotation 3- Microbiological assay methods . . . . . . . . 4- Biochemical methods . . . . . . . . . . . . . 5- Chromatographic methods . . . . . . . . . . . a- Paper chromatography b- Column chromatography .Amino Acids of Dates . . . . . . . . . . . . . . . . . \nmm www 00 O\O\O\ mus A-ND WTHODS O O O O O O O O O O O O 0 O O O O O 0 Moisture Determination . . . Total Nitrogen Determination Amino Acid Determination . . 1- Paper chromatography 2- Column chromatography . . . a- Moore and Stein l95h-Method b- Beckman/Spinco Amino Acid Analyzer Method 1- Preparation of the sample for the free amino acid determination 2- Preparation of the sample for the total amino acid determination O O O O O O O O O O O O O O O O O O O O O O O O O t:\O\O\O\O \O 4 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . l6 1- Moisture and Nitrogen Content of Dates . . . . . . l6 2" Amino ACidS Determination o o o o o o o o o o o o 0 16 a- P aper Chromatogapm e o o o o o o o o o o o o 16 b - COlum Chromat 0 g aphy o o o o o o o o e o o 0 16 SW 0 o o o o o o o o o o o o o o o o o o o o o o o o o 30 REFERENE S O O O O O O O O O O O O O O O O O O O O O O O O O 31 iii LIST OF TABLES Table Page I Moisture and Nitrogen Contents of Dates. . . . . . . . . 16 II Free Amino Acids Content of Dates, Var. Hallawi. . . . . 20 III Free Amino Acids Content of Dates, Var. Khadhrawi. . . . 21 IV Free Amino Acids Content of Dates, Var. Saiyir . . . . . 22 <; Total Amino Acids Content of Dates, Var. Hallawi . . . . 23 VI Total Amino Acids Content of Dates, Var. Khadrawi. . . . 2’4 VII Total Amino Acids Content of Dates, Var. Saiyir. . . . . 25 VIII Porportions of Essential Amino Acids in Dietary Allowances and in Dates (Threonine = 1.0) . . . . . . 27 IX Amino Acids of Dates Reported by Various Investi- gatorSoe00000000000000.0000... 28 iv LIST OF FIGURE S Figure Page 1 Paper Chromatogram of the Free Amino Acids of Saiyir Var. Dates. Whatman No. l. . . . . . . l7 2 Free Amino Acids of Saiyir Var. Dates as Separated by the Moore and Stein (1951+) MethOd-o . O O O O O . O . . O 0 . . . O 0 O O O 0 18 INTRODUCTION Dates have been cultivated since antiquity. The earliest written record pertaining to dates goes back to 3500 BC. The oldest date grow- ing area was ancient Mesopotamia, which is present day Iraq (19). To- day dates are one of the principal foods in some parts of the Middle East. Iraqi dates have been reported to contain 1.9 — 3.0% proteins, 0.0 - 2.5% fats, 67.3 - 70.6% carbohydrates, 1.3% minerals, 13.8 - 26.1% moisture, and small quantities of vitamins B1, and B2, ascorbic acid, and nicotinic acid (8). The free amino acids content of dates was first determined by Grobbelaar at al. in 1955. They identified 17 free amino acids. Rinderknecht in 1959 using Grobbelaar's technique in preparing the date extracts found that the free amino acids content of dates varies ‘with the stage of maturity of the fruit (green, yellow, red, and brown), rising to a maximum in the red stage and decreasing during the brown stage. He identified 12 amino acids. Rinderknecht did not find threo- nine, lysine, valine,,B-a1anine, and homoserine which had been reported by Grobbelaar g§_al, Also Rinderknecht found arginine in high concen- tration whereas Grobbelaar found only trace amounts of this amino acid. Only one sample of dates has been analyzed for total amino acids and this analysis includes only ten amino acids (20). The scarcity and discrepancy of the available data on the amino acid composition of dates and the fact that dates comprise a considerable part of the human diet in some areas of the world led to the present study. Iraqi dates were chosen for this study because Iraq is a very 1 important date producing country. It produces about h00,000 tons of dates each year, representing 50% of the total world production. Of this production about 32% is exported. REVIEW OF LITERATURE Dates (Phoenix dectylifera) There are about 200 varieties of dates growing in Iraq, and they differ in shape, color, taste, and time of ripening. The most important varieties are: Hallawi, Khadhrawi, Saiyir, Dairi, and Zahidi. The development of dates may be divided into three steps: l- The fruit stem develops rapidly with very little enlargement of the fruit. 2- The green fruit comes to full size, but no accumulation of sugars occurs during this stage. 3- Sugars accumxlate rapidly, the fruit increases in weight, and the skin of the fruit takes on a translucent appearance. The most important sugars in dates are: sucrose, glucose, and fructose (2, 26). Browning and Darkening of Dates Ripe and preserved fruit exhibit a brown to dark-brown color. Vinson in 1921; attributed this color to the tannins lying beneath the skin of the fruit. Rinderknecht in 1959 (22) found that the tannins located in the epiderm and the first layer of the hypoderm might be oxidized by a polyphenolase during ripening and yield an insoluble dark product. However, continued darkening after thermal destruction of this enzyme can be observed too. Recently, Maier in 1963 (11) isolated from California dates a cafi'eoylchilu'mic acid which he considered to be a sub‘trate of the browning enzymes. Classification of amino acids There are 22 amino acids that occur frequently in protein hy- drolyzates. Meister in 1957 (12) classified the twenty most common anfino acids as follows: 1- Aliphatic: Monoaminomonoca rboxylic : Glycine, alanine, isoleucine, leucine, and valine. Hydroxymonoaminomonocarboxylic : Serine, and threonine. Monoaminodicarboxylic (amides): Asparagine, and glutamine. Diaminomonocarboxylic : Arginine, and lysine. Sulfur containing: Cystflene, cystine, and methionine. 2- Aromatic: Phenylalanine, and tyrosine. 3- Heterocyclic: Tryptophan, histidine, proline, and hydroxproline. Of these 20 amino acids, eight are essential for adult humans. Those are: lysine, valine, threonine, leucine, isoleucine, tryptophan, phenylalanine, and methionine. The absence of any of these amino acids Will result in malnutrition. The symptoms of amino acids deficiency are: deficient muscle development and retarded growth rates in children, low resistance to tuberculosis and rheumatic fever in some population groups, anemia among women of childbearing age and children, toxemias, pre- mature labors among pregnant women, and low appetite (3, 9). Qualitative and Quantitative Determination of Amino Acids Greenstein at 93:. (6) described a number of methods which can be used for quantitative and qualitative determination of °<-amino acids. These methods are: 1- Chemical methods. Mixtures of amino acids are separated chemically and deter- mined either gravimetrically or colorimetrically by treatment with a. reagent which forms a colored solution with the amino acid. The most popular colorimetric method is the ninhydrin method, which involves the reaction between «ac-amino acids and triketohydrindene hydrate (ninhydrin). Amino acids form a violet color in this reaction, except proline and hydroxproline which give a yellow-brown color with ninhydrin. 2- Physicochemical methods. a- Ttitrimetry. This method involves the determination of the amino acid concentration in aqueous solution by titration with strong acids or alkalies. b- Isotope dilution. The principle of the method is the following. An amino acid containing a heavy or radioactive isotope is added to a mixture of amino acids. The labeled amino acid is isolated from the mixture and the label determined quantitatively. From the ratio of added to re- covered label, the concentration of the amino acid is calculated. c- Spectrophotometry. The ultraviolet and infrared spectra are also used in amino acid determination. d- Optical rotation has been used in amino acid determination. 3- Microbiological assay methods. These methods are based on the fact that the growth of certain microorganisms is a function of the concentration of specific amino acids. A- Biochemical methods. Amino acids can be released by enzymatic action and subsequently determined by one of the previously described methods. 5- Chromatographic methods. a- Paper chro matography. Martin, Consden, and Gordon in l9fl4 employed two dimentional paper chromatography for the separation and determination of amino acids. For the quantitative analysis of the amino acids by paper chromatography, color densitometry can be used, or the amino acids can be eluted and de- termined:éolorimetry. b- Column chromatography. Starch was used in columns by Stein and Moore in 1948, but it was later replaced by ion exchange resins. Ibwex 50 is used for the separation of both neutral and acidic amino acids. The elution of the amino acids depends on the presence of cations capable of competing with the cationic form of the amino acids. Automatic amino acid analyzers were introduced in 1958 and exten- sively used since. The retention on and recovery from an anion exchange column of an amino acid can be illustrated as follows: R.NH2 + HOOC.CH2.CH2.CH.NH3.COO' ------ R.NH; ('OOC.CH2.CH2.CH.NR§.COO') R.NH; (‘ooc. CH2.CHZ.CH NigtOO‘) + HCI ...... ENE-$.01" + H000. CH2. CHZ.CH NH3.COO‘ Amino acids of dates The amino acids content of dates has not been extensively studied. Grobbelaar g§_gl, in 1955 (7) attempted a complete analysis of the free amino acids of dates. Using paper chromatography, they identified the following amino acids: 8-amino butyric acid, fl-alanine, alanine, arginine, aspartic acid, biakiain, glycine, glutamic acid, homoserine, 5- hydroxy- pipecolic acid, isoleucine, leucine, lysine, pipecolic acid, serine, threonine, and valine. later Rinderknecht in 1959 (22) also using paper chromatography reported the presence of the following free amino acids in dates: - amino butyric acid, alanine, arginine, aspartic acid, biakiain, citrulline, cystine, glycine, glutamic acid, 5-hydroxypipecolic acid, proline, and serine. He also indicated that there is a sharp rise in the glutamine, and arginine content during the red stage and a decline during the brown stage of the development of dates. Piez gp_gl, in 1956 (21) using both paper chromatography and ion exchange resin,.hn determined the following amino acids as mg./l g. dates 1.8 5-hydroxypipecolic acid, 0.13 hydroxy proline, 0.7 proline, and 0.005 pipecolic acid. Earlier, 1954, Garther and Dobzhanky (5) reported the presence an ofAunknown ninhydrin-positive substance in the urine of people who consumed dates. This substance seemed to be excreted quantitatively. It was suggested by Grobbelaar in 1955 that this unknown product could be hydroxypiperidine carboxylic acid. MATERIALS.AND METHODS Four 1-pound boxes of semidry dates of each of the 3 most im- portant commercial varieties in Iraq were used in this investigation. These varieties were: Hallawi, Saiyir, and Khadhrawi. Moisture Determination One 100 g. sample was randomly selected from 2 pounds of boxed pitted dates. The sample was ground in a meat grinder and 3 g. of the ground material were used for the determination of the moisture of each variety. Trying was done in a vacuum oven at 70°C to constant weight (13). Total Nitrogen Determination Five grams of ground sample prepared as described above were used for total nitrogen analysis by means of the Kjeldahl-Cunning-Arnold method. Triplicate determinations were made for each variety (13). Amino Acid Ibtermination 1- Paper Chromatography Paper chromatography was used only for the identification of the free amino acids of dates. Fifty grams of dates were soaked for 1 hour in 100 m1. of 70% ethanol. They were then homOgenized for 5 minutes in a Waring Blendor. The slurry was filtered and the filter cake was washed with approximately 10 ml. of 70% ethanol. The filtrates 'were passed through a column of Dowex 50KB, l.h cm. in internal diameter and 40 cm. in height, and washed with 50 ml. of water followed by 50 m1. of 1N ammonium hydroxide solution for the elution of the amino acids. The eluate was concentrated in a flash evaporator 9 10 to a volume of 5 ml. Ten drops of the extract were applied to a spot in the corner of Whatman #1, (22 x 18 inch sheet) paper h inches from each edge; one drop was applied at a time and dried with a jet of warm air. The paper was irrigated descendingly overnight with butanol/ acetic acid/water (h/l/5 volume/volume) in the long direction, then dried in a forced air oven at hOOC for 2 hours and trimed one inch from both top and bottom. Then it was sprayed with borate buffer pH 8.3 (300 m1. of 0.1 M boric acid plus 60 ml. of 0.1 N NaOH) after covering the area run by the sample with aluminum.paper. .After spraying, the paper was dried at #000 for half an hour. The second solvent was made by mixing 30 g. of mecresol, 15 g. phenol, and 7.5 m1. of borate buffer pH 8.3; this was the mobile phase. The aqueous phase was made by mixing 250 ml. of the borate buffer of pH 8.3 with 8 ml. of the mobile phase and was placed in a tray at the top and bottom.of the chromatographic cabinet. The paper was placed in the cabinet for irrigation in the short direction. It was allowed to equilibrate with the aqueous phase for h hours and then irrigated with the second solvent the same way as with the first solvent. The paper was then dried at #000 for 2 hours and sprayed with a mixture of 15 ml. glacial acetic acid, 50 m1. of 0.1% ninhydrin in ethyl alcohol, and 2 ml. of collidine. It was then placed for 5 minutes in a 100°C oven for color development (10). The amino acids were identified by applying known amino acids on a similar sheet of paper and chromatogramed under the same conditions. Only the Saiyir variety was used for the paper chromatography analysis. Several chromatograms were prepared for this variety. 2- Column Chromatograpl’w A- Moore and Stein 1951: Method. Four hundred and fifty grams of Dowex 50x8, 2OOJ+00 mesh was washed with 2-3 liters of ltN HCl, rinsed with distilled water and then washed with 2N NaOH (about #00 m1.) until the filtrate was alkaline. The resin was then suspended in 500 ml. of 1N NaOH and heated over a steam bath for 3 hours. The heated mixture was allowed to stand for 1 hour, then the supernatant liquid was decanted and replaced with hot 1N NaOH. This pro- cedure was repeated 5 times. The resin was then thoroughly washed with distilled water (about 5 liters), filtered and kept as the moist sodium salt. One hundred ml. of the resin was transferred to a Buchner funnel and was washed first with 1 liter of 1N NaOH, followed by 1 liter of 0.2N sodium citrate-acetate buffer, pH 5.0. The resin was mixed with 100 m1. of the same buffer. This suspension was used to prepare a water jacketed column of 150 cm. in height, 1 cm. in internal diameter. Hundred ml. of 0.2N NaOH containing BRIJ 35* solution was passed through the column overnight at 15-20 inches water pressure. Then 200 ml. of 0.2N sodium citrate buffer, pH 3.1 was passed through the column before being used. The buffers were prepared as described by Moore and Stein, 1951+ (7). Ten ml. of the sample was prepared as for paper chromatog'aphy and was adjusted to pH 2.5 with 5N HCl. It was applied to the * BRIJ 35 is an ether of polyethylene glycol and acts as a detergent (15). A B- 12 column and washed down with 3 portions of 0.3 ml. aliquots of citrate buffer pH 2.2. The column was mounted over a fraction collector, the water in the column jacket was thermostatically adjusted to 30°C and the 0.2N sodium citrate buffer, pH 3.1 was forced into the column by applying air pressure of 1+0 inches of water. After 90 ml. of this buffer had passed through, the temperature around the column was raised to 50°C, and after 210 ml. of the buffer had passed through, its pH was gradually increased by mixing it with 0.2N citrate buffer, pH 5.1 (17). Two ml. fractions were obtained from the column. To each fraction 1 ml. of the ninhydrin reagent (prepared by dissolving 20 g. of ninhydrin and 3 g. of hydrindantin in 750 ml. of methyl cellusolve, and made to 1 liter volume by AN Na acetate buffer pH 5.5) was added. The tubes were shaken well and heated for 15 minutes in a boiling bath, then diluted with 50/50 ethanol/ water. The color was determined spectrophotometrically at 57011.: (id-L0 for proline and hydroxyproline) after cooling the tubes to room temperature. The blank reading was usually chosen from tubes before or after the peak (16). The amino acid identification was accomplished by comparison with known amino acids separated under identical conditions as the unknowns. Beckman/Spinco Amino Acid Analyzer Method This method is principally the same as the Moore and Stein method. 13 Many steps of the analysis are performed automatically. The analyzer is provided with three columns, the temperature of which is regulated automatically, a gradient elution system, a ninhydrin reaction system with the effluent, three color measuring devices, and an automatic recorder. From a 100 3. sample representing 2 pounds of boxed dates, two A g. samples were used for the free amino acids analysis and two 0.# g. samples were analyzed for the total amino acids for each of the three varieties (25). 1.. Preparation of the sample for the free amino acid deter- mination: The #.0 g. sample was mixed with 40 m1. of 1% picric acid in all glass tissue grinder and the macerate was centri- fuged in a clinical centrifuge at full speed. Twenty- five ml. of the supernatant was passed through a Dowex 2X10 column, 2.0 cm. in height and 2.5 cm. in internal diameter. The resin bed and the walls of the chromotogram tube were washed with five 3.0 m1. portions of 0.2N HCI, and the effluent was concentrated to a volume of about 1 ml. in a flash evaporator and transferred into a test tube with water. The solution was adjusted to pH 7.2 - 7.5 with 1N NaOH. Two tenths of l.ml. of freshly prepared 0.5N solu- tion of sodium sulfate was added to the concentrate and allowed to stand in the air for h hours at room tempera- ture; this resulted in the auto oxidation of the glutathione 14 which would interfere with the separation of aspartic acid (18). The sample was then adjusted to pH 2.2 - 2.3 with NaOH solution and subjected to the spectro-photometric analysis. Preparation of the sample for the total amino acid determination: For the total amino acid analysis the proteins and peptides of dates were hydrolyzed according to the Spackman method (25) and the Bandemer and Evans method (1). In the Spackman method #00 mg. of ground dates were placed in a pyrex tube, 1 m1. of conc. ROI and 1 m1. of water were added, the mixture was frozen in a dry-ice-alcohol bath, the tube evacuated by means of high vacuum pump and the neck of the tube sealed under vacuum. The tube was then placed for 22 hrs. in an air circulating oven adjusted at 110°C for digestion. After digestion the top of the tube was removed and the hydrolyzate was filtered. Both the tube and filter paper were then washed with water, and the hydro- chloric acid was evaporated from the sample in a water bath almost to dryness. The dried film of the hydrolyzate was removed by 0.2N Na citrate buffer, pH 2.2 and made to 10 m1. volume. In the Bandemer and Evans method the 400 mg. sample was also mixed with 1 ml. HCI and 1 ml. water, but the tube was not sealed; instead, it was placed in an autoclave for 15 6 hours at 15 pounds pressure. After digestion, the sample was filtered and was made up to 10 ml. volume by adding the citrate buffer, pH 2.2. Determination of the amino acids by the automatic analyzer: Two or three ml. of the prepared sample were applied on the two columns of the analyzer; one for the basic amino acids and one for the neutral and acidic amino acids. The basic amino acid column was 15 cm. in height and 1 cm. in internal diameter, and was operated at 50°C. The elution of the basic amino acids was achieved with 0.35N Na citrate buffer, pH 5.28. The neutral and acidic amino acids column was 150 cm. in height and 1 cm. in internal diameter, and was also operated at 50°C. The elution of the neutral and acidic amino acids started with 0.2N Na citrate buffer, pH 3.25, and was continued with 0.2N Na citrate buffer of pH n.25. Thiodiglycol was added to the buffers to help in minimizing the conversion of small amounts of methionine to methionine sulfoxide during the addition of the sample to the column and during the time that the sample was standing on the long column while the basic amino acids analysis was being carried out on the short column. The use of the detergent with the eluting buffer has been found to permit faster flow rates without concominant broading of the peaks on the effluent curves. The complete separation in the analyzer took 22 hours. For the quantitative determination, measured quantities of all the amino acids known to be present in dates were run through the analyzer and a mathematical formula was prepared for the calculation of the unknowns. RESULTS.AND DISCUSSION 1- Mbisture and Nitrogen Content of Dates The average moisture and nitrogen contents of the three varieties of dates analyzed are shown in Table 1. Table 1. Moisture and Nitrogen Contents of'Dates. Variety .Average moisture % .Average nitrogen.% (wet basis) Hallawi 17.1 0.286 Khadhrawi 13.h 0.260 Saiyir 19.6 0.275 2- .Amino Acids Determination a- Paper chromatography. The free amino acids identified by paper chromatography are: aspartic acid, glutamic acid, glycine, valine, leucine, and proline. The location of these amino acids on the paper chromato- graphy is shown in Figure l. b- Column chromatography. By means of the Stein and Moore method, thirteen peaks were ob- tained on the chromatogram of the free amino acids of the Saiyir variety dates (Figure 2). or these peaks the following were identified: alanine, aspartic acid, glutamic acid, glycine, l6 Solvent 2. l7 Solvent l ) I . I gAspartic ()rlutaminc C) Glycine 9 Valine QLeuCinc .r’roline Figure 1. Paper chromatogram of the free amino acids of Saiyir var. dates. Whatman No. l. Solvent 1: Butanol-acctic acid-water (h:l:5) ”t 2: m-crcsol-phcnol (2:1), pH 8.3 borate buffer. AbSorption, 570 can) ;=__. 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The two methods which were used here for the protein hydrolysis, m ,am; 1" results. the autoclave method and the sealed tube methodA Since the autoclave method is the easiest of the two, it should be prefered for this analysis. Regarding the results of the analysis, it was found that the difference in'water content among the 3 varieties studied was greater than the difference in nitrogen content. All the essential amino acids were present in dates, except tryptophan which escaped the analysis. The proportions of these amino acids in comparison to the dietary proportions recommended by Rose (23) for the adult man appears in.Table VIII. 27 Table VIII. Proportions of’Essentia1.Amino.Acids in Dietary Allowances and in Dates (Threonine = 1.0). .Amdno.Acid Allowance Hallawi Sa2:::S Khadhrawi Isoleucine 1.h 0.8 0.9 0.8 Leucine 2.2 1.6 1.7 1.6 Lysine 1.6 1.0 1.1 1.1 Phenylalanine 2.2 1.1 0.9 0.9 Methionine 2.2 O.h 0.3 0.2 Threonine 1.0 1.0 1.0 1.0 Valine 1.6 1.2 1.2 1.2 From this comparison, and disregarding tryptophan, the limiting amino acid of dates appears to be methionine. 0f the three varieties analyzed, variety Hallawi contains the highest proportion of methionine. Table IX indicates the amino acid composition of dates as reported by previous investigators (7, 22, 23) and in this work. Rinderknecht reported 12 free amino acids, Grobbelaar gt.a1, 17 free amino acids, "The.Amino.Acid Content of Foods," 10 amino acids are reported as present. In this work 16 amino acids have been identified in both the free and bound state. Some small peaks in our flowing chromatograms were not identified. The differences between our results and those of the pre- vious investigators may be due to stage of maturity, variety, origin of the 28 Table IX. .Amino Acids of Dates Reported by Various Investigators. Free A. A. Total A. A. This This Ref. 22 Ref. 7 work Ref. 23 work K-amino butyric acid + + - - - b-alanine _ + _ _ _ Alanine + + + _ + Arginine + + Aspartic acid + _ Biakiain + + _ _ _ Citrulline + - - - - Cystine + - - - - Glycine + + + _ + Glutamic acid + + + _ + Histidine - - + + + Homoserine - + - - - 5-hydroxypipecolic acid + + - - - Isoleucine - + + + + Leucine - + '+ + + Lysine - + + + + Methionine - - + + + Phenylalanine - - + + + Pipecolic acid - + - - - Proline + - + _ + Serine + + _ + Threonine - + + + Tryptophan - — - + - Tyrosine - - + - + Valine - + + + + 29 dates used, and analytical methods. However, no efforts were made here for the identification of biakiain, citrulline, and pipecolic acid. .According to the data obtained, the total quantity of the free amino acids is about l/6 of that of the bound amino acids in all three date varieties analyzed. One amino acid, namely, phenylalanine, is 'present in higher concentration in the extract than the hydrolyzate. Since phenylalanine is not destroyed during acid hydrolysis, the paradox can be explained by the presence of a ninydrin-positive comp pound which is present in the extract and emerges from.the column with phenylalanine, but which is destroyed during the hydrolysis. Two com- pounds have been reported to have effluent volumes similar to that of phenylalanine: the dipeptide, leucyl-glycine and o-tyrosine. If the dipeptide is present, hydrolysis of the extract will reduce the phenyla- lanine peak and increase the leucine and glycine peaks. Such a hydrolysis has not been tried. Taurine has also been identified in the three varieties, but it was impossible to determine its quantity due to the appearance of an unidentified compound which emerged from.the column at the same time as taurine. SUMMARY Dates of three varieties (Hallawi, Saiyir, and Khadhrawi) produced and processed in Iraq were analyzed for moisture, total nitrogen, free amino acids and total amino acids. Although paper chromatography was used for the identification of some of the free amino acids of the dates, the overall analysis was essentially based on gradient elution column chromatography performed by means of an automatic amino acid analyzer. The following amino acids were present both in the free state and in the acid hydrolyzate of dates: alanine, arginine, aspar- tic acid, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. Tryptophan and cystine require special analytical methods, and they were not determined here. The ratio of free to bound amino acids differed significantly be- tween individual amino acids. However, for all amino acids col- lectively, this ratio was approximately 1 to 6. All the essential amino acids, except tryptophan which escaped this analysis, were found to be present in dates. The limiting amino acid, from the nutritional viewpoint, appears to be methionine. 30 12- 13- 11+- 15- 16- REFERENCES Bandemer, S. L., and Evans, A. J. The amino acid composition of some seeds. Jour. Agri. and Food Chem. _1_1_, 13,4, 1963. Dowson, V. H. W. Dates of Mesopotamia. Third Annual Date Growers' Inst. 1926. Floodin, N. W. Amino acids and proteins, their place in human nutrition problems. Jour. Agi. Food Chem. 1;, 222, 1953. Fox, S. W., and Foster, J. F. Introduction to protein chemistry. 1957. Gartler, S. , and Dobzhanslw, T. H. Excretion in human urine of an unknown amino acid derived from dates. Nature. .1111, 553, 1951+. Greenstein, P. J. , and Milton, W. General analytical procedures for amino acids. Chem. of Amino Acids. 1, 2MB, 1961. Grobbelaar, N. , Pollard, J. K., and Steward, F. C. New soluble nitrogen compounds (amino .r inLino-acids and amides) in plants. Nature. 172, 703, 1955. . Iraqi dates organization. Analysis of Iraqi dates. 1961+. Leverton, R. M. Amino acids. Food, the Yearbook of Agriculture. 1959- Levy, A. L. and Chung, D. Two-dinentional chromatograer of amino acids on buffered papers. Anal. Chem. 22, 396, 1953. Maier, V. P. lukth Am. Chem. Soc. Meeting. 1963. Meister, A. Biochemistry of the amino acids. 1957. Methods of analysis. Assn. Offic. Agi. Chemists. 1960. Moore, 8., and Stein, W. H. Chromatography of amino acids on starch columns. Jour. Biol. Chem. 118, 53, 19kg. Moore, S. , and Stein, W. H. Chromatography of amino acids on sul- fonated polystyrene resins. Jour. Biol. Chem. 122, 663, 1951. Moore, 8., and Stein, W. H. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. Jour. Biol. Chem. 211, 907, 1951+. 31 17- 18- 19- 20- 21- 22- 23- 2h- 25- 26- 32 Moore, 3., and Stein, W. H. Chromatography of amino acids. Jour. Biol. Chem. 211, 893, 195A. Moore, 8., Spackman, D. H., and Stein, W. H. Chromatograptw of amino acids on sulfonated polystyrene resins. Analytical Chemistry. 32, 1185, 1958. Nixon, R. W. Recent observations of date culture in Iraq. Seventh Annual Date Growers' Inst. 1930. Orr, M. L., and Watt, B. K. Amino acid content of foods. U.S.D.A. Home Econ. Res. Dept., No. h. 1957. Piez, K. A., Irreverre, F., and Wolf, H. L. The separation and determination of cyclic imino acids. Jour. Biol. Chem. 223, 687, 1956. Rinderknecht, H. The free amino acids pattern of dates in rela- tion to their darkening during maturation and storage. Food Research. 23:, 298, 1959. Rose, W. C. Amino acids requirements of men. Fed. Proc. 8, 511-6, 19h9. Schweigert, B. S., McIntire, J. M., Elvebjem, C. A., and Strong, F. M. The direct determination of valine and leucine in fresh animal tissues. Jour. Biol. Chem. lgfi, 183, 191th. Spackman, D. H. Instruction Manual Handbook. Beckman/Spinco model 120. Amino Acid analyzer. Bean instrument, Inco. Palo Alto, Calif. 1963. Vinson, A. F. The chemistry of the date. The First Date Growers' Institute. 192h. “GPO-”r maWigwam‘ Free Amino Acids of Dates: Var. Khadhrawi . nl. v. n 3 R E N N U E T A” T"). 31 (llllllllllllll