. A STUDY OF THE NON-CAFFEINE NITROGENOUS COMPOUNDS OF COFFEE
Dissertation Presented in Partial Fulfillment of theaRequirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University
By GERALD EMERSON UNDERWOOD, B. S., M. Sc. The Ohio £>tate University 1951 The author wishes to express his gratitude to his advisor, Professor F. E. Deatherage, under whose guidance this work was conducted, and to the Nestle Company for establishment of the Fellowship which made the project possible.
1 TABLE OF CONTENTS Page Introduction...... 1 Literature Review ...... 4 Experimentation...... 7 I. Study of proteins present in green coffee...... 7 Solubility classes of coffee proteins...... 8 Isolation iof water-soluble protein...... 11 Isoelectric point of water-soluble protein.. 13 Estimation of £roteolytic enzymes...... 16 II. Fractionation of nitrogen compounds present in coffee on basis of solubility...... 22 Fractionation of green coffee.;...... 22 Fractionation of roasted coffee...... 29 III. Study of amino acids present in coffee.... 33 Qualitative identification of amino acids... 33 Quantitative estimation of amino acids...... 37 IV. Use of an ion exchange resin for hydrolysis of proteins...... 58 Hydrolysis of casein...... 60 Hydrolysis of water-soluble coffee protein.. 66
Discussion of Results...... 72
Summary...... 79 Bibliography...... 80 Autobiography...... 84
ii A STUDY OF THE NON-CAFFEINE NITROGENOUS COMPOUNDS IN COFFEE
INTRODUCTION
Coffee 1b tine name given to the seed of a small evergreen tree which is cultivated in tropical countries. The plant belongs to the genus Goffea, order Rublaoeae. The raw coffee seeds or "beans’1 are roasted by heating with hot combustion gases in rotating cylinders. The end of the roast is accompanied by a rapid rise in temper ature to about 200° C .■ The roasting process is stopped oy cooling rapidly, often.by quenching with water. This roasting produces the flavor and aroma characteristic of the beverage, aiso caned corree, which is prepared by grinding the roasted beans and extracting with hot water. The use of coffee as an article of diet is fairly recent; it was wholly unknown.to the Greeks and Romans.
In 1754-> a- Franciscan monk took a plant to Rio de Janeiro and cultivated it in the garden of the monastery. This one plant was the means of Introducing coffee into Brazil. Today, Brazil produces more than two-thirds of all the coffee consumed in the world; Colombia ranks second among coffee-producing countries. The United States leads the world in the consumption of coffee. It is estimated that the people of this country consume about twenty pounds per capita each year. Despite its tremendous importance economically and its widespread use, comparatively little fundamental chemical knowledge about coffee is available. There is still no general agreement regarding the changes that" take place in the roasting process. The chemical com pounds responsible for the desirable aroma and flavor of roasted coffee are not definitely established; neither is it understood why roasted coffee becomes stale. The recent introduction of soluble coffee extracts on the market has stimulated fundamental research on coffee with a view to solving these problems. In any product where odor and flavor are as import ant as they are in coffee, it would be expected- that nitrogen-containing compounds would play a major role.
The three most important sources of nitrogen in the coffee bean are caffeine, trigonelline, and protein. Caffeine has been thoroughly investigated and trigonelline has recently been the subject of some comprehensive work.
However, the protein fraction, although reported as repre senting 10-14$ of green coffee, has apparently never been examined. It was felt that a study of the coffee proteins, with emphasis on the amino acids present, would contribute to a more complete understanding of the chemical compo sition of coffee. Through such studies we may be able to bring closer a solution-to the problems mentioned above.
2 Therefore, -this Investigation has been primarily con cerned with a study of the protein fraction of coffee; both green and roasted coffee beans were studied.
3 LITERATURE REVIEW
Nothing significant has been published concerning the protein fraction of coffee. However, it will be worthwhile to examine briefly the available literature on other non-caffeine nitrogenous materials found in coffee. Trigonelline, the betaine of nicotinic acid, was first isolated from coffee beans by Polstorff (34-) in. 1909. dorter (14-) verified. the fact that the compound Isolated by Polstorff was really trigonelline. Slotta and Neisser (39) devised a method for analysis of trigonelline in coffee. In a subsequent paper (4-0), they reported the analysis of fourteen different coffees from all parts of the world. They found from 0.8 to 1.2^ of trigonelline in the raw coffees from various sources, and 0.3 to 0.6^ in the same samples after roasting. Trigonelline repre sents about 5% of the water-soluble portion;of roasted coffee and has been reported (31) to have a bitter taste about one-fourth that of caffeine. Hughes and Smith (19) found that nicotinic acid is produced during the roasting- of coffee by the decomposi tion of trigonelline present in the raw beans. However, the actual amount of nicotinic acid formed during the roasting represents only about 1-32& of the amount of trigonelline lost. A dark roast contains more nicotinic acid than a light roast. The nicotinic acid is almost completely extracted in the preparation of the beverage for drinking; the amount in a cup of coffee is about one milligram. This amount of nicotinic acid would be ex pected to contribute substantially to the requirements of this vitamin when large amounts of coffee are consumed. In fact, De Oastro (12), in discussing nutrition in Brazil, states that there is no pellagra zone in the country, the disease being a clinical rarity there. Extensive coffee consumption may account for this fact since the average Brazilian diet seems quite low in nicotinic acid and con tains the classical "pellagra-producing" maize. However, Teply, et al (45) found that when coffee extract was fed to blacktongue dogs on a synthetic nicotinic acid-low diet, sufficient nicotinic acid was provided but a biotin deficiency was apparently produced. Bertrand and Weisweiller (3) isolated pyridine from roasted coffee in 1913- Other workers (41, 21) have confirmed this finding. In a recent publication (20), Hughes and Smith have reported that pyridine is also formed by the destruction of trigonelline. The pyridine content increased in amount during roasting; the production of pyridine closely paralleled that of nicotinic acid and the amounts found were of the same order. These authors give a list of many substances which have been reported
5 as occurring in coffee, including ammonia, methylamine, t rime thy lam ine, pyrrole, pyrazine, and N-methyl pyrrole. No comprehensive studies have been reported on these, materials; some of them may result from thermal decompo sition of proteins. Indications are that they occur in only minute amounts. However, even if present only in traces, it is probable that they contribute to the charac teristic flavor and aroma of coffee. EXPERIMENTATION
I. Study of Proteins Present in G-reen Coffee Since no previous work on the protein fraction of coffee had been reported, it was necessary to carry out several general experiments to. learn something of the nature of the proteins present. Therefore, the green beans, after grinding, were extracted with various sol vents In an attempt to classify the proteins with respect to the traditional solubility groups (9). Then that portion of the proteins which was water-soluble was iso lated, purified, and characterized. This water extract was also tested for the presence of proteolytic enzymes. These preliminary experiments were carried out on two different coffees— Santos and Medellin Excelso (Colombian). Coffees are divided Into two major groups, Brazils and Mllds. Santos Is considered the best of the Brazils, while the Colombian coffees are the most widely used of the Mild group. Mild coffees generally have more body, more acidity, and a more desirable aroma and appear ance. Most of the later work was confined to the Santos coffee. This represents the class of greatest commercial importance and It was felt that more could be accomplished in these investigations if we limited ourselves to a single type of coffee. A summary of the methods used and the results
7 obtained in these preliminary experiments is contained in: the following pages.
Solubility Glasses of Goffee Proteins
Method After extraction of the green coffee with the proper solvent, the protein was precipitated by use of j trichloracetic acid (18). This reagent precipitates pro teins, but not proteoses, peptones, etc. To secure an approximate idea of the amount of protein extracted by the various solvents, the precipitate was filtered, dried, and weighed; and the percentage protein in the coffee was calculated. Experimental procedure The green coffee beans were ground in a hammer mill to approximately 40 mesh size and stored in evacuated cans until used. In a typical determination, an 8 g. sample of green Colombian coffee was placed in a 75 ml. centrifuge tube. To this was added 65 ml. of distilled water, a stopper was Inserted, and the contents were shaken vigor ously for a few minutes. After centrifuging, the super natant liquid was decanted and filtered through a Buchner funnel. The residue in the centrifuge tube was treated similarly with a fresh 50 ml. portion of water. A third extraction with 50 ml. of water (total of 165 ml.) was found to remove the last of the water-soluble protein, since further extracts gave no precipitate with trichlora cetic acid. To the combined filtrates was slowly added, with stirring, an equal volume of a 3>% aqueous solution of trichloracetic acid. The flask was set aside for four hours; at the end of this time, the precipitate had all collected at the bottom. The supernatant liquid was care fully decanted and discarded. The precipitate was washed three times with 93% ethanol, centrifuging and decanting the alcohol after each washing. After three additional washings with ethyl ether, the material was filtered on suction and transferred to a tared crucible. It was dried 1 hour at 105° 0., then cooled and weighed. Similar extractions were carried out using the following solvents: (a) 80% ethanol, (b) 10^ sodium chlor ide, (c) 1% sodium hydroxide. Furthermore, all the extrac-: tions were repeated, using Santos rather than Colombian coffee. To obtain approximate values for the amount of protein extracted by each solvent, it was assumed that the precipitate was all protein. The percentage of protein was reported In terms of the dry green coffee. The moisture content of both green and roasted coffee samples was deter mined by drying for 2 hours In a vacuum oven at a tempera ture of 100° C. and a pressure of 4 inches of mercury. The results are summarized in the following table. Table 1. Amount of Soluble Protein Present in.Green Coffee Extracting % p r o t e i n . Sample Wt. liquid Wt. of ppt._ .(dry basis)
Colombian 8 g. 80^ ethanol 0 mg. 0 Santos 8 80^ ethanol O 0
Colombian 8 water 221 2.9 Santos 8 water 227 3.0 Colombian 8 io^ NaCi 223 3.0 Santos 8 ~L0% NaCl 232 3.1 Colombian 8 NaOH 301 4.0 Santos 8 1% N a O H 4.9 . ,.361...... The precipitate collected from three separate determinations of the water-soluble protein of Oolomhian coffee showed a total weight variation of 11 mg., corres ponding to a variation of 0.15^ protein among the three samples. This indicates fairly good reproducibility by this method. Summary It was found that most of the protein which could be extracted from coffee was water-soluble. An additional amount, however, was soluble in dilute alkali; and there was more alkali-soluble protein in the Santos than in the Colombian coffee. Approximately 3% of each green coffee was water-soluble protein; an additional 1% of the Colom bian and 1.9^ of the Santos was alkali-soluble protein.
lO There was no appreciable globulin fraction (insoluble In water but soluble in dilute neutral salt solutions) and no prolamines (soluble in 80% etbanol). It should be noted, however, that a portion of the protein classed as water- soluble may actually belong to a globulin fraction. It is possible that some of this protein would be insoluble in pure water but was made soluble in the dilute salt solution formed by the soluble Inorganic salts present in-the coffee.
Isolation of Water-Soluble Protein
Method The green coffee was extracted with water, centri fuged, and the extract filtered. The filtrate was treated with hydrochloric acid, and the precipitated protein was washed with ethanol and ether. One portion of the protein was continuously extracted with ether, then dried; another portion was not ether extracted, but merely dried. The nitrogen content of such samples prepared from both Santos and Colombian coffees was determined. Experimental procedure A 50 g. sample of ground green coffee was vigorously shaken with 400 ml. of water. The mixture, was centrifuged and filtered on suction.- To 300 ml. of filtrate was added, all at once, 6 ml. of concentrated hydrochloric acid. After mixing thoroughly, the mixture was allowed to stand for 5
11 minutes. The protein precipitated immediately and settled rapidly. The supernatant liquid was decanted, the residue centrifuged, and again the liquid was decanted. The residue was washed 5 times with 95^ ethanol (total volume of 300 ml.), centrifuged after each washing, and the wash liquid dis carded. The residue was then washed three times with ethyl ether, filtered on suction, and washed twice with ether while on the filter. The nearly white solid was transferred to an evaporating dish and dried 5 hours at 105° C. There was obtained 0.7-0.8 g. of solid; the Colombian was nearly white, the Santos slightly brown in color. Additional samples of each protein were similarly prepared, except that after drying for 1 hour, the samples were finely ground in a mortar, and continuously extracted for 15 hours with ether. These samples were then dried for 15 hours at 105° 0. All four of these protein.samples were analyzed for nitrogen content, using the Kjeldahl method (33). The results are summarized in Table 2.
Sample % Nitrogen: Colombian protein 15.14 Santos protein 15.05 Ether-extracted Colombian protein 15.42 Ether-extracted Santos protein 15.38
12 Summary
The water-soluble protein was isolated from the green coffee, purified, and analyzed for nitrogen content. The protein from both Santos and Colombian coffees was found to contain approximately 15.4-^ nitrogen, corresponding to a "protein factor" of 6.5. A more rapid method of iso lation gave a protein of about 9 Q % purity.
Isoelectric Point of Water-Soluble Protein
Method The green coffee was extracted with water and the filtered extract poured into cold ethanol. The precipi tated protein was washed with cold ethanol, followed by cold ether. The protein was then continuously extracted with absolute ether, dried in air, and finally under vacuum. The product was only about 60% protein but was completely water-soluble and apparently not denatured. A weighed portion of this protein concentrate was dissolved in distilled water and a portion of the solution pipetted into each of a series of standard buffer solutions. Frequent observations were made to ascertain that buffer solution in which the protein was least soluble. Since a protein is least soluble at its isoelectric point, this method gave a measure of the isoelectric .point of the water-
soluble coffee protein (17).
13 Experimental procedure
A 150 g. sample or green Santos coffee was extracted with 1000 ml. of* water. The extract was mixed with Filter Gel and filtered on suction. A 500 ml. portion of the fil trate was cooled to 0° G., and then poured into 2500 ml. of 9 3 % ethanol, also cooled to 0° G. The mixture was thoroughly- shaken, and a white solid "began to separate almost at once. After standing overnight in the refrigerator at 8° C., the solid had settled to the bottom. The supernatant liquid was decanted and 500 ml. more of cold 9 3 % ethanol was added to the residue. After mixing and settling, this was filtered on suction. The solid residue was washed three times with cold ethanol, then twice with cold ethyl ether. The brownish solid became nearly white on drying. After drying overnight at room temperature, the solid was ground in a.mortar and then continuously extracted with absolute ether for 8 hours, using a Soxhlet extractor. The residue was then dried at 36° C. and finally in the vacuum oven at 50-60° for 2 hours. The brownish powder weighed 5.3 g. and had a nitrogen content of 9 * 0 % (Kjeldahl method). Assuming that the nitrogen was present as protein, this meant that the sample was about 60^ protein. The entire sample was readily soluble in water. The solution gave a voluminous precipitate on addition of trichloracetic acid.
14 A 0.4 g. sample of -the protein.:concentrate, prepared as described above, was dissolved in 50 ml. of water. Sep arate 2 ml. portions of this solution were pipetted into a series of standard acetate buffer solutions (5), ranging from pH 1.1 to pH 5.2.; After addition of the protein solu tion, each tube was inverted and righted three times, then allowed to stand. This preliminary test Indicated that the isoelectric point was somewhere between pH 4.2 and 4.8, since a precipitate was formed only in the tubes in this range. Therefore, two identical series of acetate buffers were prepared, pH 4.0-5.0. To one of these was added the solution of Santos protein, to the other a solution of Colombian protein, similarly prepared. Results observed are summarized in Table 3: o = no change in appearance; / 9 opalescence in solution; x - precipitate.
Table 3. Isoelectric :POlnt of Water-Soluble Protein Tube No. 1 2 3 4 5 6 pH ..... 3.95 4.19 4.39 4 . 5 8 4 . 7 6 A . 9.5 - Sample Time
Santos / X'-r X X XXXX o 15 min.
Santos XXXXXXXXXXXXX o 30
Santos XXX XXX xxxxx XXXX / 60 Colombian / X XX XXXXX 15 Colombian / X XX XXXXXXX XX 30
Colombian _/ X XXX xxxxx XXXX XX 60 15 : One hour after addition of the protein, the pH of tubes 3, 4, and 5 was checked electrometrically, using a glass electrode*. These determinations were.in excellent agreement with the pH values noted above. From the above results, it was apparent that the isoelectric point (least solubility) of each protein was. between pH 4.58 and pH. 4.76, and somewhat closer to the former. Summary Samples of water-soluble protein were Isolated from both the Santos and Colombian coffees. Judging from solu bility behavior, this protein was not denatured. The iso electric point of each of these proteins was found to be at
pH 4.6-4.7.
Estimation of Proteolytic Enzymes
It was repeatedly noted that when an aqueous extract of green coffee was allowed to stand for several hours, the amount of protein precipitated from it was much less than when the protein was precipitated immediately after extrac* tion. It was postulated that this rapid decrease in protein content of the extract might be due to the presence of an active protease in the green coffee. It was thought that If such an enzyme were present, the information concerning its activity would be Interesting and perhaps useful. Beckman pH Meter, model G-. 16 Experiments were designed to measure quantitatively the activity of* any protease . present. Method The breakdown of a protein to its various degrad ation products should result in the setting free of addit ional amino groups. Such groups can he estimated by the method of Van Slyke (^T)» This method, briefly, consists in allowing nitrous acid to act on the compound; the nitrogen in the primary amino groups is converted to free nitrogen and .the volume of nitrogen is measured. This, in turn, gives a measure of the free amino groups present in the compound. By adding a disinfectant such as toluene (to prevent bacterial action) to the green coffee extract, it was possible to follow the enzymatic '.decomposition of the protein by merely running a van Slyke amino nitrogen determination occasionally. An increase in amino nitrogen would indicate that proteolysis was occurring, and that a protease was present. As a further check to determine whether protein degradation was due to a protease in the coffee or to bacterial action, the amount of protein preci pitated from an extract by trichloracetic ;acid was measured. A portion of this extract was stored under toluene and another portion of the same extract placed in a stoppered flask without toluene. The amount of protein precipitated'
17 from these two samples after standing several hours was compared^ with, the amount of protein originally precipitated from the extract. Any decrease In the sample containing toluene was due to a protease In the coffee; any extra decrease in the amount of protein in the flask containing no toluene was probably due to bacterial action. Experimental procedure A 50 g. sample of green Colombian coffee was shaken with 500 ml. of distilled water. The solid was allowed to settle and the liquid filtered on suction. The filtrate was adjusted to pH 7 by addition of solid sodium carbonate. A 100 ml. portion was saturated with toluene and stored in a 125 ml. g. s. bottle at room temperature. Another portion, similarly treated, was stored at 37° 0. An extract of green Santos coffee was also prepared and two samples incubated as described above. A third 100 ml. portion of the Santos extract was shaken with 0.5 g . of sodium benzoate,' which was used as a preservative In place of the toluene. This sample was left at room temper ature. Each of these five samples was analyzed for amino nitrogen content over a period of eight days. The results, expressed as volume of nitrogen at S. T. P. per ml. of sample, are collected below.
18 Table 4. Measure of Activity of Proteolytic Enzymes Preser- Vol. of l 2" per ml. after Sample Temp. vative O hrs. 36 hrs. Ill hrs 200 hrs
Oolombian 25° 0. toluene 0 .20 0.21 ---- 0.20 Colombian 3 7 ° C. toluene 0.20 0.19 0.18 0.18 Santos 25° 0.' toluene 0.21 0.21 ---- 0.21 Santos 3 7 ° c. toluene 0.21 0.20 ---- 0.21 Santos 25° 0. sodium 0.21 0.21 0.19 0.21 benzoate To further check the stability of* the soluble coffee proteins, a 100 s. sample of green Santos ooffee: was shaken vigorously with 500 ml. of distilled water. After settling, this was filtered. A 50 ml. portion of the filtrate was immediately treated with 50 ml. of 3 % trichloracetic ;acid solution. After standing 80 minutes, this was filtered through a dry filter paper and washed with 40 ml. of 95% ethanol. The precipitate was trans ferred to an evaporating dish, dried 1 hour at 105° C.» cooled, and weighed. Another 105 ml. portion of the filtrate was placed in a 125 ml. Erlenmeyer flask, and 10 ml. of toluene was added^ A 55 ml. portion of the filtrate was placed in another flask, without toluene, and both flasks were tightly stoppered. After standing at room temperature for two days, 50 ml. samples were taken from each flask, treated with trichloracetic ;acld, and the amount of precipitate
19 measured as above. After 10 days, another 50 ml. sample was taken from the flask containing the toluene. The amount of protein precipitated in .each case is indicated in Table 5.
Table 5. Weight of Protein Precipitated as a Measure of ______Protease Activity______Wt. of protein ppt'd. after ______Sample______0 h r s . 46 hrs ._____24-0 hrs . Untreated green coffee extract 136 mg. 1 mg. ----- Green coffee extract & toluene 136 mg. 130 mg.____14-2 mg. Data from both tables indicate that no active protease was extracted from the green coffee. It is also apparent that the protein in an aqueous extract of the green coffee was rapidly decomposed, but that the protein in the extract was stable for several days when a suitable preservative was added. Summary Aqueous extracts of green coffee were prepared and tests made to determine whether a proteolytic enzyme was present. This was done by adding a preservative to the extract and periodically determining the amino nitro gen content. A further check was made by measuring the amount of precipitate obtained by adding trichloracetic; acid to portions of the extract at intervals. Thesb experiments indicated that there was no active protease present in the green coffee extracts, that the breakdown 20 of proteins In tiie extracts was probably due to bacterial action, and that the protein in the extract was stable for several days in the presence of toluene.
21 II. Fractionation of Nitrogen Compounds Present In Coffee on Basis of Solubility
To establish a basis for further work, it was considered necessary to divide the nitrogen compounds of the coffee bean into smaller groups. Since it had already been demonstrated that an appreciable part of the protein fraction could be extracted from the green coffee beans, the decision.was made to use solubility as the basis for securing partial separation of the nitrogen compounds. Each fraction could then be investigated separately, thus somewhat simplifying the problem. The following experiments describe the scheme of separation, followed, and summarize the analytical results obtained.
Fractlona11 on of Green Coffee
Method The green coffee was analyzed for total nitrogen and for caffeine content. Samples of this coffee were then extracted with water. The total nitrogen, caffeine nitrogen, and amino nitrogen of the extract were determined. A portion: of the extract was treated with trichloracetic acid and a nitrogen determination on this precipitate gave a measure of the proteins present in the extract. The filtrate from the trichloracetic acid precipitation was also analyzed for total, caffeine, and amino nitrogen.
22 Another portion of the aqueous extract was treated with phosphotungstic acid (48). This reagent will precip itate proteins, proteoses, alkaloids, amines, and the basic amino acids; it should precipitate nearly all nitro gen compounds which would be expected to be present except simple peptides and the neutral and acidic amino acids. The nitrogen content of this precipitate was determined and also the total, caffeine, and amino nitrogen values of the filtrate. Finally, most of these fractions were hydrolyzed with hydrochloric acid and amino nitrogen determinations made on these hydrolysates. A similar fractionation-was carried out, using dilute alkali for the extraction in place of water. The. total nitrogen content of each of these fractions was determined. Experimental procedure A lO g. sample of green Santos coffee was placed in a 75 ml. centrifuge tube. The sample was then extracted with four 50 ml. portions of distilled water* by a method analogous to that described previously (see page 8). Before extraction/ the water was saturated with toluene to prevent bacterial decomposition. The combined extracts were filtered on suction and diluted to a volume of 200 ml. Portions of this extract were analyzed for total nitrogen, amino nitrogen, and caffeine nitrogen. The total nitrogen
23 was determined “by the iKjeldahl method; amino nitrogen was determined by means of a micro Van Slyke apparatus; all caffeine determinations were made at the Nestle Company Laboratories in Marysville, Ohio. A 67 ml. aliquot of the aqueous extract was treated with 22 ml. of 1 5 % trichloracetic acid solution and allowed to stand for two hours. The material was then filtered through quantitative filter paper and the precipitate washed with two 10 ml. portions of 2 , 5 % trichloracetic;acid solution. The combined filtrates were diluted to a volume of lOO ml. and analyzed for total, caffeine, and amino nitrogen; the precipitate was analyzed for total nitrogen. Another 67 ml. aliquot of the extract was t&eated with 2 ml. of sulfuric acid, and then with 20 ml. of 20% phosphotungstic acid solution (made by dissolving 20 g. of phosphotungsticmeld in a solution of 100 ml. of water to which was added 3 ml. of sulfuric acid). After standing 24 hours, the extract was filtered through quant, 1tative paper. The precipitate was washed with two lO ml. portions of 2.5^ phosphotungstic acid solution. Agaln> the combined filtrates were analyzed for total, caffeine, and amino nitro gen, and the total nitrogen of the precipitate was determined TO secure a general idea regarding the combined' amino acids which were present, samples of the green coffee, of the water extract, and of the precipitate and filtrate.
24 from the -trichloracetic acid treatment of the water extract were hydrolyzed with hydrochloric acid. The amino nitrogen content of these hydrolysates was then . determined. The procedure followed in preparing the hydrolysates is described below. Green coffee: a 1 g. sample of the green, ground Santos coffee was mixed with 20 ml. of 6 N. hydro chloric acid and heated under reflux on an oil bath at 135° 0. for 12 hours. The mixture was then filtered on suction and the insoluble material washed several times with hot water. The combined filtrates were evaporated to dryness by heating on a water bath at a pressure of 11 mm. of mercury. The residue was treated with 20 ml. of water and again taken to dryness. This treatment was repeated twice more; the residue was then tS-ken up im water, neutralized with solid sodium carbonate, diluted to a volume of 25 ml., and stored under toluene. Water extract: 25 ml. of the aqueous extract of green Santos, prepared as described above, was treated with an equal volume of concentrated hydrochloric;acid. This mixture was refluxed 12 hours, the excess acid removed by repeated vacuum distillation to dryness, and the resi due made up to a volume of 25 ml. with water. Trichloracetic ;acid filtrate: 25 ml. of the filtrate obtained by treating a portion of the aqueous
25 extract with, trichloracetic acid and filtering was mixed with 25 ml. of hydrochloric-acid. The mixture was refluxed 12 hours, the excess acid removed, and the residue again made up to a volume of 25 m l . Trichloracetic ;acid precipitate: , a sample of green coffee was extracted with water and the soluble proteins precipitated by use of trichloracetic acid, as described above. The precipitate was filtered and washed, then the precipitate and paper were mixed with 50 ml. of 6 N. hydrochloric acid. After refluxing for 12 hours, the excess acid was removed and the residue made up to a volume of 25 ml. In carrying out the amino nitrogen determinations, 2 ml. aliquots were used in each.case. Temperature and atmospheric pressure were recorded and observed volumes of nitrogen were corrected for a blank determination carried out on the reagents alone. This corrected vol ume of nitrogen at the observed temperature and pressure can then be converted to milligrams of nitrogen by calcu lation or by reference to suitable tables. Results of these analyses are collected in Table 6. These results and those in succeeding tables are reported on the basis of the dry coffee.
26 Table 6 . Fractionation of Water-Soluble Nitrogen Compounds ______In G-reen Coffee Amino N; after Sample Total N. Caffeine N. Amino N. hydrolysis green Santos 2.28# 0 . 3 2 # ----- 1.47# o 01 water extract 1.28 • 0 . 1 6 0.81 TOA* pp t.from water extract 0 . 5 1 ____ — — — — 0.43 filtrate from H O o TOA ppt. 0 . 7 7 0 . 3 0 • 0.37 PTA* ppt.from water extract 1.21 ------— . . — ------_ _ _ _ filtrate from PTA ppt. 0.07 0 0.09 -----
TCA indicates trichloracetic acid; PTA indicates phosphotungstic acid. A similar fractionation of tbe green coffee ws,s carried out, using 1# sodium hydroxide solution rather than distilled water as the extracting medium. Total nitrogen content of the various fractions was determined by the- KJeldahl method. Results are reported in Table 7.
Summary It was found that 56# of the total nitrogen in the green coffee beans was soluble in water. All the caffeine was apparently water-soluble. Of the soluble nitrogen, 40# was precipitated by trichloracetic acid; using a protein factor of 6.5, it was estimated that 3.3#
27 Table 7. Fractionation of Alkali-Soluble Nitrogen Compounds in Green Coffee Sample Total N.
1% NaOH extract" 1.53 TCA ppt. from alkaline extract 0.77 Filtrate from TCA ppt. 0.75 PTA ppt. from alkaline extract 1.43 Filtrate from PTA ppt. 0.11
of the ?green ooffee was made up of water-soluble proteins. Treatment of the aqueous extract with phosphotungstic acid precipitated 9 5 ^ of the soluble nitrogen; the remainder was accounted for as free amino nitrogen. Determinations of amino nitrogen content of acid hydrolysates of the various fractions indicated that the trichloracetic acid precipitate was probably composed entirely of proteins; the filtrate from the trichloracetic.acid precipitation of the aqueous extract, after proteins had been removed, showed 48^ of the total nitrogen present as amino nitrogen. Apparently, in addition!to proteins, there are considerable amounts of other amino acid-containing compounds present in the water extract— probably proteoses * peptones, peptides, and possibly compounds related to Schiff bases. Practi cally all the water-soluble nitrogen can be accounted for
28 by adding -together the amino nitrogen of the hydrolysate, the caffeine nitrogen, and the trigonelline nitrogen. Other studies (40) have shown that the trigonelline com- tent of green Santos is approximately corresponding to a nitrogen content of 0.10^. Analyses reported in Table 7 showed that of the total nitrogen of the green beansrwas soluble In X% sodium hydroxide solution. The extra nitrogen, which was soluble in dilute alkali but not in water, was found to be protein .in nature, since it was precipitated by trichlora cetic acid. Using the protein factor of 6.5, it. was found that.5 .0 ^ of the green coffee was composed of alkali- soluble proteins.
Fractionation:of Roasted Coffee
Method A portion of the Santos green coffee beans, from the same source as that, used previously, was subjected to a medium roast. There was approximately ± 7 % roasting loss. These roasted coffee beans were ground and separate samples extracted with water and with dilute alkali. The nitrogen compounds in the extracts were fractionated as was done in: the case ;of the green coffee. Experimental procedure The roasted coffee beans were ground on a Wiley Mill
29 to pass a 20-mesh. screen. This coffee was then fraction ated accord ins to the same scheme used for the green coffee (see page 23). Results of this fractionation are summar ized in the followins tables. As before, the data is reported on the basis of the dry coffee.
Table 8 . Fractionation of Water-Soluble Nitrogen Compounds ______in Roasted Coffee______Amino N. after Sample Total N. Caffeine N. A m i n o N. hydrolysis roasted Santos 2.37^ 0 . 3 3 ^ ------1.38^ water extract 0.59 0 . 3^- 0 . 0 8 0.20 TCA ppt. from water extract 0.01 ------— _ — — 0.01 filtrate from rH 0 TCA ppt. 0.58 * O .09 0.19 PTA ppt. from water extract 0.50 ------— — ------filtrate from PTA ppt. 0.09 0.01 0 .08
Table 9. Fractionation of Alkali-Soluble Nitrogen ______Compounds in Roasted Coffee______Sample Total N. 1% NaOH extract 0.93 TCA ppt. from alkaline extract 0.26 Filtrate from TCA ppt. 0.67 PTA ppt. from alkaline extract 0.80 Filtrate from PTA ppt. 0.12
3 0 Summary Only 25^ of the total nitrogen in tlae roasted coffee beans was soluble in water. Once again, all the caffeine present was found to be water-soluble. Apparently the water-soluble proteins of the green coffee were dena tured and rendered insoluble in the roasting process since there was practically no precipitate obtained by treating the aqueous extract of the roasted coffee with trichlor acetic-acid. Approximately Q 3 % of the soluble nitrogen, was precipitated by treating the extract with phosphotungstic acid; most of that not precipitated was found to be present in the form of free amino groups. There was about the same amount of nitrogen found in the filtrates from the phospho- tungstic acid treatment in the aqueous extracts of both the green and roasted coffees; this was also true of the two alkaline extracts. Acid hydrolysis of the various fractions obtained from roasted coffee caused marked increases in amino nitrogen values. Hydrolysis of the water extract caused an increase of amino nitrogen from 14^ to 3 ^ % of the total nitrogen present in the extract. This indicated that appreciable quantities of amino acid compounds, of smaller molecular weight than proteins, were extracted from the roasted coffee. This was also the case with the green. coffee. However, less of these Intermediate products,
3 1 which were soluble in trichloracetic acid but precipitated by phosphotungstic acid, were round in the roasted coffee extract than in the corresponding green coffee extract. There is some loss of trigonelline in the roasting process and the content in roasted Santos represents about 0 .06% nitrogen (40); it has been demonstrated that this compound is readily extracted from the roasted coffee by water. Once more, the sum of the amino nitrogen of the hydrolysate, the caffeine nitrogen, and the trigonelline nitrogen is nearly equal to the total nitrogen of the water extract. This would seem to Indicate that there is little of the basic amino acids in the hydrolysate prepared from the water extract. Analysis of the alkaline extract of the roasted coffee showed that 39^ of the total nitrogen was soluble in this medium. The additional nitrogen compounds, soluble in alkali but not in water, appeared to be mainly protein. In addition, there was an Increase in the intermediate fraction— soluble In trichloracetic -acid solution but insoluble in phosphotungstic acid solution.
32 III. Study of Amino Acids Present in Coffee
Previous studies reported in tills dissertation ,had indicated that a large proportion of the nitrogen in the coffee bean was present as amino acids, combined in the form of proteins or other compounds. It was believed that a study of the amino acids present in the raw. and roasted beans and in water extracts of both green and roasted coffee might shed some light on the changes that take place in the roasting process. Therefore, the following investigations were undertaken.
Qualitative Identification of Amino Acids.
In order to learn which amino acids were present in the coffee, the method of paper chromatography was used. Since the first publication by Consden, Gordon, and Martin (lO), suggesting the use of paper chromatography for identi fication of the amino acids present in a mixture, literally hundreds of additional papers on the general subject of paper chromatography have appeared. Briefly stated, the underlying principle of paper or partition chromatography may be summarized as follows: one solvent (water) Is held by the inert support (filter paper), while the second solvent moves past the first. There are repeated distri butions of the solute (amino acid) between the two solvents. Since different amino acids will have different partition
33 coefficients in the two solvents, some amino acids will move farther along the filter paper than will others with the result that separations of the amino acids from each other will be achieved. It has been found that adsorption as well as partition plays a part in determining the dis tance a given amino acid will move. The positions of the separated amino acids can be determined by use of suitable color reactions. The variations in technique have ranged from the use of tiny strips of paper suspended in test tubes (3 5 )» to the use of some 900 filter paper discs placed in a vertical stack (27). Excellent reviews of the subject, with numerous references, have been published (43). Since the theory and multiple applications, of the method have been thoroughly discussed in other publications, it was not considered necessary to include a detailed review of that material here.
Method A sample of the green coffee was hydrolyzed and the amino acids present in the hydrolysate determined by the technique of paper chromatography. This method also gave an approximate idea regarding the comparative quantities of several of the amino acids present. Experimental procedure A sample of green Santos coffee was hydrolyzed: with 6 N. hydrochloric acid by the method described earlier
3 4 •; “v (see pagi|r*25). Various concentrations of this hydrolysate were then chromatographed on large sheets of 'Whatman .No. 1 filter paper; this paper has been shown to be one of the best for general chromatographic work (2 2 ). In general, our technique was patterned after that of Gonaden, etral. For most of the work, a 20 gallon stone jar was used as a container. The top was covered by means of a thick pane of glass. The individual drops of hydro- lyBate were placed in a horizontal line across a 34 cm. x 52 cm. sheet of filter paper. The drops were about 7.5 om. from the top of the sheet and 4 cm. apart. The paper was suspended In the jar for downward chromatography by means of a glass trough in which the proper solvent was placed. The trough was similar In construction to the one described by Longenecker (24), and was held in position by nichrome wire. The paper was saturated with water vapor by placing a beaker of water in the bottom of the Jar, replacing the cover, and allowing the system to equilibrate overnight. The solvent was then placed in the trough and allowed to pass down over the paper. After about 24 hours, the paper was removed and the solvent permitted to evaporate at room temperature. The paper was then sprayed with a 0.1^ aqueous solutioniof ninhydrIn (3 6 ) and the paper again allowed to dry at room temperature. . Within 24 hours, maximum
35 development of the color was achieved and the various amino acids could he identified "by comparison with spots from known amino acids which were run simultaneously. By means of this technique, it was possible to find the following amino acids in the green coffee hydrolysate: glutamic acid, leucine, aspartic acid, glycine, valine, phenylalanine, alanine, serine, threonine, and tyrosine. These: amino acids are arranged in the order of decreasing concentration in the hydrolysate, as indicated by comparison of the size'and intensity of the spots with those secured from standards. The presence of tyrosine was confirmed by the Miilon.reaction- (26) and tryptophan:was found to be present in the non-ihydroly zed coffee by use: of the Adamkiewicz test (1)- lh addition, it was possible to demonstrate the presence of the basic;amino acids, arginine, histidine, and lysine. This was done by treating a portion of the hydrolysate with phosphotungs tic:; acid to precipitate the basic acids, filtering off this precipitate, and releasing the amino acids by shaking with excess barium hydroxide. The material was then filtered and the filtrate chromato graphed as above. No positive tests were found for cystine, methionine, isoleuolne, norleucine, prollne, or hydroxy- proline. However, as pointed out by Dent (13)* some of these acids are difficult to demonstrate by this technique unless they are present in large amounts.
36 Summary By1 us© of paper chromatography, the presence of 14 of the naturally -occurring amino acids was demonstrated in. a hydrolysate prepared from green coffee beans.
Quant1tati ve Estimation of Amino Acids
Having determined which amino acids were present in the coffee bean, the next step was.to determine how much of these various amino acids was present. The method used for separation of the amino acid, mixtures produced by hydro lysis of various fractions was ion exchange chromatography. Within the last few years, numerous articles have appeared concerning the use of ion exchange materials in the separation and analysis of amino acid mixtures. Cleaver, et al (8 ) have given a general discussion of the behavior of amino acids in ion exchange reactions. They point out that in a solution of pH 7 containing amino acids, those which are monoamino and moncarboxylic will be present largely as dipolar ions with a net charge of zero; any dicar- boxyllc amino acids will be present largely as ions with a net negative charge; and the diamino acids will exist mainly as ions with a net positive charge. It would be expected that Under such conditions only the basic ;amino acids would be adsorbed by the hydrogen form of a cation exchange resin,v which behaves as an Insoluble acid. It was
57 found experimentally, however, that all amino acids which were studied were to some extent adsorbed by the acid resin. Apparently, as Cannan has pointed out (6 ), more is involved in the interaction.between ion exchangers and amino acids than the simple exchange of ions for other ions possessing a charge of like sign. He suggests that the phenomena of differential adsorption and partition as well as true ion exchange all appear to be Interrelated in the process of; the separation of amino acids by ion exchange chromato graphy . The ion exchange separations of amino acid mixtures have been mainly of three types: removal of acidic amino acids, using a basic,; anion exchange resin (7 , 1 1 ); removal of basic ;amlno a c i d s , using an acidic,; cation exchange resin (4); and complete fractionation of the amino acid mixture of a protein hydrolysate. Cannan (6 ) gave a good description of an ideal method for utilizing ion exchangers to separate amino acids into groups on the basis of charge. This idea was later extended by Winters and Kunin (50); as a result of their work, they were able to devise a scheme for the separation .of an amino acid mixture into the three charge groups (acidic,; basic, and neutral) and also to sepa rate the basic mmino acids from each other. Partridges(32) reported experimental work which led to the separation of a hydrolysate of egg album in. i into seven fractions when.,
38 passed through a synthetic : c a t i on.. e x change resin; Moore and Stein have secured excellent analytical results by passing-protein hydrolysates through starch columns (28). More recently (29), these men have reported certain advantages when the starch is replaced by a cation; exchange resin: more amino acids can.be separated on a single column, inorganic isalts do not have to be removed, the ion exchanger has a greater capacity than starch, and the time necessary to complete analysis is much less. The resin they used was Dowex 50 (2), which is described by the manufacturer as "a high capacity cation exchange resin of the sulfonated styrene divinylbenzene copolymer type". The resin is prepared by polymerization of styrene followed by sulfonation; the product is sold in the form of spher ical beads of various sizes. These beads contain.nearly half their weight as water and may be considered as homo genous gels with a very large surface area. Actually, very little Information has been published on the use of Dowex 50 for separation of amino acid mix tures. In their only published paper on the subject, Moore, and Stein.istated that they placed 3 mg. of an amino acid mixture on a column of Dowex 50; the column dimensions were 0.9 cm. x 55 cm. The amino acids were eluted by means of hydrochloric acid solutions of increasing normality. Small fractions of the effluent were collected and. analyzed and
39 the amount of* amino nitrogen found was plotted against the total volume of effluent collected. By this procedure* they were able to secure individual peaks for 17 amino acids and ammonia. In a later paper, which was delivered at the 1950 meeting of the American Chemical Society in Chicago but has not yet been published*, they suggested that better results were obtained by using the Dowex;50 in the sodium form and eluting with a series of buffers of Increasing pH. They indicated that it might be possible to separate nearly all the common amino acids on a.single col umn. Recovery of the basic amino acids was variable and low but the acidic and neutral acids were recovered almost quantitatively. With this limited information available attempts were made to fractionate the hydrolysates from various fractions of the coffee on a column of Dowex 50. The details of procedure and results obtained are reported below. M ethod Acid hydrolysates were prepared of green coffee, of roasted coffee, and of water extracts of both green and roasted coffee. Aliquots of these four hydrolysates were chromatographed on :an: ion exchange column; The resin, Dowex 50, was used In the sodium form, and the amino acids were eluted from the column by use of acetate buffers. Small
This paper has Just appeared in J. Biol. Chem. 192. 663 (1951). 40 ; fractions of the effluent were collected and each fraction was spot-tested on ninhydrin-impregnated paper. A study i ■ of these spots gave an indication of the separation achieved and made it possible to combine and analyze the fractions which were found to contain a given amino acid. Prelimi nary experiments on the column were carried out using known: amounts of pure amino acids. This was done in order to determine the separation possible and the percentage recovery of amino acids on the column. The quantity of amino nitrogen present in the effluent fractions was measured by use of the ninhydrin reaction. This reagent reacts with heated solutions of the alpha-amino acids to give a blue color; the intensity of this color, measured spectrophotometrlcally, gives a measure of the amino acids present. As noted previously, the Dowex: 50 column.is not suitable for analysis of the basic amino acids. To secure some information regarding the amount of these present in the hydrolysate, they were determined as a group. This was done by treating a separate aliquot of the hydrolysate with phosphotungstic acid. In addition to the basic amino acids, this reagent will precipitate other baBic substances such as ammonia and amines; it is also a precipitant for alkaloids, such as trigonelline and caffeine. The amount of nitrogen contributed to the hydrolysate by such materials was estimated by running a KJeldahl determination on the phosphotungs tic acid precipitate. Experimental procedure A sample of 200-400 mesh. Dowex -50* was converted to the sodium form toy treating with successive portions of 0.2 N. sodium hydroxide, The excess alkali and the resin "fines" were removed toy repeated washing with distilled water: after thorough stirring, the resin was allowed to settle for a few minutes and the cloudy supernatant liquid was then decanted. The resin was added to the column in the form of a thick slurry. The preparation of the column was completed toy passing through 150 ml. of pH 3.5 buffer under a pressure of 200 mm. of mercury. In the finished column (see Figure 1), the 120 cm. x 1.5 cm. Pyrex tutoe was filled to a depth of 110 cm. with the resin. The dry weight of this resin (24 hours at 105° 0 .) was subsequently found to toe 96 g.. The chromatographic operation was carried out by adding the amino acid mixture to the column; and success ively eluting with different buffers. The buffers used were prepared from sodium acetate and hydrochloric:acid, and all were 0.2 N. with respect to sodium. To prevent bacterial decomposition of the amino acids on the column, the buffers were saturated with toluene. Preliminary experiments indicated that the order of emergence of the
*Obtained from the Dow Chemical Company
4 2 \ ; fieser i/oir* neutral and acidic :amino acids found in the green coffee hydrolysate (see page 3 6 ) was as reported in Table 10. This order was established by collecting small fractions of the effluent and determining the amino acids present in the various fractions by means of paper chromatography. 1
Table H O • Order of Emergence of Amino Acids from Dowex 50 Golumn
.. J2H 3.5 PH 4.3 .... _ . PH 5.2 aspartic acid leucine tyrosine threonine phenylalanine serine glutamic acid glycine alanine valine
The preceding experiment indicated that the first four amino acids through the column were least completely separated from each other. Therefore,to test the quanti tative applicability of the method, 1 mg. each of aspartic acid, threonine, and serine and 2 mg. of glutamic acid were dissolved in 5 ml. of water and added to the column. The acids were then eluted with 370 ml. of the pH 3.5 buffer under a pressure of 200 mm. of mercury. The first 150 ml. was discarded and the remainder was collected in
4 4 approximately 2 ml. fractions. A large sheet of filter paper was sprayed with a 0.1% aqueous solution of ninhydrin and allowed to dry in air. Then small drops from each fraction were placed in consecutive order on this paper, by means of a small wire loop. Upon drying overnight at room temperature, the characteristic blue color had appeared in those spots which contained amino acids. By examination of the spots, it was possible to identify four definite peaks corresponding to the maximum concentration of the four amino acids. On the basis of these spots, the collected fract ions were combined into four separate portions, representing the different amino acids. Results of the separation pro cedure are summarized below.
Table 11. Separation of Four Amino Acids on a Dowex 50 Column Amino acid Total effluent volume ----- 0-165 ml. aspartic acid 165-196 threonine 196-220 serine 220-249 ----- 249-273 glutamic acid 273-345 ----- 345-370
In order to measure the percentage recovery of the
45 various amino acids, use was made of the reaction of nin hydrin with the amino acid solutions. The procedure was patterned after that described by Moore and Stein (30), who found that the method gives quantitative results if corrections are made for the different color yield obtained from different amino acids. The color yields for the different amino acids were reported in relation to leucine which was assigned an arbitrary value of 1.00. A standard curve, plotting leucine concentration against optical den sity, was constructed to serve as a reference for determin ation of all the amino acids. Using this curve as a basis, the fractions collected from the ion exchange column were analyzed to determine the percentage recovery of the four amino acids. Results are summarized in the following table.
Table 12. Recovery of Four Amino Acids on a Dowex 50 Column Amino acid Mg. on column Mg. re c ove red % recovery aspartic 1.00 1.05 105 threonine 1.00 0.95 95 serine 1.00 1.03 103 glutamic 2.00 1.99 100
In a subsequent run,; a 2 mg. sample of leucine was added to the column and recovered in 97%> yield. With these indications that the method gave nearly quantitative recovery of synthetic amino acid mixtures, attention was
46 turned to the analysis of the various hydrolysates.
A 10 g. sample of the green ground Santos coffee was mixed with 200 ml. of 6 N. hydrochloric .acid and refluxed on an oil hath at 135° C. for 12 hours. The hydrolysate was filtered on suction;, the residue washed several times with hot water, and the excess acid removed from the com bined filtrates by repeated distillation in vacuo. The residue was taken up in water, neutralized with sodium carbonate, diluted to a volume of 250 ml., and stored under toluene in the refrigerator. On a freshly-packed ion exchange column, prepared as described above, was placed 5 ml. of this hydrolysate. This represents.2^ of the 10 g. sample or the hydrolysate from 200 mg. of coffee. To the reservoir was added 500 ml. of 3.5 buffer. The hydrolysate was all ibrced into the column first, then the buffer was added. The chromatogra phic operation was carried out under a pressure of 200 mm. of mercury and the flow rate through the column was about 47 ml. per hour. The hydrolysate was highly colored and some of this color passed through the column. However, by the time the first amino acid appeared, the effluent liquid was water-clear and colorless so that the color of the hydrolysate caused no difficulty in colorimetric determin ations of the amino acids. . '
47 After the first 100 ml. of liquid was through the column, separate 2 ml. fractions were collected. This pro cedure was expedited toy use of a receiver which automatically siphoned its contents over when it contained approximately 2 ml. A drop of toluene was added to each fraction as it was collected and the vials were stoppered and' stored in the refrigerator until analyzed. Completion of the chromatographic operation required atoout 26 hours. Since the fractions were collected manually, it was necessary to interrupt the procedure overnight and to toegin again the next morning. In a complete run, 700 ml. of pH 3.5 buffer, 250 ml. of pH 4.5 buffer, and 250 ml. of pH 5.2 buffer were put through the column in that order. After the first 550 ml. was through, fractions were col lected in 5 ml. portions rather than 2 ml. portions. At the end of a run, the resin was removed from the column and placed in a beaker. It was washed three times with 0.2 N. sodium hydroxide solution, the excess alkali was removed by repeated washing with distilled water, a final washing was made with the pH 3.5 buffer, and the column was repacked. The collected fractions were each spot-tested by the technique previously described and the fractions were divided into groups after a study of these spots. Although earlier experiments using known amounts of the amino acids had Indicated fairly good separation of the first four amino
48 acids on the column, it was found that in the hydrolysates there were such large relative amounts of glutamic and aspartic acids that separation of the first four amino acids was incomplete. Aspartic acid and threonine appeared to come through together and serine and glutamic acid were not completely separated from each other. In addition, there was some overlapping of glycine and alanine. The method used to establish the proportion of amino acids present in the three pairs was paper chromatography, using buffered paper as suggested by McFarren (25). By compar ison of the size and color of the unknown spots with those from a standard amino acid mixture, it was possible to estimate very closely the proportion of aspartic ;acid to threonine, of glutamic acid to serine, and of glycine to alanine in the hydrolysate. The collected fractions were divided into six separate portions as shown, in Table 1 3 . Only very faint positive tests were found in the region where tyrosine should appear. Apparently, only a small amount of this amino acid was present and it was contained in such a large volume of liquid that analysis for it by this method was: not practical. Each of the portions was diluted to a suitable volume and analyzed by use of the ninhydrin reaction as previously described. The values obtained from those portions containing two amino acids were divided on the basis of the results found
49 by examination of the paper chromatograms. Results of the analyses, In terms of the dry coffee, are reported in Table 14.
Table 13. Separation of Green Coffee Hydrolysate on Dowex 50 Column Amino acid Total effluent volume
------0-110 aspartic acid-threonine 110-144 serine-glutamic acid 144-326
------326-366 glycine-alanine 368-506 valine 506-635
— — 635-810 leucine 810-920
------920-1010 phenylalanine 1010-1115
------1115-1200
50 Table 14. Amino Acid Analysis of Green Ooffee Hydrolysate % in Amino acid Mg. green coffee Mg. nitrogen Alanine 0.95 0.48 0.149 Aspartic acid 2.57 1.28 0.270 Glutamic -acid 4.88 2.44 0.464 Glycine 1.32 0.66 0.246 Leucine 2 .67 1.34 0.286
Phenyl alanine 1.24 0.62 0.105 Serine 0.64 0.32 0.085 Thre on ine 0.46 0.23 0.054 Valine 1.04 0.52 0.124 Total 15.77 7.89 1.783
Another 50 ml. portion of the hydrolysate■was treated with 15 ml. of 20% phosphotungstlc ;acid and Kjeldahl determinations were carried out on the precipitate (basic; nitrogen), on the filtrate (non-basic nitrogen), and on another portion of the hydrolysate (total nitrogen). The total nitrogen of the hydrolysate, in terms of the dry sample, was found to be 2.18%; the basic;nitrogen was 1.07^ and the non-basic ;nitrogen was 1.10^. In the 200 mg. sample used for the analyses reported in Table 14, these results indicate that there should have been 2.2 mg. of non-basic nitrogen present in the hydrolysate. A total of 1.78 or 81^ of this total was accounted for in the data
51 reported.
A similar experiment was carried out on the roasted coffee, again hydrolyzing 10 g. of coffee and making the hydrolysate up to a final volume of 250 ml. A 5 ml. portion was chromatographed hy the method previously described and another portion of the hydrolysate was treated with phos- photungatic :acid and the nitrogen content of the various fractions determined. Results are reported on the basis of the dry roasted coffee.
Table 15. Amino Acid Analysis of Roasted Coffee Hydrolysate % in Amino acid Mg.. roasted coffee Mg. nitrogen Alanine 0 .92 0.46 0.144 H H in
Aspartic acid 2.30 • 0.241 Glutamic acid 4.20 2.10 0.400 Glycine 1.17 0.58 0.218' Leucine 2.36 1.18 0.255 Phenylalanine 1.09 0.54 0.092 o (VI
Serine 0.54 • 0.072 Threonine 0.38 0.19 O .045 Valine 0.94 0.47 0.112 Total 13.90 6.94 1.577
Further analyses on the hydrolysate showed 1.98^ 52 total nitrogen, 0.9 Sfo "basic j nitrogen, and. O . 9 9 % non-basic nitrogen. Of the 1.98 mg. of non-basic^nitrogen added to the column; 1,58 mg. or 80^ was accounted for in terms of the nine amino acids reported.
Hydrolysates prepared from the aqueous extracts of both green and roasted coffee were analyzed by the same technique. An attempt was made to prepare the hydrolysates so that they would contain approximately the same quantity of amino acids in a 5 ml. aliquot as did the hydrolysates prepared from the coffee samples themselves. The amount of coffee to extract in order to obtain such an amino acid content was determined by reference to the amino nitrogen values in the different hydrolysates, as reported in Tables 6 and 8. The green coffee extract was prepared by extracting two lO g. portions with water as previously described (see page 25). The combined extracts were evaporated to a volume of lOO ml. on the steam bath, and an equal volume of concen trated hydrochloric acid was added. This mixture was refluxed for 12 hours, filtered, the excess acid removed, and the neutralized residue made up to a volume of 250 ml. A 5 ml. portion.of this hydrolysate, corresponding to the extract from 400 mg. of green coffee, was chromatographed on the ion exchange column. A 50 ml. portion of the 53 hydrolysate was treated with. 20 ml. of 20^ phosphotungstlc acid and the nitrogen content of the precipitate, the filtrate, and another portion of the hydrolysate deter- mined. Results are summarized below.
Table 16. Amino Acid Analysis of Hydrolysate from Aqueous Extract of Green Coffee % in Mg. Amino acid Mg.' green coffee nitrogen
Alanine 0.93 0.23 0.146 Aspartic:acid 2.68 0.67 0.281 Glutamic acid 4.96 1.24 0.472 Glycine 1.27 0.32 0.236
Leuc ine 2.59 0.65 0.277 Phenylalanine 1.23 O .31 0.104 Serine 0 .66 0 . 1 6 0.088 Threonine 0.47 0.12 0.055 Valine 0.95 0 .24 0.113 Total 15.74 3.94 1.772
The total nitrogen of the hydrolysate was 1.25^; the basic^nitrogen was 0.70^; the non-basic -nitrogen was 0.55^. This means that there was 400 mg. x .0055 or 2.20 mg. of non-^ basic nitrogen added to the column. Of this amount, 1.77 mg. or 81^ was recovered.
Six lO g. samples of the roasted Santos coffee were
54 extracted with, water*, the combined extracts evaporated to a volume of lOO ml., and an acid hydrolysate prepared as described for the water extract of the green coffee. A 5 ml. portion, representing 1200 mg. of the roasted coffee, was chromatographed and.a 50 ml. portion was treated with 55 ml. of 20$ phosphotungstlc ;acid. The extra phoaphotung- stic .acid was necessary because of the large amount of caffeine present in the extract. Results of these deter minations follow.
Table 17. Amino Acid Analysis of Hydrolysate from Aqueous Extract of Roasted Coffee $ in Mg. Amino acid Mg. roasted coffee nitrogen
Alanine 0.25 0.02 O .056 -tf H O
Aspartic 'acid 1.62 • 0 .170
Glutamic acid 5.37 0.28 0.521 Glycine 1.18 0.10 0.219
Leucine 1.08 0.09 0.116 t o o a •
Phenylalanine 0.02 0.025 Valine O .28 0.02 O..Q55 Total 8 .06 0.67 0.920
N o serine or threonine was found in this hydrolysate. Other results showed 0 , 3 7 % total nitrogen, 0.48$ basic ^ nitrogen, and 0.094$ non^basic nitrogen. Of the 1.15 mg. of non-basic nitrogen added to the column, 0 . 9 2 mg. or 81$
55 was accounted for. Summary A procedure was described for the use of ion exchange chromatography in analyzing a mixture of amino acids. This method was applied to the acid hydrolysates prepared from green coffee,- roasted coffee, and water extracts of both green and roasted coffee. The quantities of nine amino acids present in each of these hydrolysates was estimated. The basic nitrogen of each hydrolysate was determined by treatment with phosphotungstlcjacid. In each instance, the nine amino acids reported accounted for approximately Sl.% of the non-basic nitrogen present in the hydrolysate. The amount of these amino acids.in the various hydrolysates, calculated as milligrams of nitrogen per gram of the dry green or roasted coffee bean, respectively, are summarized in Table 18. As^these results indicate, about half of the total amount of these nine amino acids present in the green bean was soluble in water. The corresponding ratio in the case of the roasted coffee was about 1:10. In relation to the other amino acids, there was a definitely higher proportion of aspartic acid, glutamic 3acid, and glycine in the extract of the roasted coffee than there was in the roasted bean itself; the relative amount of leucine was about the same, while alanine, phenylalanine, and valine were distinctly
56 less In -the ex-tract. No serine or -threonine was found in the roasted coffee extract. The relative amounts of each of the amino acids in the green "bean and the roasted bean were found to be comparable.
Table 18. Comparative Amino Acid Content of Coffee Hydrolysates Mg. nitrogen iper gram.of dry bean Amino acid Gr. bean Water ext. R. bean Water ext.
Alanine 0.7^5 0.365 0 . 7 2 0 0 . 0 3 0 Aspartic acid 1.350 0 . 7 0 2 1.205 0.142 G-lutamic acid 2. 320 1.180 2 . 0 0 0 0 . 2 6 8 Glycine 1.230 0.590 1.090 0 .182 Leucine 1.430 0 . 6 9 2 1.265 0.097 Phenylalan ine 0.525 0 . 2 6 0 0.460 0 .022 Serine 0.425 0 . 2 2 0 0 . 3 6 0 Thre on ine 0.270 0 . 1 3 8 0.225 Valine 0.6 2 0 0.282 0 . 5 6 0 0 . 0 2 8
Total 8.915 4.429 7.885 0.769
57 IV. Use of an Ion Exchange Resin for Hydrolysis of Proteins
The classical methods of protein hydrolysis are unsatisfactory in several respects. The use of strong acids, such as hydrochloric or sulfuric, results in the complete destruction of tryptophan- and the partial decomposition of some of the other amino acids, notably serine and threo nine. In addition, large amounts of a dark brown,substance called humIn are formed; apparently at least part of this humin is due to the condensation of the indole nucleus of tryptophan with small amounts of aldehydes that are produced during the hydrolysis (15). The hydrolysates obtained by alkaline hydrolysis are colorless and free of humin. How ever, when the hydrolysis is conducted in an alkaline medium, the amino acids undergo racemizatioh, there is some deamin ation, arginine is converted to ornithine and urea, and the cystine and cysteine are destroyed. Enzyme hydrolysis is unsatisfactory because of the long time required to secure complete hydrolysis and because of the fact that the enzymes are themselves proteins. The hydrolysate is thus contami nated by products split from the enzymes. In the course of the investigations on coffee pro teins, the limitations of current methods of protein hydro lysis were noted and a new method was sought. Until recently the only simple hydrolyzing catalysts known were strong acids and bases. These are apparently effective 58 through, the high concentrations of* hydrogen Ions and hydroxyl ions, respectively, which they provide. Recently, however, a new group of hydrolyzing catalysts have been:, discovered. Steinhardt (42) found that when.proteins were hydrolyzed at 65° 0 . by certain sulfonic acids such as f dodecylsulfonic or cetylsulfonic acids, the amide and pep tide bonds were broken .over 100 times as fast as when they were hydrolyzed with hydrochloric acid. Schramm and Primosigh (38) extended this work and verified the fact that the high molecular weight sulfonic acids exert a cata lytic effect in the hydrolysis of proteins. They carried out their experiments in dilute hydrochloric acid solutions, and added catalytic amounts of the sulfonic acids. Under conditions of their experiments, working at 65° 0., hydro lysis of the proteins was found to be only about 50^ complete. It has also been observed that insoluble cation exchange resins in the hydrogen form act as catalysts for certain reactions. Sussman (44) reported the application of acid-regenerated cation exchangers as catalysts for esterification, acetal synthesis, ester alcoholysls, acetal alcoholysis, alcohol dehydration, ester hydrolysis, and sucrose inversion. Thomas and Davies (46) carried out the hydrolysis of several esters using a synthetic sulfonated resin as catalyst. Levesque and Ci*aig-(23) studied the kinetics of the esterificatiom of butanol and oleic :acid
59 using an acid-form cation exchange resin as catalyst. However, apparently nothing has been reported with regard to the use of an ion exchange resin for the hydrolysis of proteins. Since these materials had been shown to be effective catalysts in other hydrolysis reactions, it was decided to study their action on proteins. Numerous preliminary experiments were carried out in order to determine whether protein hydrolysis could actually be achieved by use of an ion exchange resin. The resin used in these experiments was Dowex 50; its properties have been discussed in an earlier section of this paper (see page 39). This resin,: of the nuclear sulfonic type, behaves as an Insoluble strong acid in aqueous solutions and it was believed that it might furnish sufficient hydrogen ions to accomplish the hydrolysis. The fact that the resin removes most of the amino acids from the solution as - they are formed would also be expected to favor the hydrolysis. The resin as received was in the hydrogen form and could be used without further treatment. It contained considerable moisture and the weights of resin used are reported in terms of this damp material; drying for 24 hours at 105p 0 . gave a moisture content of 46^.
Hydrolysis of Casein ;
The protein used for exploratory experiments was a
60 ■H* commercial sample of* vitamin-free casein which was found to contain 'L'5.65% nitrogen,:. 7-45^ moisture, and 0*74;^ ash. This gives 14.87^ nitrogen on a moisture-free, ash-free basis. Method A sample of casein was mixed with several times its weight of resin and an excess of water, then refiuxed. Portions of the mixture ;were withdrawn at intervals and the biuret test (3 7 ) applied to determine whether the hydro lysis had occurred. Additional samples were then hydrolyzed in a similar manner, the hydrolysate extracted from the resin with hydrochloric acid, and Van Slyke amino nitrogen determinations run on these extracts to learn how much hydrolysis had occurred. These values were compared with a hydrolysate obtained by hydrolyzing the casein with 6 N. hydrochloricacid. Finally, an effort was made :to obtain the casein:, hydrolysate in a crystalline form by extracting the resin, after completion of the hydrolysis, with barium hydroxide. The excess barium was removed with sulfuric acid and the filtrate evaporated to dryness. Details of procedure and results obtained from these various experiments are collected in the following pages.
Obtained from General Biochemicals, Inc.
61 ' Experimental procedure In a 250 ml. round-bottom flask were placed 1 g. of casein, 5 of 200-400 mesh Dowex. 50, and 100 ml. of distilled water. This mixture was shaken vigorously, then refluxed on an oil bath, which was kept at a temperature of 135° 0. Both the resin and protein were insoluble and collected at the bottom of the flask. There was some "bumping'1 but no stirring was used. By means of a pipet, samples of the mixture were withdrawn occ;as;ionally and the biuret test applied. Results of the experiment are shown below.
Table 19. Rate of Casein Hydrolysis with Dowex. 50 Reflux time Biuret reaction
10 hours / 22 hours / 46 hours / 70 hours —
Although a negative biuret test does not necessarily indicate complete hydrolysis of a protein, it was obvious that::some hydrolysis had occurred. The solution remained essentially water clear throughout the run.
In order to place the results on a quantitative
62 basis, a hydrolysis was carried out similar to that described above except that no material was removed for biuret tests. After reflUxing 84 hours, the mixture was washed into an. evaporating dish and evaporated to dryness on the steam bath. The residue was extracted with three 40 ml. portions of 6 N. hydrochloric acid to remove adsorbed hydrolytic products from the resin.. The extractions were made by stirring the resin with the acid, allowing the resin to settle, then decanting off the acid. KJeldahl determin ations on the extract and on the resin indicated that the extraction of the hydrolysis products from the resin was about.98^ complete. The combined extracts had a slight yellow color but upon neutralization with solid sodium hydroxide, became almost colorless. The neutralized extract was adjusted to a volume of 250 ml. by addition of water, and amino nitrogen determinations were made. Other runs were made in which the experimental conditions were varied, in order to find the optimum con ditions for carrying out the hydrolysis. Thus, the effects of increasing the reflux time, of stirring the mixture, and of doubling the amount of resin were studied. These amino nitrogen values were compared with the value obtained by hydrolyzing a sample of the casein:with hydrochloric acid. A 1 gv sample of casein was hydrolyzed with 20 ml. of 6 N. hydrochloric acid by heating at 135° G.
63 for 24 hours. The hydrolysate was neutralized with solid sodium hydroxide and made up to volume. The solution was dark brown and considerable insoluble black humin settled out. Results of the amino nitrogen determinations on these various samples are summarized in Table 20.
Table 20. Extent of Casein Hydrolysis with Dowex. 50 Experimental conditions______Reflux time Amino N. 5 parts resin 84 hours 8.3^ 5 parts resin 132 hours 8 .6 5 parts resin 168 hours 8 .6 5 parts resin; mixture stirred 94 hours 8.3 10 parts resin 90 hours 8.9 6 N. hydrochloric acid 24 hours 10.8
These results indicated that the hydrolysis of the casein was about 8 0 ^ complete after refluxing 4 -*-5 days in the presence of 5 parts of Dowex 50. Increasing the amount of resin or extending the time of the reaction did not appreciably increase the degree of hydrolysis. However, subsequent studies (see page 70) indicated the hydrolysis may actually have been about 100% complete, and that the low amino nitrogen values in the resin hydrolysates may have been due to further changes which occurred in the hydrolysate.
64 In an effort to secure the resin hydrolysate of casein in crystalline form, 3 &. of casein was mixed thoroughly with. 15 g; of the 200-400 mesh Dowex 50 and 300 ml. of water. This was gently refluxed on a heating mantle for 100 hours. The mixture was then washed into an:, evaporating dish and evaporated to dryness on .the steam hath. To the residue was added 200 ml. water and 130 ml. of saturated barium hydroxide solution; this gives a defi nitely alkaline solution. The mixture was stirred thoroughly, heated, and filtered. The residue was washed with a hot solution .containing 270 ml. of water and 6 ml. of barium hydroxide. This washing was repeated a second time and the filtrates combined. To the filtrate was added 4 N. sulfuric acid, drop by drop, until the solution was faintly acid (approx. pH 5). This solution was warmed overnight on the steam plate, then filtered through paper. The precipitate was washed several times with hot water. Kjeldahl deter minations indicated that approximately 9 7 % of the original nitrogen was present in the filtrate. The clear filtrate was evaporated to dryness on the steam bath; most of the water was removed from the residue by adding a little 0. P. acetone and again:taking to dryness. The residue was powdered and dried at 105^ C. for 20 hours. The hydrolysate was then:in the form of a light brown powder and weighed 3.087 S«; it- contained Q . 8 % ash. Apparently f
6 5 r the barium was not quantitatively removed. Summary Considerable protein hydrolysis can be achieved by merely mixing the protein with Dowex.50, adding water* and refluxing the mixture. Using a sample of casein and 5 times the weight of the resin, a negative biuret reaction was secured after 70 hours. When the Dowex 50 was used, about 80^ as much amino nitrogen was formed as was present in a hydrolysate prepared by the classical hydrolysis of the protein with 6 N. hydrochloric ;acid. Doubling the weight of the resin or increasing the reflux time past 100 hours did not appreciably increase the amount of hydrolysis. At the end of a hydrolysis in which Dowex 50 was used, most of the hydrolytic products were firmly held by the resin. These were removed by eluting with strong hydrochloric acid or with saturated barium hydroxide solution* Hydrolysates obtained by use of the resin were nearly colorless in contrast to the dark, humin-containing hydrolysates obtained by hydrolyzing with hydrochloric acid. The preparation of a crystalline casein hydrolysate was also described.
Hydrolysis of Water* Soluble .Coffee,Protein
The use of an ion exchange resin for the hydrolysis of proteins had shown considerable promise in preliminary studies on casein. Therefore the method was extended to a
66 study of the water-soluble proteins extracted from green Santos coffee. An attempt was made to secure a more complete quantitative measure of the amount of hydrolysis than was done in the investigations on casein. The protein from the green coffee was prepared as previously described (see page 11). It was found to have a nitrogen .content of 1 5 .4-0$. Method Separate hydrolysates of the protein were prepared, using 6 N . hydrochloric:; acid for one and Dowex 50 for the other. Bach hydrolysate was analyzed for its amino acid content by the method described in detail in an earlier section (see page .42). This method involves separation of nine of the .amino acids on an ion exchange column and determination of the basic;acids as a group by precipitation with phosphotungstlc ;acid. In addition, ammonia was deter mined in each hydrolysate by the classical aeration.proce dure and the tryptophan; lcontent was separately estimated on the honr-Jaydrolyzed protein.
Experimental The hydrochloric;aoid hydrolysate of the^ coffee protein .was prepared by refluxing 400 mg. of protein with 8 ml. of 6 N. hydrochloricoacid on an oil bath at 135? 0- for 12 hours. After hydrolysis, the excess hydrochloric acid was removed by distillation in vacuo, the residue was
67 taken up In water, neutralized with, sodium carbonate, and diluted to a volume of 100 ml. The Dowex 50 hydrolysate was prepared by mixing 400 mg. of the protein with 4 o ml. of water and 2 g. of the resin. The mixture was heated at reflux for 100 hours and amino acids were removed from the resin by the use of barium hydroxide as previously described (see page 6 5 ). The hydrolysate was finally made up to a volume of 100 ml. Again, the hydrolysate obtained from the resin was nearly colorless while that from the hydrochloric acid contained considerable humin. It has been suggested (29) that the amount of humin produced In an acid hydrolysate of a protein can be kept very low by use of a large excess of acid. To be certainithat the low humin formation.in the resin hydrolysate was not due to the excess of liquid phase present, another portion of the protein was hydrolyzed for 12 hours with 200 times its weight of 6 N. hydrochloric acid. This procedure did appear to somewhat decrease humin formation, but the hydrolysate still became very dark and some Insoluble humin was produced. A 5 ml. portion of each hydrolysate was chromato graphed on the Ion exchange column and the eluant fractions analyzed for the various amino acids. Also, a 50 ml. port ion-'.of each hydrolysate was treated with phosphotung- stic acid to precipitate the basic constituents. Separate
68 determinations of* ammonia were made on 5 ml. portions of each, hydrolysate. This was done by making the solution "basic with potassium carbonate and aerating into a standard solution of sulfuric acid. Titration of the excess acid with 0.0020 N. sodium hydroxide gave a measure' of the ammonia present. The nitrogen content of the phosphotung- stic acid precipitate was then determined and corrected •» for the ammonia content. The use of barium hydroxide for removing the amino acids from the.resin was found to cause a large loss of the ammonia present. Therefore, the ammonia nitrogen of the resin hydrolysate was determined by aerating a hydrolysate In which the amino, acids were sremoved from the resin by use of hydrochloric acid rather than barium hydroxide. Results of these analyses are reported in Table
2 1 . By using the method of Graham, et al (16), the tryptophan content of the coffee protein was found to be ±.7%, thus accounting for an additional 0.93 nig. of nitrogen in Table 21. Tryptophan is destroyed by acid hydrolysis or by refluxing with distilled watery hence was not found in either hydrolysate. On Inspection of Table 21 it is observed that ammonia nitrogen is slightly higher for the hydrochloric acid hydrolysate, Indicating perhaps more degradation or deamination In this procedure. Furthermore, a notably low
69 h
Table 21. Analysis of Cbffee Protein Hydrolysates Milligrams of nitrogen HC1 hydrolysate Resin hydrolysate Alanine 2 .86 2.74
Aspartic acid 5.12 4.93 Glutamic acid 8.74 1.97 Glycine 4.53 4.36 Leuc ine 5.58 5.44 Phenylalanine 2.06 2* 07 Serine 1.56 1.46
Thre on ine 0.95 0.94 c* CVI Valine 2.57 • Basic;amino acids and humin 15.39 14.70 Ammonia 4 .89 4.50 Total recovered 54.25 45.52 Total in hydrolysate 61.60 61.60 glutamic acid value was obtained in the resin hydrolysate; it is probable that the glutamic acid in the resin hydro lysate formed pyrrolidone. carboxyllc acid (49). The prolonged boiling and slightly acid medium would provide the ideal conditions for loss of water fhom glutamic ’acid, leading to formation of the pyrrolidone carboxyllc acid. This reaction could also at least partly account for the low amino nitrogen values in the resin hydrolysates of casein as. reported in Table 20. Casein contains a high
70 percentage of glutamic acid, but the amino nitrogen is no longer free in pyrrolidone carboxylic acid. Paper chroma tograms of the casein hydrolysates showed amounts of most of the amino acids to be approximately equivalent in the resin and HOI hydrolysates, but much less glutamic acid in the resin hydrolysate. Further, it was found that upon brief digestion with hydrochloricnacid, glutamic acid appeared in the resin hydrolysates in an amount comparable to that found in the original HC1 hydrolysate. Summary Samples of coffee protein were hydrolyzed with Dowex 50 and with 6 N. hydrochloric acid. Each hydrolysate was analyzed for several amino acids and for ammonia. All amino acids were found to be present in the two hydrolysates in comparable amounts with the exception:of glutamic acid. There was much less of this acid in the resin hydrolysate; this was probably due to its conversion.to pyrrolidone carboxylic sicid. Slightly more ammonia was present in the HC1 hydrolysate, possibly Indicating some decomposition in this medium. There was no humin formation in the resin hydrolysate.
71 DISCUSSION OF RESULTS
This investigation-has been primarily concerned with the protein fractionvof the coffee bean. Approximately 3$ of the green coffee was found to be present as water- soluble protein; an additional 1-2$ protein was extracted with dilute alkali. There was more alkali-soluble protein present in Santos, a Brazilian coffee, than there was in Colombian, a Mild coffee. The water-soluble protein was isolated from the green coffee by extracting the green beans with water, adding hydrochloric ;acid to precipitate the protein, then successively extracting the protein with alcohol and ether. The Isoelectric -point of the water-soluble protein was determined by adding aliquots of a solution of the non denatured protein to a series of buffers. A protein is least soluble at its isoelectric point; in the case of the coffee protein., this was found to be at a pH of 4.6-4.7. Further experiments on the water extracts of the green coffee indicated that there was no active protease present, that the breakdown of proteins in the extracts was probably due to bacterial action, and that the protein in the extract was stable for several days in the presence of toluene. To establish a basis for further study, the nitrogen compounds of the green and roasted coffee were divided into fractions on the basis of solubility. These and subsequent 72 studies were confined to Santos coffee* It was found that 56^ of the total nitrogen in the green beans was soluble in water; all the caffeine was water-soluble. When the extraction was made with sodium hydroxide rather than water, 6 7 % of the total nitrogen was present in the extract. The extracts were further fractionated by use of trichlora cetic acid and phosphotungstic acid. The extra nitrogen, which was soluble in dilute alkali but not in water, was found to be protein in nature, since It was precipitated by trichloracetic acid. Several of the.fractions were hydrolyzed with hydro chloric acid; in all cases, this caused a large Increase In amino nitrogen values. This was also true with the filtrate obtained after treatment of the water extract with trichloracetic acid, i. e., after proteins were removed. This indicated that, In addition to proteins, there were considerable amounts of other amino acid-containing com pounds present in the extract. Only 2 3 % of the total nitrogen. In the roasted coffee beans was soluble In water; all the caffeine was soluble. There was a negligible amount of precipitate obtained by treating the aqueous extract of the roasted coffee with trichloracetic acid; apparently the water-soluble proteins of the green coffee were denatured in the roasting process. As with the green coffee, acid hydrolysis of the various
73 fractions caused marked increases in amino nitrogen values. However, less of these intermediate products, soluble in trichloracetic acid solution but insoluble in phosphotung- stic acid solution, were found in the roasted coffee extract than in the corresponding green coffee extract. An alkaline extract of the roasted beans was found to contain 39%> of the total nitrogen. The nitrogen:compounds which were soluble in alkali but not in water were mainly protein.: In all the extracts of both green and roasted coffee, phosphotungstlc acid precipitated nearly all the nitrogen present; the small amount not precipitated was accounted for as free amino nitrogen. This may be in the form of amino acid-containing compounds of low molecular we ight. Since there were apparently large amounts of combined amino acids present in the coffee bean, further studies were made on this phase of the problem. Largely by means of paper chromatography, 13 amino acids were identified in an acid hydrolysate of green coffee: alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, leucine, lysine, phenylalanine, serine, threonine, tyrosine, and valine;tryp tophan:.was present in the coffee before hydrolysis. Nine of these amino acids were determined quantitatively by use of lonrexchange chromatography. G-lutamic acid was present in largest amount, followed by leucine and aspartic: acid,
74 then glycine, phenylalanine, valine, alanine, serine, and threonine. Approximately the same relations were found in hydrolysates prepared from the roasted coffee and from a water extract of the green coffee.. However, in the roasted coffee extract no serine or threonine was found, and there was more glycine than leucine. In relation to the other amino acids, there was a definitely higher proportion of glutamic acid, aspartic.acid, and glycine in the extract of the roasted coffee than there was in the roasted bean itself; the relative amount of leucine was about the same, while phenylalanine, valine, and alanine were distinctly less in the extract. The relative amounts of each of the amino acids in the green bean and the roasted bean were found to be comparable. In the course of Investigations involving the hydro lysis of the coffee proteins, it was noted that existing methods of protein hydrolysis are unsatisfactory in several respects. Since cation exchange resins were known to cata lyze other hydrolytic reactions, It was believed that they might also catalyze protein.hydrolysis. In preliminary experiments with casein, it was found that by merely mixing the protein with a cation exchange resin and an excess of water, then heating, it was possible to secure considerable hydrolysis of casein. The resin used was the hydrogen.form of Dowex 50, a nuclear sulfonated resin.
75 Subsequently, two hydrolysates of the water-soluble coffee protein were prepared: a resin hydrolysate and a hydrochloric acid hydrolysate. These were analyzed by use of the ion exchange column in order to determine the rela tive amounts of the different amino acids in the two hydro lysates. All the amino acids determined were found to be present in the two hydrolysates in comparable amounts with the exception of glutamic acid. There was much less of this acid in the resin hydrolysate, probably because of its.con version to pyrrolldone carboxylie acid. In the acid hydro lysates of both casein and the coffee protein, there was a large amount of humln formed; in contrast, the resin hydrolysates were clear and nearly colorless. There was slightly more ammonia in the acid hydrolysate of the coffee protein, possibly Indicating decomposition.which did not occur with the resin. In considering possible areas for future research, it is suggested that the water-soluble amino acid-containing compounds of the roasted coffee bean be further Investigated. There Is an appreciable amount of this fraction present and it is apparently not in the form: of proteins. It should be established whether this fraction represents protein decomposition products such as proteoses, peptones, and t peptides, or polymerized materials of the Schiff base type. It might also be useful to learn whether the compounds are
76 present as such, in the green, bean or are produced during the roasting process. Another fraction which should be further studied is that small group of nitrogen compounds present in the water extract of the roasted coffee, which are not precipitated by phosphotungstic acid. These appear to be comparatively simple primary amino compounds of some sort; they are apparently not strongly basic :since they are soluble in phosphotungstic acid solution. It might also be profitable to extend the quantitative estimation- of amino acids in the various hydrolysates, especially for the individual diamino acids. With regard to the use of an ion exchange resin: for the hydrolysis of proteins, it is possible that this may represent the most significant contribution of the various studies reported in this dissertation. Since exist ing methods of protein hydrolysis do have definite limit ations, any new method should be thoroughly Investigated. Areas for further research in this field include a study of the effects obtained when different resins and different proteins are used; results obtained when strong acids and alkalis are used in conjunction with the resins; and the effect of conducting the resin hydrolysis at various tem peratures and pressures. Possible future applications include the use of resins for the preparation of protein hydrolysates which are suitable in human nutrition;
77 investigations on the kinetics of* protein.hydrolysis; and the use of* the resins for studies of* protein structure. Since the hydrolytic conditions when the resin is used are quite mild and the hydrolysis proceeds slowly, it might be profitable to stop the hydrolysis after varying time inter vals and study the products which had been produced. Such studies may provide information regarding the original structure of the protein molecule. In addition, when the various resin structures which give most rapid hydrolysis have been established by a process of trial and error, it may be possible to actually construct a resin which will give very rapid and complete hydrolysis at a low temperature. Such studies regarding the effect of structure might lead to a more complete understanding of the steps involved ini the action of enzymes.
78 SUMMARY
Approximately 3 % of* the green coffee hean .was present as water-soluble protein. Additional protein was soluble in dilute alkali. The water-soluble protein was isolated; its isoelectric p o int was a t pH 4.6 -4.7 . Indications were that there was no active proteolytic enzyme present, in water extracts of* the green bean. It was found that 3 & % of* the total nitrogen in green Santos coffee and 2 3 % of the total nitrogen in the roasted beans was soluble In water. In both, an addit ional amount, mainly protein in nature, was soluble in dilute -alkali. There was a large amount of non-protein;material In. the extracts, which was found to contain free amino groups after hydrolysis. Fourteen amino acids were identified In the green bean; nine of these were estimated quantitatively in the green and roasted coffee and in the water extracts of each. A new method of protein; hydrolysis, using an ion exchange resini was found to give almost complete hydrolysis of casein and of the water-soluble coffee proteins. BIBLIOGRAPHY
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83 AUTOBIOG-RAFHY
I, G-erald Emerson Underwood, was born in Wellsburg, West, Virginia, on November 30, 1921. I secured my public school education:at Waynesburg, Ohio. In 194-3, I received the Bachelor of* Science -degree from Mount Union College. The following year, I entered the Graduate School at the Ohio State University. I served as an assistant In the Chemistry Department, and in 1946 received the Master of: Science degree with my major work in organic ;chemistry. I was employed" for about two years by the Ohio State Research Foundation and then worked the next two years in industry as an associate chemist at the Babcock and Wilcox Research Laboratories in Alliance, Ohio. In 1950, I reentered the Graduate School at Ohio State as a Research Fellow in the Department of Agricultural Biochemistry in order to complete requirements for the Doctor of Philosophy degree.
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