A Study of the Non-Caffeine Nitrogenous Compounds of Coffee
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. 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.