CARBOHYDRATES OP THE COFFEE BEAN; ISOLATION OP A KANNAN DISSERTATION Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By MURRAY LANE LAVER, E. S. A. The Ohio State University 1959 Approved by: Adviser Department of Chemistry ACKNOWLEDGMENT The author wishes to express his sincere thanks to Professor M. L. Wolfrom for his continued interest, counsel and encouragement during the course of this work. The assistance of Dr. Alva Thompson pertaining to the development of the chromatographic techniques and his general laboratory guidance is gratefully acknowledged. This work was generously supported by the Nestle Company, White Plains, New York, under contract with The Ohio State University Research Foundation (Project 530) and continued by Westreco, Inc., Stamford, Conn., under Project 878 with The Ohio State University Research Foundation. Acknowledgment is also made to Dr. A. R. Mishkin and the staff of Westreco, Inc., Marysville, Ohio, for supplying starting material and offering counsel throughout the work. 11 TAELS OP CONTENTS Page I. INTRODUCTION AND STATEMENT OF PROBLEM 1 II. HISTORICAL REVIEW 2 A. Carbohydrates of the Coffee Bean 2 3. Hannans 11 1, Ivory Nut Hannans 13 III. EXPERIMENTAL 23 A. Isolation of the Polysaccharide Fractions of Roast Coffee 23 1. Nature of the Starting Haterial 23 2. Extraction with 80/20::Ethanol/ Water 23 3. Extraction with 2/1::Penzene/ Ethanol 2k k, Extraction with Water 25 5. Extraction with Dilute Ammonlum Oxalate 25 6. Holocellulose Preparation 26 7. Extraction of the Holocellulose of Roast Coffee with 10,2 Potassium Hydroxide 27 B. Isolation of the Polysaccharide Fractions of Specially Extracted Spent Coffee Grounds 27 1. Nature of the Starting Material 28 2. Extraction with Absolute Ethanol 28 iii lv Page 3. Extraction with 2/1::Bensene/ Ethanol 29 4. Extraction with '/later 29 5. Extraction with Dilute Ammonium Oxalate 30 6. Holocellulose Preparation 30 7. Extraction of the Holocellulose of Specially Extracted Spent Coffee Grounds with 10/® Potassium Hydroxide 31 C. Hydrolysis of the (10.® Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee „ 32 1, Hature of the Starting Material 32 2. Hydrolysis with ?2,£ Sulfuric Acid 33 3, Hydrolysis of Cellulose with Anhydrous Sulfuric Acid 36 4. Hydrolysis of (10/o Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee with Anhydrous Sulfuric Acid 41 D. Derivatives of the Constituent Sugars of the (10,« Potassium Hydroxide)- Insoluble Holocellulose of Green Coffee 46 1. Isolation of Galactaric (Hucic) Ac id 46 2, Isolation of Penta-(2,-acetyl^?-D- ^alactopyranose 4? 3. Isolation of Tetra-£-acetyl-L- arabinose Diethyl Dithioacetal 48 V Page a. Partial Hydrolysis with 1.5,-3 Sulfuric Acid ^8 b. Synthesis of Tetra-0.- acetyl-L-arabinose Diethyl Dithioacetal ^9 E, Quantitative Assay of the Constituent Sugars of the (10/£ Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 51 P. Separating Homogeneous Polysaccharides from the (10/£ Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 56 1, Acetylation v.lth Trifluoracetic Anhydride 56 a. De-acetylatlon and Hydrolysis of the Chloro­ form-insoluble Acetate 58 b. De-acetylation and Hydrolysis of the Chloro­ form-soluble Acetate 59 2. Extraction w ith 18,» Sodium Hydroxide 60 a. Determining an Hydrolysis Procedure for Praction-A 62 b. Hydrolysis of Praction-A with Pormio Acid 63 o. Purification of Fraction -A by He-precipitatlon from Sodium Hydroxide 6? d. Purification of Fraction -A by Complexing with Fehling Solution 69 vi Page e. Isolation of D-mannose PhenylhyIrazone from Praction-A 71 f. Hydrolysis of Fraction- E with Formic Acid 71 g. Hylrolysis of Fraction- C ivith Formic Acid 72 h. Hydrolysis of Fractlon- D with Formic Acid 72 G, Determining the Structure of Praction-A 75 1. Ilethylation of Praction-A 75 2. Hydrolysis of lie thy la ted Praction-A in Formic Acid 78 3. Attempting to Isolate a Crystalline Derivative of 2,3,6-Tri-£-me thy1-D- mannopyranos e 81 a. Attempting to Prepare 2 ,3,6-tri-Q.-methyl-£- phenyl-D- mannopyranosylamine 81 b, Attempting to Prepare the Phenylhydrazide of 2,3,6- Tri-£-me thy1-D- mannonolac tone 83 IV. DISCUSSION 85 A. Isolation of the Polysaccharide Fractions of Roast Coffee 85 vil Page B. Isolation of the Polysaccharide Fractions of Specially Extracted Spent Coffee Grounds 85 C. Hydrolysis of the (10/* Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 93 1. Hydrolysis with 72.i Sulfuric Acid 93 2. Hydrolysis of Cellulose with Anhydrous Sulfuric Acid 9^ 3. Hydrolysis of (10,t Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee with Anhydrous Sulfuric Acid 97 D. Derivatives of the Constituent Sugars of the (10* Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 98 E. Quantitative Assay of the Constituent Sugars of the (10/S Potassium Hydroxide) -Insoluble Holocellulose of Green Coffee 99 F. Separating Homogeneous Polysaccharides from (10/S Potassium Hydroxide)- Insoluble Holocellulose of Green Coffee 103 1. Acetylation with Trifluoroacetic Anhydride 103 2. Extraction with 18/S Sodium Hydroxide 105 a. Determining an Hydrolysis Procedure forFraction-A 106 vill Page b. Hydrolysis of Praction-A with Formic Acid 108 c. Purification of Fraction-A by Re­ precipitation from Solium Hydroxide 109 d. Purification of Fraction -A by Conplexing with Fehling Solution 110 e. Isolation of D-Kannose Phenylhydrazone from Fraction-A 111 f. Hydrolysis of Fraction- 3 with Formic Acid 112 g. Hydrolysis of Fraction- C with Formic Acid 112 h. Hydrolysis of Fractlon- D with Formic Acid 113 1. General Discussion Pertaining to the Fractionation of the (10,£ P o ta s s ium Hyd ro x ide)- Insoluble Holocellulose of Green Coffee by Extraction with 18,2 Sodium Hydroxide Solution 11^ G# Determining the Structure of Fraction-A 118 V. SUMMARY 122 A. Isolation of the Polysaccharides of Roast Coffee 122 ix Page 3. Isolation of the Polysaccharides of Specially Extracted Spent Coffee Grounds 122 C. Characterization of the (10,2 Potassium Hy 1 r o x 1 de} - In s o 1 uh 1 e Holocellulose of the Green Coffee Eesn 122 D. Separating Homogeneous Polysaccharides from the (10/a Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee 123 E. Determining the Structure of Fraction-A 125 LIST OP FIGURES Figure Page 1. Chromatogram of (10;£ Potassium Hydroxide)- Insoluble Hollocellulose Hylrolyzate of the Green Coffee Bean 37 2. Hydrolysis of Cellulose with Anhydrous Sulfuric Acid ^+0 3. Hydrolysis of (10/S Potassium Hydroxide)- Insoluble Hollocellulose with Anhydrous Sulfuric Acid ^3 Analytical Chromatogram Processed for Direct Photometry 52 5. Standard Curves for Galactose and Hannose 6. Standard Curves for Glucose and Arabinose 55 7. Hydrolysis of Praction-A in Formic Acid 65 8. Hydrolysis Curves in Formic Acid 68 9. Hydrolysis of Practlon-C in Formic Acid 73 10, Hydrolysis of Fractlon-D in Formic Acid 7^ 11. Hydrolysis of Ilethylated Fraction-A in Formic Acid 79 x LIST 0? TABLES Table X Hg Values of Methylated Sugars 82 2 A Compilation of the Data on the Fractionation of Green Coffee, Hoast Coffee, and Specially Extracted Spent Coffee Grounds {dry weight basis) 88 xi LIST OF CHARTS Page I Flow Sheet for the Fractionation of the Polysaccharides of Roast Coffee (g./lOO g., dry weight basis) 86 II Flow Sheet for the Fractionation of the Polysaccharides of Specially Extracted Spent Coffee Grounds (g./lOO g., dry weight basis) 87 III Flow Sheet for the Extraction of (10/» Potassium Hydroxide)-Insoluble Holocellulose of Green Coffee viith 18% Sodium Hydroxide 107 xli I. INTRODUCTION AND STATEMENT OP PROBLEM Comparatively little fundamental chemical knowledge of coffee is available even though this food is widely used and is of great economic importance. The chemistry of the roasting process is little understood, and the chemical compounds responsible for the desirable aroma and flavor of roasted coffee are not definitely established. Since carbohydrates constitute 50-60$ of the coffee bean, it would be expected that these substances would play a significant role in the roasting process. Therefore a fundamental study of the chemical nature of the carbohydrates was begun some years ago by this laboratory and this is a continuance of that work. 1 II. HISTORICAL R3VI3W A j Carbohydrates of the Coffee Bean Payen (1) was the first investigator to report on the (1) A. Payen, Corapt. rend., 22, 233 (1846). carbohydrates of the green coffee bean. He found glucose (probably sucrose), starch, dextrin, and vegetable acids to comprise 15.5^ of the green coffee bean. The most notaole early work was that of Schulze and associates (2,3,4) who (2) 3. Schulze, E. Steiger and W. Maxwell, Hoppe- Seyler's Z. physiol. Chem., ^4, 227 (1890). (3) E. Schulze, Hoppe-Sezler1s Z. physiol. Chem., 387 (1892). (4) E. Schulze, Chem. Ztg., 2Sl» 1263 (1893). partially hydrolyzed the ether hot alcohol -, and 0.2,£ sodium hydroxide - extracted residue of the green coffee bean with sulfuric acid. Prom the sulfuric acid - soluble hydrolyzate Schulze isolated D-galactose and D-mannose. The D-galactose crystallized as the free sugar and was further characterized as galactaric (mucic) acid, as the oxime and 3 as the phenylhyirazone. D-raannose was obtained by precipitation with lead acetate an! characterized as the phenylhyirazone. This contradicted the work of Reiss (5) (5) H. Heiss, Ber., 22, 609 (1889). who had isolated the same compound from ivory nut but considered it different from mannose and called It "seminoDe". Schulze's results showed that "seminose" was in reality mannose. In his first paper (2) on the green coffee bean Schulze and co-workers found insufficient evidence for pentoses. In his second paper (3) Schulze worked with the residue from the 5/« sulfuric acid hydrolysis. He hydrolyzed this with 75/2* sulfuric acid and the resulting sirup remained slrupy for months but finally D-mannose crystallized.