2.51;:‘:3:_:_:__E_S__‘:_::=3: r: ¢~i r(.. 3:" - s. 3k 3%.} fix " ;£i -; fl‘-' .'..'\'i..:~ \ “‘ ' I IQW‘I‘J — . ". 7" ' l ' ' I I L5 U a“; ' x .l I‘ -"-' ' " SVEIWIF?§4 3 _‘,- £32an 1. s1 ' :‘L. t’f ll flllllllmzllfljlflllll 11"ng lllljlflljflflll LIBRARY Michigan State University ' )V1ESI_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this Checkout from —_——. your record. FINES wiH be charged if book is returned after the date stamped be10w. ABSTRACT SUGAR IN TART CHERRIES AND THEIR CHANGES DURING MATURATION by Spiros Minas Constantinides Sugars in the tart cherries (Prunus cerasus L. var. Montmorency) make up approximately 50% - 60% of the total dry matter in the edible portion of the fruit. This study was made to determine qualitatively the type of sugars present and the quantitative changes in the main sugar constituents during the ripening of the fruit. Using the techniques of paper chromatography, thin- layer chromatography and column chromatography, seven sugars ‘were found to be present. Glucose and fructose make up almost 100% of the total sugars. The remaining five sugars seem to be oligosacharides of the reducing type and exist in minute quantities, less than 0.005%. The Dubois method and the paper chromatographic technique were used to determine glucose and fructose quan- titatively. The accuracy by this method was 100%‘1 2. Glucose and fructose were found to exist durbng the ripening period in a ratio of 1.1 to 1, with the ratio tending to become less when shrivelling of the fruit began. Spiros Mines Constantinides On wet basis, the total sugars increased as the fruit ripened and this increase paralleled the increase in dry matter. On dry basis, the total sugars reached their highest concentration when the cherries developed a full red color, and then decreased and remained relatively constant during the rest of the harvest period. Maximum sugar forma- tion preceded the date of commercial harvest by two weeks. SUGAR IN TART CHERRIES AND THEIR CHANGES DURING MATURATION by Spiros Minas Constantinides A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science 1963 r/ ,I' . “qr-“129’! :1 U 1’ i I, / filly/03 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Professor Clifford L. Bedford for his guidance and encourage- ment throughout the course of this work and during the prepara- tion of this manuscript. The author is also deeply indebted to Professor Pericles .Markakis for his beneficial suggestions and advice, to Pro— fessor L. R. Dugan for his assistance in permitting him to use his laboratory for the thin layer chromatographic work involved, and to Professor D. H. Dewey for his critical evaluation of this manuscript. The author is most grateful to his wife Niovi for her inspiration and encouragement throughout this study, also to the State Scholarships Foundation (I.K.Y.)Greece, for the financial support of this project. ii ACKNOWLEDGMENTS . . . . LIST OF FIGURES . . . . LIST OF APPENDICES . . INTRODUCTION . . . . . REVIEW OF LITERATURE . MATERIALS AND METHODS . TABLE Preparation of Sample for Methods for Analysis Paper Chromatography Thin—layer Chromatography . . . . . . Column Chromatography . . RESULTS AND DISCUSSION Qualitative Analysis OF CONTENTS Analysis . . Quantitative Analysis of Glucose and Fructose SUMMARY AND CONCLUSIONS . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . APPENDICES . . . . . . . . . . . . . . . . . . iii Page ii iv \o <> c— ta < T!- H.mu 1‘ 3.1.1:... bill! a. I L. . QIJ: a; LIST OF FIGURES Figure Page 1. Absorption curves of the phenolic condensation products of glucose and fructose . . . . . . 25 2. Effect of phenol concentration on the absorbency of the phenolic condensation products of glucoseeeoeeeeoeoeeoeeeoe 26 3. Effect of phenol concentration on the absorbency of the phenolic condensation products of fmCtoseeeeeeeoeeeoeeeeeee 27 A. Standard curves for glucose and fructose . . . 28 5. Changes of glucose and fructose during ripening of tart cherries (wet basis) . . . . . . . 29 6. Relation between total sugars and dry matter in tartCherrieseeeeeooeeeeoeoe 30 7. Changes of glucose, fructose and total sugars (glucose and fructose) in tart cherries . . 31 iv INTRODUCTION A great deal is known about the general physiology of the fruit, yet relatively little of the detailed chemistry of the fruit has been worked out. Even less chemical work has been done on the cherry fruit. This is not surprising for, until the advent of modern chromatography in its vari- ous forms, searching for all but the most obvious components of the cherry was like looking for the proverbial "needle in the haystack." Even now the enzyme chemistry of the cherry and other fruits is still almost a closed book. It should be remembered that the phase in the life of the fruits in which the food industry is most interested, the mature fruit harvested in summer, is essentially one of dying. The object of the fruit storage industry is to pro- long this dying phase as long as possible. The biochemistry of the stored fruit then is the biochemistry of a complex system that is running down. Nevertheless a complete knowl- edge of the components of the system, and especially of those factors which control the running down processes, should provide the necessary basis for the horticulturist to grow a fruit which is flourishing and that will keep healthy during storage. -2- This study deals with the sugars in the free state, including oligosaccharides present in tart cherries. The basis for this work is the belief that sugars form a substrate for metabolic processes yielding energy. The level of energy of the fruit might be expected to deter- mine the length of its life on detachment from the tree. In recent years the discovery of the Kreb's tricarboxylic acid cycle and of the energy released in the phosphorylating mechanisms involved therein has emphasized the importance of organic acids in the respiration process of plants. A sugar analysis of cherries from an academic stand- point is of equal importance; to compare with sugars present in other fruits; their changes during ripening; to study flavor that arises in conjunction with the acids and other constituents present; to study the sugar-acid ratio of cherries during the development of the fruit, and lastly the techniques applied and developed in the present work may be proved effective for other plant tissue analysis as well. In this study an attempt was also made to introduce a quantitative picture of glucose-fructose change during ripening of the tart cherry. The mechanism of the interplay within the carbohydrates themselves clearly involves other metabolic systems. Much more detailed work is necessary, and work going further into intermediate metabolism must be carried out, before a clear picture can be drawn of the -3- carbohydrate metabolism of fruits developing on the tree and in storage. REVIEW OF THE LITERATURE Traditional Methods The methods generally employed for the determination of the major sugar constituents of fruits have not greatly changed over the last thirty years. Copper reduction methods (macro or micro) have been used for the determination of reducing sugars, and sucrose has been determined by measur- ing the increase in reducing power after hydrolysis under standard conditions. The separate determination of glucose and fructose is carried out enzymatically or by first oxi- dizing the glucose by methods (iodine or alkaline hypoidite) which leaves fructose untouched, determining the residual reducing power due to fructose and estimating the glucose by difference (#7). Most of the analytical methods suggested for sugars are empirical and depend largely on a pre-existing knowledge of the identity of the sugars. The above mentioned methods are based on the reduction of alkaline copper solution by sugars, the reactions involved are complicated usually in- complete, not stoichiometrical, and are not specific for sugars as such. Among the many chemical methods available for the determination of reducing sugars, copper reduction methods predominate (33), (27), (L0). Other'methods used -h- -5- by many workers for the determination of sugars are the ferricyanide reduction methods, iodometric methods, and the colorimetric methods (2a). Chromatographic Methods Of the separation techniques available to the chemist, none surpasses the chromatographic method (9), (28), (8), (18). Chromatography can at least be traced in its development through the work of Runge, Schonbein, Day, Coppelsroeder and Tswett to the more recent work of Martin and Synge (31). Introduced by Partridge (36),(37) paper chromato- graphy of carbohydrates and related substances employs many of the methods and solvents used in the resolution of amino acid mixtures on paper. Kowkabany (25) reviewed the basic method of paper chromatography of sugars, giving solvents and spray reagents suitable for carbohydrate analysis. Many colorimetric methods for quantitative analysis from the chromatogram have been reported. Flood, gt_gl. (13) describes a method for quantitative analysis of sugars from the chromatogram using periodic acid to oxidize the sugars, formaldehyde given off. Hawthorne (17) developed a similar method using the micromodification of Willstatter and Schudel method (stoichiometric oxidation by hypoiodite and titration of the excess iodine with thiosulfate). Hirst, -6- gt_§l. (19) used warm sodium periodate solution to oxidize the sugars and determined the formic acid produced. McFarren g§_§1. (33) developed a method by which sugars can be determined quantitatively by measuring the maximum densities with a densitometer. McCready,g§_gl. (32) used a similar method based on reflectance. Laidlaw, g§_gl. (26) reported a technique for quantitative elution.of'the chromatogram using water. Dimler, gt_§l. (11) used a procedure which is based on using the anthrone reaction for the colorimetric measurement of the constituents resolved by paper chromatography and eluted from.the papers. DuboiS, g§_§1. (12) introduced a new colorimetric method for simple sugars, oligosaccharides, polysaccharides based on the reaction of reducing sugars with phenol and sulfuric acid. All the above quantitative methods have been used on pure sugar solutions and have not been tried on plant tissues to a great extent. Column chromatography is also considered a very versatile tool of analysis for carbohydrates (7). Lew (29) ‘used clays with filter aid to separate sugars. Binkley, g§_gl. (5), (6) used Magnesol (hydrated Mg acid silicate) and Celite ‘to separate acetylated sugars. Rough (20) separated sugars 'using cellulose as the adsorbent and a mixture of butanol, ‘water and ethanol as the solvent. Whistler, g§_§1. (A6) describes a method for sugar separation using Charcoal and -7- Celite, with different concentrations of ethanol as the developers. Alm (1) introduced a gradient elution procedure for sugar separation using column chromatography. Other workers Bacon (h), Ash (2) and Nancy (35) used the above methods very effectively. Another helpful chromatographic technique is the thin-layer chromatography. Stahl (#1) showed that sugars not detected by paper chromatography can be detected by the above technique. Oligosaccharides were separated using the same method by Weill, et a1. (#5). Further studies need to be made to determine whether thin-layer chromatography can be used as a quantitative method as well. Sugars in Cherries The sugars in tart cherries and cherries in general are comprised of glucose and fructose. In an early work reported by Widdowson, et a1. (#7) h.7% glucose and 7.2% fructose were found in the edible portion of the sweet cherries. The chemical changes that occur in the fruit during the period of ripening have been reported only by a few 'workers. Taylor, et a1. (AB) found that there was an in— crease in the soluble solids and the total solids content of the tart cherry fruit as the harvest season progressed. {The same workers (Ah) reported an increase of sugar content from 8.6% to ll.h% during the period of June 30 to July 28, ‘using the Munson-Walker method for sugar analysis. -3- In sweet cherries, Genevois (1h) reported traces of xylose in addition to fructose and glucose. Gherghi (15) found higher sugar content after color formation. Serini (39) reported that glucose and fructose were the only sugars present in sweet cherry juice held for 24 days at 1° C. Willing (48) working on different varieties of German sweet cherries, and using Bertrand's method for sugar analysis, found glucose, fructose and traces of sucrose. Glucose and fructose increased during ripening with glucose slightly higher. In general very little work apparently has been done on determining the types of sugars in cherries. MATERIALS AND METHODS Preparation of Sample for Analysis The fruits for this study were obtained in the summer of 1962 from a block of thirteen year old Montmorency cherry trees growing on the Michigan State University Horti- cultural Farm, East Lansing. Random samples of approximately two kilograms of cherries were gathered from the trees at weekly intervals for a period of fifty-one days, beginning at the time the fruits had a pale pink color, June 20, and ending late after the commercial harvest period, August 9, ‘when the fruit showed signs of shrivelling. Full bloom of the trees was between the 3rd and 5th of May, 1962. During the picking, care was taken to keep the stem on the fruit, to avoid bruising the fruits, and to keep to a certain degree of uniformity of sampling. The cherries 'were immediately taken to the laboratory where they were cleaned from the leaves and other unwanted material, sorted, put in one pound waxed paper cartons and frozen at 0° F. For sugar analysis the edible portion of the frozen cherries was cut up into slices and the pit taken out. One hundred grams were weighed out and them immediately dropped into a Waring Blendor containing about 100 m1 of boiling 95% ethanol. The cherries were blended for ten minutes at -9- -10- a high speed, then transferred quantitatively to 1000 m1 volumetric flasks with hot 83% ethanol. The flasks were placed for half an hour in a hot water bath of about 700 C, cooled to 20° C, made to volume with 83% ethanol and then stored at 32° F for 24 hours. The cherry extract was filtered through E & D No. 512 filter paper and 900 ml were collected. This was transferred to a Buchler flash evaporator concentrated to about 50 ml at 380 C. The concentrate was made up to 100 ml with water, and centrifuged at 2,500 r.p.m. for 20 minutes. The extract was then placed in 100 ml reagent bottles and stored at 00 F to be used later for chromatographic work. No other clarification or preparation was necessary. Methods for Analysis Paper Chromatrography The close chemical similarity of the various sugars to one another has always made the problem of sugar analysis a fundamentally difficult one. The progress of biochemistry, physiOIOgy, botany and food technology has been impeded for many years by the lack of quick and accurate methods by which the various carbohydrates might be differentiated from one another and estimated quantitatively. The recent develop- ments of chromatographic adsorption methods of analysis have provided investigators in many fields with an exceedingly powerful and useful tool for separation and purification. -11- Although they are not generally used as quick routine procedures, they do provide means of separating sharply the constituents of many mixtures composed of substances that are chemically similar. Egpegimgntal Qualitative Analysis Descending chromatographic procedure was employed using Whatman No. l chromatographic paper. The prepared cherry samples were applied on a line 3 inches from the tap of the paper and similar quantities of known sugar (5%) were also applied about 1 inch from the sample spots. Four lambda quantities were applied, the spot was dried with a cold air current from a hair drier after the application of each lambda. The solvent used for chromatographing was n-butanol, acetic acid and water (A:L:5) (37). This solvent gave better separation than other solvents tested (18), (23). It was prepared at least seven days prior to use and was stored at 32° F to obtain good separation of the two phases. It was found essential that no water droplets be present in the solvent phase. The aqueous phase was placed in the bottom and the solvent phase in the trough of the chromato- graphic cabinet. The aqueous phase and the papers were placed in the cabinet five hours prior to the introduction of the solvent phase. After its introduction the chromato- grams were allowed to develop for 48 hours to 56 hours. The -12- direction of flow was always along the machine direction of the paper. The solvent front was allowed to drip down the lower edge of the paper. After removal from the chromato- graphic cabinet, the papers were suspended in the drying hood at 25° C - 28° C for 2h hours to remove the water and solvent. After drying they were sprayed with the spray reagent and heated to 105° C for ten minutes or until colored spots appeared. The spray most often used was the benzidine spray (21). This was made up of 0.5 grams benzidine dis- solved in 10 m1 glacial acetic acid, 10 m1 of 10% trichlo- roacetic acid and 80 m1 of absolute alcohol. Other spray reagents tried were the ammoniacal silver nitrate for reducing sugars (36), naphthoresorcinol for ketoses (37), dinitrosalicylate reagent (22), b- naphthylamine spray (16), aniline hydrogen phthalate (38). None of these sprays were as sensitive as the benzidine spray. The identity of the sugars was determined by com- parison with the known sugars and also by the use of the differenting sprays. Quantitative Analysis Two lines were drawn lengthwise 5 cm from the edge of the paper. Two more lines were drawn 2 inches from the top and 3 inches from the top. Four lambdas of the sample containing unknown concentrations of glucose and fructose were spotted 6 cm apart in the center portion of the paper -13- along the 3 inch line. Three lambdas of the known sample of glucose and fructose (5% glucose plus 5% fructose) was spotted 5 cm from the edge of the paper. The chromatograms were developed as described under qualitative analysis. A blank paper was also developed to serve as a control. Then the two strips containing the known sugars were cut from the paper and sprayed with the benzidine spray to locate the distances that the sugars had travelled. The strips were then matched with the center portion and the center was cut into sections corresponding to the location of the sugars. Each section was again cut into small pieces about one square centimeter and transferred to 50 ml beakers which were covered with plastic petri dishes. Ten milliliters of redistilled water (20° C) was added to each beaker, which was then covered and allowed to stand for about 30 minutes with occasional shaking. During this time the sugars became equally distributed throughout the liquid and solid phase (water and cellulose). The eluate was then filtered care- fully through glass wool to retain the cellulose lint of the paper, and the concentration of the sugars was deter- mined using the Dubois method (12). Two milliliters of sugar solution containing 20 - 40 gammas were pipetted into 1.5 x 18 cm tubes. For the de- termination of glucose 0.1 ml 80% phenol was added, for fructose 0.3 ml was added. Then 5 ml of concentrated sulfuric acid was added rapidly (10 - 20 seconds), the -14- stream of acid being directed against the liquid surface rather than against the side of the test tube in order to obtain good mixing. The tubes were allowed to stand 10 - 20 minutes in a water bath at 25° C - 30° C, and then the absorbence of the characteristic yellow orange color was measured at 487 millimicrons for glucose and at 489 milli- microns for fructose, with a Beckman D U Spectrophotometer (slit width 0.015 mm, phototube 2, and sensitivity 5). A standard curve was prepared for each sugar under examination. Thin-layer Chromatography A method for chromatographic adsorbtion analysis on thin layer of adsorbent was described as early as 1936 by Izmailov and Schrauber (30). Through the work of Kirchner, Miller and Keller the chromatostrip method became well known and it was also used by other workers for the analysis of terpenes (30). It is amazing that the elegant technique of chromatography on open columns, that is thin layers of an adsorbent, was not applied to other lipids after it had become such a prominent method in terperne research. Actu- ally the method remained in obscurity until 1956 when Stahl described equipment and procedures for the preparation of chromatoplates and demonstrated the potential usefulness of thin layer chromatography in the fractionation of sub- stances other than terpenes. Thin layer chromatography had 5U «Hm. and. -15- suddenly gained recognition and the technique is now being applied to the analysis of a great variety of substances. Egpgrimentgl Qualitative Analysis The method used was that as described by Stahl (Al) and Mangold (30). For five plates 30 grams of Celite 535 with 10-15% calcium sulfate added, or Kieselguhr G, were mixed uniformly with about 60 ml of 0.02 M aqueous sodium acetate. The mixture formed was made into a slurry which could easily be spread on the spreader. The slurry had to be of uniform consistency and free of air bubbles. The time required for preparing the slurry should not exceed 1 - 1% minutes. The mixture is poured into the applicator. As the applicator is moved smoothly across the row of glass plates on the board to the right, the slurry is permitted to run out of the slit. The entire operation must be finished quickly less than three minutes for the slurry hardens. The five plates were spread on the plastic board 22 x 113 cm with retaining ledges 1.8 cm wide along a short and a long side. The board was placed on a laboratory bench so that the long edge faces the worker. A glass plate 5 x 20 cm and five carefully cleaned 20 x 20 cm glass plates of uniform thickness, and another plate 5 x 20 cm ‘were placed closely together in a row on the board. A drop -16- of water under each plate will prevent the glasses from sliding on the board. The applicator which would hold the slurry was placed on the small plate in the open position to the left of the worker. The chromatoplates were air dried for 10 — 20 minutes to allow the binder to set and.then were placed in a rack and activated by heating in an oven to 100° - 120° C for 1 - 2 hours. The adsorbent layer thus produced was about 250 - 275 microns thick and very uniform in appearance. Since the adsorbent layer absorbs water from the air and become deactivated the chromatoplates were stored in dessicators. The chromatoplates were developed in a cabinet con- taining a solvent of 65 ml of ethyl acetate plus 35 ml of a mixture of 2 volumes of isopropanol and one volume dis- tilled water. The mixture was prepared fresh daily. The cabinet was saturated with the solvent and then the plates (two in each cabinet) were introduced. The solvent was allowed to run up the chromatoplates until the solvent front was about 5 cm from the top. The plates were then taken out and dried at 20° C in a hood. For the develOpment of the spots each plate was sprayed with 10 ml of a freshly prepared mixture consisting of 9 ml ethanol 95%, plus 0.5 ml concentrated sulfuric acid and 0.5 ml anisaldehyde and then heated for 10 minutes at 100° C. The presence of sugars was indicated by the appearance of pink colored -17- spots on a blue background. The identification was made as in paper chromatography. Column Chromatography Column chromatography has been described as a specialized type of adsorption. Solutions are run through a bed or column of adsorbent, and with subsequent percola- tion of the proper solvents down the column, the least strongly adsorbed compounds move down the column faster than the more strongly adsorbed compounds. This results in a series of bands or zones that can be separated by mechanical extrusion of the absorbent in the fraction, or alternatively can be fractionally eluted by the solvent. The adsorbent used must be selected in accordance with the required char- acteristics. Many different substances have been used; carbon, alumina, silica gel, kieseguhr, magnesia, clays of different types, sucrose, starch and cellulose powder (7). 0‘ E 6 mental Qualitative Analysis The minor carbohydrates constituents of the cherry were in such minute quantities that it was not possible to detect them using the crude fruit extract. Therefore column chromatography was used. A slight modification of the methods of Whistler, 23 g1. (46), and Ash (2) was used. Equal parts by weight of Charcoal Darco G 60 and Celite No. 535 were mixed well and -18.. water was added to form a slurry. The mixture was intro- duced into a 3.5 cm x AS cm chromatographic column up to height of 30 cm. A one centimeter thick Celite 535 bed was formed at the bottom of the column before the mixture was added. The same bed was placed on top of the column. The column was joined to a 2000 ml Erlenmayer suction flask connected to an aspirator. The column was never allowed to dry. Before the application of the sample, the column was washed with 500 ml of water, then a 10 ml sample of the cherry extract was introduced slowly on to the Celite bed. As soon as the sample disappeared into the column, 5000 ml of water (I) were passed through the column at a flow rate of about one ml per minute and collected. Following the elution of the column with water, 5000 m1 of 15% ethanol (II) was passed through the column, and the elutate collected in the same way as above. Eluates (I) and (II) were flash evaporated separately at 38° C and concentrated to about 5 - 10 ml. The eluate of (I) con- taining monosaccharides was paper chromatographed and the sugars present determined. The elutate (II) containing oligosaccharides was introduced into another smaller column 2 cm x 30 cm, with the adsorbent up to a height of 20 cm. The column was then developed successively with 100 m1 of water, 3%, 4%, 5%, 5%. 7%, COncentrated to about 2 - 3 ml and chromatographed on paper. RESULTS AND DISCUSSION Qualitative Analysis Paper chromatographic studies on the crude fruit extracts of all eight samples showed the presence of only glucose and fructose. It was found that this procedure was sensitive enough to detect the presence of one gamma of sugar. When more than AOO gammas of sugar were applied on the spot for chromatography, overlapping occurred and no clear separation was obtained. Thin—layer chromatography brought out three more sugars besides glucose and fructose. These sugars were thought to be oligosaccharides but could not be identified by comparing them to known sugars available. It was this method which uncovered the fact that other sugars also exist besides glucose and fructose. ‘ Using column chromatography in combination with paper chromatography indicated the presence of seven sugars in the cherries. In the elution of the column with water only glucose and fructose were removed. The remaining five sugars were eluted with 15% ethanol. Higher concentrations of ethanol (20% - 30%) were no more efficient in eluting the sugars, and lower concentrations of ethanol required -19- -20- excessively large volumes of elutant to obtain recovery of all sugars. When the 15% ethanol was concentrated and passed through the second column, it was found that 7% ethanol would elute all five sugars. They were named I, II, III, IV, V in order of decreasing Rf values, I being toward the tap of the chromatogram and V toward the bottom. With the 5% and 6% ethanol as developing solvents only sugars IV and V were eluted. At lower alcohol con- centrations only very faint traces of I and III could be eluted. The low Rf values indicated that all five sugars were oligosaccharides. The color development obtained with the different sprays used, indicated that they were aldoses and reducing type of carbohydrates. No sucrose was found in any of the elutants. 0n the basis of the area of the five spots of the above oligosaccharides, it was roughly calculated that the concentration of each sugar was about 0.001 - 0.005% in the fresh fruit. The author plans to continue working on the identi- fication of these oligosaccharides. Quantitative Analysis of Glucose and Fructose Colorimetric tests for reducing sugars and poly- saccharides have been known for a considerable time. Volu- metric procedures involving the use of potassium fericyanide, -21- ceric sulphate, copper sulfate, sodium hypoiodite are applic- able to the determination of small amounts of reducing sugars after separation by partition chromatography. However, it has been shown by many workers that these methods require considerable skill and are time consuming and sensitive to slight variations in the conditions. The anthrone reagent (11) is excellent for sugars solutions, but it is not satisfactory for the analysis of sugars separated by partition chromatography because of its reaction with the residual traces of the developing solvent. Phenol in the presence of sulfuric acid can be used for the quantitative colorimetric micro determination of sugars and their derivatives (12). Simple sugars, oligo- saccharides, polysaccharides and their derivatives including the methyl esters with free or potentially free reducing groups, give an orange yellow color when treated with phenol and sulfuric acid. The reaction is based on yellow con- densation products being formed with the hydroxyfurfurals and phenol. The color produced is permanent and it is un- necessary to pay special attention to the control conditions. The developing solvents do not interfere with this procedure. The absorption curves were determined for glucose and fructose. These are given in Figure 1. In each case ‘40 gammas of sugar was used and 0.1 ml and 0.3 ml of phenol 3Olution respectively. The maximum absorbancy for glucose 'flas at 487 millimicrons and for fructose at 489 millimicrons. -22- As the amount of phenol is increased, the absorbance increases to a maximum and then decreases. This is illus- trated in Figures 2 and 3. Reproducible results were ob- tained by operating at either side of the peak or at the peak as long as the amount of phenol added is controlled. Maximum absorbancy was obtained when 0.1 m1 of 80% phenol was used with glucose and when 0.3 m1 phenol was used with fructose. The standard curves for glucose and fructose, rang- ing from 10 gammas to 60 gammas are shown in Figure 4. The curves for five replications formed almost perfect straight lines. Since no reference was found for the use of this method for sugar extracts from fruit tissues, a study to determine whether this method could be applied was made. A recovery test with four different preparations of the sample was made. In each preparation 5 grams of glucose and 5 grams of fructose (crystal form) were added to accurately weighed samples after being blended in 95% Jhot ethanol. The added sugars were well mixed in the sample, sand the same procedure of preparation was followed as de- ascribed before in the methods section. Six determinations were made for each preparation. The percent recovery found teas 97.8% for glucose and 100.4% for fructose, with one Standard deviation being 4.89% - 0.28 for glucose, and 5-02% - 0.38 for fructose. (Appendix Table VII). -23- The results of the quantitative analysis of glucose and fructose on a wet basis of all eight pickings are pre- sented in Figure 5, and Appendix Table IV. Eight sugar analyses were made on each preparation and two preparations were made for each picking. Individual data for prepara- tion A are given in Appendix Table II and for preparation B in Table III. Figure 6 and Appendix.Tables V and VI show the re— lation of total dry matter to total sugars (glucose and fructose) on a wet basis and dry basis. The changes of glucose and fructose content of the fruit on a dry basis is illustrated in Figure 7 and given in Appendix Table VI. Appendix Table I gives the formulas used for the calculations involved. The results clearly bring out that glucose and fructose calculated on a freSh wet basis increase during the ripening of the fruit. The ratio of glucose and fructose remains about the same throughout the ripening, 1.1 and 1.0 until shrivelling of the fruit begins. The considerable precipitation between 17th July to 25th July, (0.5 " rainfall) and July 30th to August 9th (0.4 " rainfall) could probably account for the slight drop in glucose and fructose on a wet basis. On a dry basis, the ratio of glucose to fructose :remains the same. The sugar concentration increases rapidly -24- from 50% June 20th (lst picking) to 62% June 27th (2nd pick- ing) and then drops to a relatively constant level, 50% during the remainder of the harvest period. The rise which coincides with the rapid growth development of the pericarp indicates that the sugars are the components of the dry matter that are rapidly formed up to 53 days after full bloom, then other dry matter constituents increase at a more rapid rate than sugars resulting in the decrease of the amount of sugar related to dry matter. More biochemical work is needed to uncover the intermediary metabolism of the fruits. More sensitive techniques of detection should be introduced, and the modern techniques of enzymology and labelling should be used in every step of the research. -25- 0. 50 L— FRUCTOSE (489 m ) /— P !‘ GLUCOSE 0.1.0 .. ‘ ._ (487 mp) 0 >4 0.30 __ O z <: E O (0 CD <: O 0.10 __ L l l L _ I l 450 500 WAVELENGTH - MILLIMICRONS FiEQJre l.--Absorption curves of the phenolic condensation products of glucose and fructose emoosaw mo monsoonm coaummcmcaoo owaoemnm on» no headphones on» so cowpmnpcmomOo Homage no oooumman.m ensmam Aozmmm mo mzuzo unmuempman.4 shaman macaw so «.5808on 00 On oe 0m . . _ i _ d a 1 A15 50.: 385.8 I A Le 00.: 330?; (TO! 00N.0 00m.0 L 004.0 00m.0 000.0 005.0 000.0 IONVHHOSHV -29- sooao Hana scum mhmu was “memes perv meanness pump we mnemoman weapon omOposmm 0mm omOozaw Ho mmwmmnoua.m shaman HHH> HH> H> > >H HHH HH H meaeoaa a .mse H .mse mm ease ma ease «H edge a «amaze am ease *ow muse open L _ _ _ _ . _ .q _ o e b. m S O pmo>nmn Hmwonossoo I.0.m .J m 00 9 Tu d l 8 J T. 0 0 [00¢ om omouoshm s / 0 IL I“. 0 w. of a J N. I. 0.m s Ith (III. mmoosao MW 1. :1 an 0 m. I 0.0 % Total sugar -30- >53 (glucose + fructose) u+a~ - 9" Dry Wet neg Basis a f i ._ 11 __ ‘0‘ 20 _—6O 10 _. _ 9_—. T a 8 .— 15 —55 _ r 7 ‘— i L L l b 6 __ Total sugar (dry basis) b _O__O_ Total sugar (wet basis) 10 L50 _ 5 L- . . % dry matter I I l L, l l J J J Date June 20 27 July 3 9 12 18 25 Aug 1 9 Figure 6.--Re1ation between total sugars and dry matter in tart cherries Amemmn know sooHp HH5H gone meme oak .moannono pump aw Aonoposnm 0cm omoozawv summon aspen 0cm omoposmm .omoosaw mo mowcmzouu.n shaman -31- HHH> HH> H» > >H HHH HH H meHxOHa a wee H wee mm aHse mH aH2. NH AHse m AHse AN ease *om muse open a _ _ i - - - - emOposnm (I mm L m nvr, ii 4 m S L 6/\ O L r... m (L 0m as L E J ‘\\\\\\ i .m emoozao L J M I 0 0 1g On a“ m .- S mmmwsm 1309 U W D. i; a m a m 1, . 9. 9 n J u” 00 a SUMMARY AND CONCLUSIONS The carbohydrate constituents of the edible portion of the tart cherry were found to be glucose, fructose, mak- ing up almost 100% of the total sugars, and five other re- ducing oligosaccharides of the aldose type. Column chro- matography has proved to be an excellent method for the separation of minute quantities of sugars present. More work is yet to be done on the identification of the five oligosaccharides present. During ripening the ratio of glucose and fructose is constant 1.1 to 1.0 with glucose a little more than fructose. This ratio tends to become 1.0 later when shrivel- ling of the fruit begins. Dry matter during ripening increases rapidly in the beginning of the ripening period and then at a slower rate. Total sugar content (glucose and fructose) on a wet basis. follows the same curve. On dry basis the total sugars in- crease very rapidly during the stage of the rapid develop- ment of the pericarp. This occurred while the cherries are changing from pink to red. This increase was from 50% tzo 62%. Then the sugar concentration drops to 58% and remains relatively constant. The author has not come across any reference where the Dubois method (12) has been applied in fruit tissues -32- -33- analysis. An important conclusion from this study is that this method had been proved to be an excellent method for micro quantities as well as macro quantities of sugars. The method is applicable on any plant tissue as long as the sugar constituents can be sufficiently separated on the paper chromatograms. Furthermore it is a fast method com- paring it to the traditional methods of sugar analysis, and probably could be used for routine work. The accuracy by this method was found to be 100% 1_2. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. LITERATURE CITED Alm, L. Gradient elution anal sis. Acta Chemica Scanda- ngvica. 6:826-36. (1952). Ash, A. S. F. and T. M. Reynolds. water soluble constitu- ents of fruit. Austrglian J. of Chemistry 8:444. (1955). . Ketose oligosaccharides in the apricot fruit. Nature 114:602. (1954). Bacon, J. S. D. A new trisaccharide produced from sucrose by mould invertase. Chgmical Society Journal 1253 p. 2528. Binkley, W. W. and M. L. WOlfrom. Chromatographic separa- tion of cans juice constituents. J. Am. Chem. 399. 68:1720. (1946). . Recovery of sucrose from cane and beet molasses. '3} Am. Chem. Soc. 762:664. (1947). Binkley, W. W. Column chromatography of sugars and their derivatives. Adyances in ngbohydrgte Chemistry. E355'9h0 (1955 0 Block, R. J., R. LeStrange and G. Zweig. Pa er Chro to- re h . Academic Press, Inc., N. Y. pp. I7U-5Ifi. (I955) Cassidy, H. G. Ad or tion and chromato h . Inter- science Puinshers Inc., N. I. (ISSI). Consden, R., A. H. Gordon, and A. T. P. Martin. Partition chromatographic method using paper. Bioch. J. 1§;224. (1944). Dimler, R. J., W. C. Schaefer, C. S. Wise, and C. E. Rist. Quantitative paper chromatography of glucose and its oligosaccharides. Aggl. Chem. 24:1411. (1952). DDbOZLS, MO, K. A. Gill-88, J. K. Hamilton, PO A. Rebers and F. Smith. Colorimetric method for determinations of s are and related substances. Anal. Chem. g§335 “56- (1956). -34- li’i. u 7..I‘-.. a. 5 2.3.4 13. 14. 15. 16. l7. 18. 19. 20. 21. 22. 23. 24. 25. -35- Flood, A. E., J. K. Jones, and E. L. Hirst. Quantitative estimation of mixtures of sugars by the paper chro- matographic method. Ngtpre. ll§9:86. ( 947). Genevois, L. Chromatographic studies of fruit glucides. Compt. Rend. g4_:1150-1. (1955). Gherghi, A. Growth and ripening of cherries. Commun. acad. re . 0 ul re Romine. ‘§:311-1 . (1956). From ChemicaI AEgtractg, 5I:ISI35. (1957 . Hammerman, D., K. W. Bartz, and A. Reife. Separation of mixtures of nine monosaccharides by two-dimensional aigggding paper chromatography. Anal. Chem. gz:1524. Hawthorne J. R. .Microestimation of sugars separated on9f1)ter paper chromatograms. Nature. 0. l 47 . Heftman E. Chromato r h . pp. 502-33. Reinhold Publishers Corp. (I852). Hirst, E. L. and K. N. Jones. Quantitative analysis of mixtures of sugars. Part III. Chemical Sapigty Journals lgége p0 1659. Rough, L. Application of paper chromatography to the separation of the sugars and their derivatives on a column of owdered cellulose. Chemical Society Journal. _252. pp. 2511-16. 1- Horrocks, R. H. Paper partition chromatography of re- ducing su ars with benzidine as a spray reagent. Nature 0 _6_l*:lphlt 0 (19149) e Jeanes, A., C. S. Wise, and R. J. Dimler. Improved technique in paper chromatography. Anal. Chem. 2231;150 (1951). Jermyn, M. A. and F. A. Isherwood. Improved separation of sugars on the paper partition chromatography. Bioch, J. 44:402-6. (1949). Joslyn, M. A. Methods in food an%%ys§s applied to plant pyoducts. .Aca em c ress. . Kowkabany G. N. Paper chromatography of carbohydrates and rela ted compounds. Advances in C boh drate Chemigtry. 2:303-53. (I955). 26. 27. 28. 29. 30- 31. 32. 33. 34. 35. 36. 37. 38. -36- Laidlaw, R. A. and S. G. Reid. Extraction of sugars from the paper at room temperature. Ngture. g§§:476. (1950). Lane, J. H. and L. Eynon. Determination of reducing sugars by means of Fehling's solution with methylene blue as internal indicator. J. Soc. Chem. Inc. 4&332T-37To (1923). Lederer E. and M. Lederer. Chrom to re h . Elsevier Publishing Corp. N. Y. W5 . Lew, B. W. Chromatography of su are and related ro- ducts. J. Am. Chem. Soc. 6 :1449-53. (1946). Mangold, H. K. Thin layer chromatography of lipids. J. of Am. Oil Chemists' Sopiety. 18:708-27. (1961). Martin, A. J. P. and R. L. M. Synge. A new form of chromayogram employing two liquid phases. Bioch. J. 35:1358. (1941 . McCready, R. M. and E. A. McComb. Quantitative deter- mination of sugars on paper chromato am by a re- flectance method. Ana . Chem. ‘gpzl 45. (1954). McFarren, E. F., K. Brand, H. R. Butkowski. Quantitative determination of sugars of filter paper chromatograms by direct photometry. Angl. Chem. 22:1146. (1951). Munson, L. S. and P. H. Walker. The unification of re- ducggg sugar methods. J. Am. Chem. Soc. g§:663-86. 19 . Nancy, R. Gradient elution of disaccharides on a stearic aiidetreated charcoal column. Angl. Chem. 39:1295. 95 . Partridge, S. M. Application of the paper partition chromatography. Qualitative analysis of reducing sugars. Ngture. 158:270-71. (1956). . Filter paper partition chromatography of sugars. Bioch. J. 4g:238-48. (1948). . Aniline hydrogen phthalate as a spraying reagent for chromatography of sugars. Nature. 161:443. (1949). 39- 40. 41. 42. 43- 44. 45- 46. 47. 48. -37... Serini, 0. Sugars and organic acids in clingstone cherries during cold storage. Ann. Sper. AEyar. Rome. 10: (0396)3. (1956). From Elem ca A stracts. 513I9345. 195 . Somogyi, M. A. A reagent for the capper iodometric determination of very small amounts of sugar. 7;. Biol. Chem. mun-76. (1937). Stahl, E. and K. Kaltenbach. Thin layer chromatogra hy of sugars. Journal of Chroggtogyaphy. 5:351. 1961). Tukey, H. B. Growth of embryo, seed and pericarp of the sour cherry in relation to season of fruit ripening. Proc. of the Am. Soc. for Hort. Sci. 31:125-144. 3 . Taylor, 0. C. and A. E. Mitchell. Relation of time of harvest to size, firmness and chemical composition of fruit of the sour cherry. Proc. of the Amer. Soc. for Hort. Science. ég:267-71. (I953). . Soluble solids, total solids sugar content and wei ht of the fruit of the sour cherry (prunus cerasus? as affected by pesticide chemicals and time of harvest. Proc. of the Agar. Soc. for Hort. u. _6_§:12h"300 1 5 e Weill, C. E. and P. Hanks. Thin-layer chromatography pf galto-oligosaccharides. Angl. Chem. 35;l736. l9 2 . Whistler, R. L. and D. F. Durso. Chromatographic separation of sugars on charcoal. J Am. Chem. Soc. 22:677. (1950). Widdowson, E. M. and R. A. McCance. The available carbo- hydrates of fruits. Bioch. J. g2:151. (1935). Willing, H. The phonology and chemistry of the develOp- ment of the fruit of cherries. Arch. Ggrtenbau. §:561-94. (1960). APPENDICES -33- Table I Calculations W (I) General formula P = (u) (3) (10) (c) c: =$’<: a: Ba GK 1! C) Si) c: to tn grams of sugar in 100 grams of fresh fruit. gammas of sugar on the standard curve corresponding to the absorbance found. milliliters of water in which paper is eluted. milliliters of cherry extract after concentration. milliliters of eluant used in the D U (2 m1). lamda of cherry extract applied on the paper. grams of cherries of the edible portion which were used. 10 ml 100 ml 2 ml 3 or A lamdas. 90 grams From formula (I) a factor F was derived: F . (E) (S) w (II) (no (a) (10) (o) P = (F) (5) (111) -39- ll, 0’ - |I l -40- om.¢ : oo.mm mam.o m 4H. m a 00.5m 0H4.0 5 Nb. d : 00.4m Om4.0 NO. 4 : mmomm me.0 0 OH.¢ a Om.©N mhm.0 00. ¢ : whomm Nmm.o m JJ. ¢ = OO.NM mO¢.O 05.4 : “Nodm @wMeO 4 H¢. é a mh.Hm NO¢.O c5. 4 : mN.#m 00m.o m Hm.d : OO.HM NOM.O ON. 4 : Om.dm m0m.0 N ON.4 mmmma.o mN.OM Nmm.o mu. 4 mwmmH.o On.¢m mom.o H HHH MH.# : mh.dN NHm.O mH.d : mb.¢N NmN.O 0 mo.# = Om.#N OHM.O 0N.¢ : m5.MN JOM.O m mh.m : mh.NN mmN.O mm.d = OO.©N #0N.O d Ob.m : mh.NN mmN.O 0N.¢ : mb.mN NON.O m ©N.# = mB.MN ©NM.O 4m.¢ : mN.hN OOM.O N mm.m NOOH.O mN.mN mmN.O mm.¢ NOQH.O OO.©N th.O H HH mo.m : OO.HN H¢N.O m ©0.m : OO.HN OéN.O h Om.N : OO.BH mHN.O hm.N = Om.hH OOH.O 0 m¢.N = Om.©H OHN.O mm.N : MN.©H ®HN.O m OB.N : 00.0H OéNoc hm. N = Om.®H NNNoo d mO.N : OO.mH mNN.O 00. N = mh.©H dNN.O m m#.N : Om.©H OON.O 05. N : OO.©H hHN.O N m#.N OON¢H.O Om.©H OON.O MH.m oouéd.o mN.HN NJN.O H H hmmdm ‘mopomm .xcman mm :m pouomm .xcwan .oadeMIllmmMI pcoo mo macae ammo mo msqws nxoam nom\ mmasmo .nuomn¢ pom mmssmu .ngomnw umwuoshm omOosHo Amanda pozv d sowpmummoua ca omoposhm cam omoosam mo acousomnu.HH magma -41- OH.m : mb.©m NH¢.O w mm.m : mfiomm 0m#.0 B wNom : 00.mm mfiéoo wN.m = OO.mm Omd.o 0 e~.m . ma.nm «54.0 00.m : 00.0m 004.0 m m0.4 : Om.mm mdéoo moon : Om.0¢ hm4.0 d N©.d : mNomm ON#.O mm.m : Om.wm 5m4.0 m h©.# : whomm Nm¢.O N#.m : 00.0m Nd#.o N 4H.m 0meH.O 00.5m wo¢.0 04.m wwwMH.O Om.®m bdd.0 H H> mo.m : MN.N4 054.0 m m©.m : MN.OJ 4n4.0 5 MNom : MNohm H5¢o0 00.“ : mNoom 00¢.O 0 b¢.m : 00.0m H©¢.O NH.m : Omoom NH#.O m ¢m.4 : mN.mm 04¢.0 H0.m : 00.04 Nm4.0 e MN.m : mN.hm Obéoo 4m.m : Om.0m 54¢.O m Hm.4 s mN.4m mm4.0 hm.m : mN.mm nm4.0 N Hm.4 mNOJH.0 mN.¢m mm¢.0 #m.m mNOJH.0 0m.mm b¢¢.0 H > mmoé : who4m H¢J.O nm.m : Onomm dm4.0 m 0m.4 : OO.mm m¢#.0 mm.m = mN.Od ©m¢.O B m©.¢ : Om.mm MN4.0 Ohoé : Om.¢m Nmm.0 0 mm.4 : 00.mm wa4.0 ow.¢ : 00.mm 50m.0 m mmoé : OO.mm Ndéoo N¢.n : 00.0m Nd¢o0 d muod 2 OO.MM NH¢.O OH.m = “b.0m NH#.O m m©.# : Omomm MN4.0 #0.m z mNoom NH#.O N mm.c 0mmma.0 mfion 0H¢.O ho.m 0mmMHoo Omoom mdéoo H >H Ida-mam noun-mm Hows-m . Medan $13.3 0:00 HO mafiafis DBMOO HO mgcg IXOfim “>103“ mmsgb e panomfi< (~an ”mg—U eflhomp< 0m090a& OmOOAHHU emseapcooa-.HH magma -42- 0N.0 a mN.N4 554.0 m ~H.0 = mm.ae 004.0 a 0H.m : 00.nm m44.0 45.m : m5.wm 5m4.0 o 0H.m = 00.nm m44.0 mm.m = n5.®m 0m4.0 m m5.m : 00.0m mm4.0 H5.m : 0m.wm 5m4.0 4 m©.m = 00.mm 054.0 00.0 a 0m.04 5m4.0 m m0.m : 00.mm 054.0 Nm.m : mN.©m m44.0 N 0©.m 5me4H.0 «5.5m 454.0 mH.© 5mNm4H.0 0m.H4 004.0 H HHH> 5nom : 0m.5m m54.0 4©.m = 00.04 Nm4.0 w 0N.m : 00.nm m44.0 No.0 : 0m.04 5m4.0 5 mm.m : 00.0m mm4.0 mw.4 : 0m.0m 544.0 0 MH.m : 0mo4m wm4.0 mm.4 = 0m.®m 544.0 m mm.m : 00.0m mm4.0 5H.0 : 0m.H4 504.0 4 wN.m : 0m.mm 044.0 5m.m : 0m.mm 544.0 n no.“ = 00.4m Hm4.0 0m.m : mN.0m 004.0 N N0.m H©w4H.0 m5.mm wm4.0 ow.m Hom4H.0 00.0m N44.0 H HH> .Hmwfipm .HO 90.0% meam e XGNH Q .HMHWHHM “090.0% Hag” e XQMNW'Q e HHW 0m “CH PCOO HO man-ad's arr—00 HO ”ficfls luau flaw wwnfl mmsemo .mmomnd pom II, mmeamo .nuomn< omouospm II, omoonHo emsmmpaoou-.HH magma -43- NH.4 : mN.NN omN.0 N5.4 : 0m.mN HmN.0 w 40.m mmHmmH.0 mN.HN 05N.0 N5.4 mmHmmH.0 0m.mN GmN.0 5 MH.4 : m5.®N m5m.0 50.4 : m5.mm 004.0 0 mm.4 = 0m.Hm 50m.0 N5.4 = 00.4m mwm.0 m MH.4 : m5.©N 55m.0 50.m : 0n.0m 4H4.0 4 mm.4 : 0n.Hm 004.0 no.4 : 0n.mm m04.0 m m0.4 : 00.0N wom.0 m0.m : mN.©m HH4.0 N 4N.4 mwme.0 0m.0m 5mm.0 mm.4 omeH.0 m5.4m 00m.0 H HHH 5m.m : mN.mH m4N.0 04.4 : m5.mm 05N.0 m mm.m mmHmmH.0 mN.wH MMN.0 HN.4 meme.O m5.NN 00N.0 5 ©5.m : mN.5N 44m.0 44.4 : 00.Nm NOM.0 0 N5.m = m5.©N 5mm.0 mm.4 : 0m.Hm 0mm.0 m 54.m = 00.mN 5Hm.0 0N.4 = mN.0m 04m.0 4 N5.m : m5.0N 0mm.0 Hm.4 : 00.Hm ©4m.0 m mo.m : 0m.0N 4mm.0 Hm.4 : 00.Hm 04m.0 N mm.m mmme.0 m5.mN 5NM.0 Hm.4 mmmmH.0 00.Hm 04m.0 H HH NM.N : 0m.NH 00H.0 5w.N : 0m.mH w5H.0 w 0M.N memmH.0 m5.NH m0H.0 H0.m mmHmmH.O mN.0H 50H.0 5 NN.N : 00.0H NON.0 mm.N = 0m.0N 4MN.0 0 0M.N : 00.5H 4HN.0 mm.N : 0m.0N mMN.0 m 0m.N : 00.mH 5NN.0 w5.N : 00.0N ONN.0 4 0m.N = 00.5H 4HN.0 45.N : «5.0H 5NN.0 m 0m.N : 00.mH QNN.0 mm.N = m5.0N 5mN.0 N 0m.N mmwMH.0 00.wH ©NN.0 H5.N mmmMH.0 0m.©H 4NN.0 H H gamma nomwmm human .mmmHn momma heunmm 11mmmmmllldmmmflm «WNHmom Amawl ammo Ho mamas ammo mo 03:45 axofim mom mmesmo .mmomp< mom mossmo .nuomnd omomwmmm mmoode Amwmmn 9030 m cofiumumampa cw omoposum 00m mmoosHm mo pcoonomuu.HHH oHnme -44- 44.4 : 00.4N m0m.0 Nm.m = m5.0N 0Nm.0 0 0m.4 m0Hm0H.0 m5.4N mHm.0 N4.m mmHmmH.0 mN.0N mmm.0 5 05.4 = mN.4m Nm4.0 4N.m : m5.5m 5N4.0 0 05.4 : mN.4m Nm4.0 m4.m : mN.0m 444.0 m H4.4 = m5.Hm N04.0 Hm.n : mN.0m Nm4.0 4 MH.4 : m5.0N m5m.0 m4.m : 0m.0m 044.0 m 00.4 : 0m.Hm 50m.0 m4.m : mNo0m 444.0 N Hm.4 000MH.0 0m.Nm 0H4.0 Nm.m 000mH.0 «5.0m 044.0 H H> 04.4 : mN.4N 00m.0 00.m = 0m.5N mHm.0 0 4m.4 m0Hm0H.0 0m.4N 0Hm.0 0N.m m0Hm0H.0 0m.0N MNm.0 5 0m.4 : 00.nm 5H4.0 m4.m : mN.0m 044.0 0 0m.4 : 00.mm 5H4.0 N4.m : 00.0m m44.0 m mm.4 : m5.Nm mH4.0 00.m = m5.04 0m4.0 4 0N.4 : 0m.Hm 50m.0 0m.m = mN.04 4m4.0 m 04.4 = . m5.Nm mH4.0 0m.m : mN.04 0m4.0 N 00.0 00000.0 m~.~m 000.0 00.0 000m~.0 m~.0m 000.0 H > mm.4 : 0m.MN 00N.0 m0.m : mN.5N 0Hm.0 0 HN.4 .m0Hm0H.0 m5.NN 00N.0 55.4 m0Hm0H.0 m5.MN m0N.O 5 0m.4 : 0m.Hm 50m.0 N4.m : 00.0m m44.0 0 N0.4 : mN.mm 0H4.0 HN.m : 0m.5m 0N4.0 m 0m.4 : 00.mm 5H4.0 HN.n : 0m.5m 0N4.0 4 0m.4 : 00.nm 5H4.0 Hm.m : mN.0m Nm4.0 M mm.4 : m5.Nm mH4.0 50.m : 0m.0m 0H4.0 N 05.4 000MH.0 0m.4m 5m4.0 HN.m 000MH.0 0m.5m 0N4.0 H >H hmwgm .HOPOMW hmwdm e MCMHD .Hmmrpm houomnm hmwsm lo XGMHQ e U Hflmmm Jaw-CW! ”:00 HO MUCH—E Damo M0 mar—HE vavfim LG“! amen—MO eQLOMD< .Hmnm WMEEQO Ont-.0094 omopozum mmoosHo I E ummcflunoo--.HHH .0000 -45- cnuxmuvqbvwnua <3C>Ch0-¢ «abunoebdnc3r4 .00.. xnuxmunu~wkoq> m 0‘ e m .00000 mamas 5008-:- O. m0Hm0H.O : : 000MH.0 .- 000000.0 : 000mH.0 0N.mm 00.Nm m5.H4 0N.H4 00.0m m5.mm 00.H4 05.04 00.Nm 0~.Hm 00.N4 m5.m4 00.n4 00.m4 00.m4 00.m4 HO mmesmmw mmooSHo 0.0000000--.HHH 000.9 HNM4U\\Ol\m HNM4U\\OK\W HHH> HH> M¢MJMIiIdeMHmillllqmwflamm .pmompw lmcH -3000 h -46- 4HH.H 0H.HH 0N.0 00.0 00.4 00.0 40.0 00.0 HHH> 000.H 4H.HH 40.0 00.0 44.0 4N.0 00.0 00.0 HH> 0NH.H 4H.0H 00.4 00.0 00.4 00.0 04.0 00.0 H> 00H.H 0m.0H 00.4 04.0 H0.4 00.0 44.0 04.0 > 4NH.H 00.0 00.4 0H.0 H0.4 00.4 0H.0 0H.0 >H 40H.H HH.0 0N.4 40.4 0H.4 00.4 00.4 00.4 HHH 00H.H NH.0 00.0 «0.4 H0.0 00.0 ~0.4 «0.4 HH 00H.H 00.0 04.~ 00.N 00.N 40.N 4m.~ H0.N H omouospm Hmpoa omOposhm omoosao m < m 4. Iwmmxomm cmoosao omwpo>< owmpo>< .hmmmum .ummmun Iihmnunurrfi moupumno mo msmpm 00H :0 human mo msmpw 1"- Amwmmn 003. 0 van < :oapmumaoum n humeesm:n.>H magma -47- Table V.--Moieture and dry matter content Percent 6::Fercent Picking Moisture dry matter I 89.3h 10.66 11 86.85 13.15 III 86.53 15.47 IV 82.89 17.11 V 82.38 17.62 VI 82.32 17.68 VII 81.22 18.78 VIII 80.86 19.14 panama 0nd mo msmpw 00H :0 Lawn» mo newuo omouonmm, ancech 4HH.H 00.00 H4.0N 00.00 00.0w 40.0w 00.0w 4H.H0 HHH> 000.H 00.00 44.0w 00.00 00.0w 00.0w 00.H0 Nm.0N HH> 0NH.H 00.00 00.0N 00.00 04.0N 04.0w 40.00 0H.00 H0 00H.a Hm.w0 NN.0~ 00.00 00.0w 00.0m 00.00 0H.H0 > 4NH.H 00.00 00.0w 0H.00 00.0w 4N.0~ 00.00 0H.00 0H 40H.H 00.00 00.0N 0N.H0 00.0w wa.m~ m4.a0 00.H0 HHH 00H.H 00.H0 00.0w 0m.N0 04.0w 40.00 0m.m0 0m.N0 HH 00H.H 0a.00 0H.0N 00.0w N4.NN 00.0w 40.0w 00.0w H .mmwwfim H38 .Mmfiwfi “0&5 5%.: 5&5 .3095 .30.; megs Amammn 0uuv m cam < :oapwamaoua n 0pweasmuu.H> oapwa 0N.0 00.4 0m.® N0.4 : 40.0 0 0H.m 00.4 Nboa wnoé : Owom 0 00.4 00.4 4Noo whod : Owom 4 4H.00 00.4 00.4 0H.0 00.00 00.4 = 00.0H 0 00.4 00.4 dmoa 0m.4 = wOoOH N 00.0 00.4 00.0 00.0 NN.0 N0.0H H 00.0 = HN.OH 00.0 : 00.0 0 HH.0 : N0.© 00.4 : HMom 0 4 H H OM.4 : Hm.® 00.00 00.“ : MM.© M O . O O . g . . = . 3m .. $0 $3 .. 3m N HH.0 H0o4 N0.0 00.0 00.4 00.0 H 00.0 H0.4 40.0 0N.0 00.0 00.0H 0 Hm.4 H0.4 N0.0 00.4 00.0 00.0 0 Hw.4 H0.4 N0.0 00.4 00.0 40.0 4 00.00 00.4 H0.4 00.0 00.00 00.4 00.0 40.0 0 00.0 H0.4 00.0H 04.4 00.0 N0.0 N Hmom H0.4 NN.OH 0Hom 00.0 NN.OH H 0 mean 0W, ma mucosa .owH om 0um>oomm omOpusgm upmonm00 vmuum 0hm>0nmm omoosaw cumuampm cmvvm uoon< . oagEmm conu< + «anew» vhmvnwpm wumwcm0m mewuw n oneneshm nawuw a oncosao ‘III {chill pmou 0uo>Ooomuu.HH> magma 00.0 H No.0 0ho>Ooom mm.0 H 00.4 0ho>ooom I aoapma>ov I :oaam«>mv 4000.0. uumvcmpm mm0~.0+ uuquaom Aw 4.00M 0 0uo>0000 40.00 0 0ho>ooom J; 0H0.0 ommho>< «00.4 omwuo>< 00.0 00.4 40.0 00.4 = H~.0H 0 0H.0 00.4 00.0H 0H.0 : 00.0H 0 00.00H 0H.0 00.4 00.0H 40.4 : 04.0H 4 H0.4 00.4 00.0 00.00 00.4 : H~.0H 0 00.0 00.4 40.0 0H.0 : 00.0H N H0.0 00.4 00.0H 0H.0 00.0 00.0H H 0 nemum camemm omoposhm 0 msmgm mamsmm omoosaw Afiamom 0uo>Oomm cacaosum uumunmum vouvw 0uo>000m cmoosaw vumvampm umvvm nevu< + mamawm vouu< + mamamm wumunmpm vumuampm madam n amouOSLm madam n omoosao ; 005:0»:0011.HH> oHnma = H- | ll|||||||||Wlllfl|||||||fl||||H||U|H|||H||||H|||||H|HI 3129310202380