Cigar Tobaccos. Chemical Changes, that Occur during Curing. C. O. Jensen. Ind. Eng. Chem. , 1952, 44 (2), pp 306â309. DOI: 10.1021/ie50506a024.
Publication Date: February 1952. ACS Legacy Archive. Note: In lieu of an abstract, this is the article's first page. Click to increase image size Free first page.
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CIGAR TOBACCOS Chemical Changes that Occur during Curing Considerable modification of the chemical composition of cigar leaf tobacco takes place during air curing. In addition to the obvious loss of water there is a decrease of from 10 to 30% in the dry weight of leaves. Greater curing losses take place in leaves attached to stalks than in leaves removed from the stalks (primed leaves). Potassium and phosphorus migrate from the leaf during stalk-curing whereas calcium and magnesium do not. During curing, starch and sugars decrease rapidly but no loss of crude fiber takes place. There i s a marked decrease of malic acid accompanied b y a large increase of citric acid. A rapid destruction of chlorophyll occurs. Changes in the nitrogenous compounds are particularly evident as shown b y the disappearance of 30% or more of the protein fraction and a large increase in water-soluble nitrogenous compounds. These soluble compounds include amides, ammonia nitrogen, thiamine, and pantothenic acids. A small fraction of the original nicotine disappears.
C. 0.JENSEN The Pennsylvania Stale College, State College, Pa.
Jeffrey ( 2 0 ) who found that optimal relative humidity was 65 to 70% a t the usual curing temperatures. T w o methods of harvesting cigar-type tobacco are employed in this country. The greater proportion of the crop is harvested by cutting the stalk, placing the stalk with attached leaves on lath, and hanging these laths, leaf tips down, in the curing barn. Shade-grown cigar-wrapper tobacco is harvested by picking individual leaves as they mature and placing the detached leaves on cords attached to sticks suitable for hanging in the curing shed, This method of harvesting by detaching individual leaves is called “priming.” Methods of curing cigar tobacco have been described by several authors (12-14, 37, 46). The gross appearance of cigar-type tobacco during air-curing changes progressively from green through yellow to brown, although the yellow stage may be hardly noticeable in stalk-cured filler types. The tobacco may reach the yellow stage in 6 days when prime-cured and to be light brown in 10 days (44). However, stalk-cured tobacco does not usually reach the uniformly brown stage for at least 3 weeks. Curing ordinarily takes 8 to 12 weeks and changes in composition take place even after the leaves are uniformly brown (45).
RESHLY harvested cigar tobacco must be cured, aged, and fermented before i t is considered acceptable for use in cigars and chewing tobacco. The manufacturer or his agent controls the processes of aging and fermentation whereas the tobacco grower is responsible for the curing process. Although the general procedure employed by each grower may be similar, the rate of curing and the quantitative changes in composition of tobacco as a result of curing may vary significantly in a given district. A much greater variation in the changes that occur during curing is found in the cigar leaf grown in different regions of the earth and even in different districts of this country, where climate, soil, strain of tobacco, and methods of curing vary. The general topic of curing of all types of tobacco has been ably discussed by Frankenburg (8). His review includes a comprehensive survey of foreign as well as American literature. The present article k concerned only with the air-curing of cigar-type tobacco and is further limited, for the most part, to a summary of investigations on strains of cigar tobacco grown in North America. Air-curing is also called “natural curing” to differentiate the process from the methods that employ high temperatures. Examples of such high temperature processes are fire-curing and flue-curing. Conditions that exist during air-curing are largely determined by the prevailing weather, as modified by the building in which the process takes place. Certain growers may employ forced air and artificial heat for a few days to create more favorable curing conditions but this is not the usual practice. Only shade-grown cigar-leaf wrappers are regularly treated with artificial heat for a short period in the early stages of curing. The purpose of heating devices, when employed in the curing of binder and filler types, is not to create an artificial climate but to restore, if possible, the propitious conditions of temperature and relative humidity that ex’st when the weather is favorable. Such heating devices ordinarily do not raise the temperature of the curing barn more than about 10” to 15” F. above the outside temperature ( 1 , 28). Descriptions of optimal conditions of temperature and humidity for the curing of cigar tobaccos have been given by Bucher and Street ( Z ) , Johnson and Ogden (22), Garner (14, 15), and others (3, 47). These can be summarized by stating that the satisfactory temperatures are those between 70’ and 95 F. with relative humidities of about 60 to 75y0. These recommendations are similar to those for burley tobacco as determined by
Loss of Water and Dry Matter The most obvious change that occurs during curing is a loss of water. The original moisture content is about 85 to 90%, and the final moisture content of the cured leaf is about 20 to 25%. When the weight of water evaporated is compared to the original water in the sample of tobacco under investigation the per cent of moisture lost is found to be over 95% (37). Johnson and Ogden ( 2 2 ) observed that primed Wisconsin tobacco leaves, under average conditions of curing, lost two thirds or more of the water in the first 2 weeks. For the same period about one half the original water was lost from leaves cured on the stalk. The progressive loss of weight as curing proceeds is caused not only by the evaporation of water but also by a loss of dry matter. Approximately 10 to 20% of the total solids disappear during the curing of primed leaf, whereas 20 to of the dry matter of stalk-cured leaf may be lost. The rate of loss of water, dry matter, and total weight from primed Connecticut leaves is shown in Table I. The values presented are taken from the data of Vickery and Pucher (39). The first study of American cigar-leaf tobacco which includes a
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
February 1952 Table 1.
Loss of Water, Dry Matter, and Total Weight of Leaves during Curing ( 3 9 )
Total weight Water Dry matter
(For 50 kilos of primed leaves) Loss in % of Fresh Weight of Harvested LeafCured Cured Cured 12 days 18 days 51 days 77.90 86.64 59.30 75.46 84.07 57.66 2.57 1.74 2.44
Table II. Loss o f Dry Weight during Air-Curing of Connecticut Sun-Grown Tobacco (77) (For 100 leaves) Loss in % of Dry Weight of Harvested Leaves Primed Stalk-cured 1909 1910 I909 1910 Whole leaves Dry matter Or anic matter A*%O Leaf web Dry matter 2 ; g n i c matter Midribs Dry matter Organic matter Ash = % gain
10.7 11.3 0.6+
13.5 13.9 0.4+
29.8 28.4 1.4
24.7 23.3 1.4
6.5 7.4 0.9+
8.5 9.1 0.6+
21.4 21.0 0.4
16.1 15.8 0.3
4.2 3.9 0.3
5.0 4.8 0.2
8.4 7.4 1.0
8.6 7.5 1.1
Migration of Ash Constituents from Leaves to Stalks during Curing (24) Analysis of 100 Leaves Primed Stalk-cured, mg./leai mg./leaf 234 77 94 31 7 12 6
comparison of the composition of cured leaf to the uncured leaf was made by Garner, Bacon, and Foubert in 1914 ( 1 7 ) . They investigated the changes that take place during the curing of Connecticut sun-grown tobacco. The changes in dry weight which occur during curing of primed leaves are compared to the changes occurring in leaves attached to the stalk (Table 11). The variability of results is well illustrated by these data which were secured on the 1909 and 1910 crops of the Halladay strain of Connecticut sun-grown tobacco. Large differences from the changes as reported by Garner, Bacon, and Foubert can be found in the literature because of variation of weather conditions during the growing and curing season, variations in the original composition of cigar leaf, lack of uniformity in the construction or curing barns and other factors. The results secured by Garner, Bacon, and Foubert allow two significant conclusions: ( a ) Translocation of ash constituents to the stalk is indicated when leaves are attached during curing; and ( b ) the loss of dry matter is much greater in stalk-cured than in primed leaves. Migration
calcium or magnesium takes place. Since no loss of calcium occurs during curing, the apparent increase of calcium in cured leaves has been used to calculate the loss of dry weight and usually checks fairly well with the actual loss of dry matter (9). Although it is possible that certain soluble organic compounds may migrate from leaves to stalks, the greater disappearance. of organic matter from stalk-cured as compared to primed leaves is accounted for, in large part, by increased catabolic reactions in the slower drying leaves. Changes in Organic Matter
The approximate changes of organic matter,as a result of curing primed leaves and leaves attached t o the stalk have been summarized in Table IV. The values given cannot be considered as typical of either wrapper, binder, or filler tobacco but are an estimated average of the composition-*ofall three. Frankenburg (8) has divided the compounds of the tobacco leaf into three groups:
(A) The static group, containing such components as inorganic substances (ash), oxalic acid, ether-soluble resins and fats, pentosans, and crude fiber. These com ounds of the static group undergo little change during curing, w i t , the exception that ash constituents may migrate when leaves are attached to the stalk. (B) The dynamic group composed of metabolizable carbohydrates, readily oxidized ether-soluble organic acids, and undetermined labile organic compounds. (C) The nitrogen group including proteins, amino acids, amides, alkaloids, ammonium compounds, and nitrates. The nitrogen group of compounds chan es considerably as a result of curing, both by transformations of one compound into another and by loss of weight. Nitrogenous Compounds. A steady loss of nitrogen takes place as the curing process advances (44). Not only is there a loss of total nitrogen but the distribution of this element among various compounds changes materially. The changes in nitrogen distribution have been determined by Garner, Bacon, and Foubert ( 1 7 ) and by Vickery and coworkers (39,43,44)for primed leaf; and by Garner et a]. (17), Haley and Haskins (18),and Frankenburg (8) for stalk-cured cigar tobacco (Table V). The most evident changes t h a t occur in the nitrogenous comyounds are 'a loss of protein and an increase of water-soluble nitrogen compounds. The hydrolysis of protein proceeds to the amino acid stage since little increase of peptide nitrogen is noted (4.4). Amino nitrogen compounds increase'but not in amounts Table IV. Changes in Organic Constituents as a Result of Curing Primed Leaves and Curing on the Stalk Amount in % of Harvested Dry Weight Composition after curing Primed Stalk-cured 24 - 2 - 7 9 0 - 1 10 0 0 2 - 2 - 2 3 - 2 - 3 11 - 1 - 1 10 - 2 - 5 15 - 6 - 6 * 84 15 -25
Composition before curing Nitrogenous compds. Pectic substances Crude fiber Starch Sugars Or anic acids E t f er extract Other organic compds. Total organic compds.
of Compounds ~
The transfer of ash constituents from leaves through midribs t o stalks had been indicated first by Johnson in 1893 (23)through a n analysis of stalks from mature plants and stalks after curing with attached leaves. He reported that during the curing period, stalks gained 30% of their original content of nitrogen, 36% of their content of phosphorus, and 8% of their content of potassium. An actual analysis of similar leaves attached to and detached from stalks was made by Mohr (24) (Table 111). A definite loss of potassium and phosphorus and a probable loss of sulfur and chlorine by stalk-cured leaves is indicated. No migration of
Changes of Nitrogenous Compounds during Curing
[Approximations from data of various authors (8, I T , 18,SO, 44)] Change in % of Amount in o/ of Initial Amount Dry Matteor, Mature Primed Stalk-cured Total nitrogen 5.0 - 10 - 30 Protein N 3 5 50 50 Alkaloid N 0.5 - 5 - 5 Nitrate N 0.5 25 +25 0.05 4-500 Ammonia N Amide N 0.05 +200 ... Amino N 0.2 +350 ...
INDUSTRIAL AND ENGINEERING CHEMISTRY
Effect of Curing on Certain B-Vitamins of Cigar Tobacco (5)
(1847-1848-1948 crops of Pennsylvania tobacco) Amount Der 100 Grams Initial D i y Weight, Mg. Gain or Mature Cured LOSS, % 34.8 + 1 Nicotinic acid 34.4 50 0.76 Riboflavin 1.5 5.1 Pantothenic acid 3.2 4- 6 0 1.6 100 Thiamine 0 8 .
Table VII. Changes in Soluble Carbohydrates during Curing of Primed Leaves ( 4 4 ) (Carbohydrates soluble in boiling water a t pH 4) Material Derived from 1000 Grams of Components Fresh Leaves, Grams Expressed 8s Time after harvest, hoursGlucose 0 41 159 303 3.19 1.60 5.75 Total 4.96 0.52 3.64 1.51 Fermentable 3.07 1.08 2.11 1.68 Unfermsntable 1.89 ? -
Organic Acids of Cigar Tobacco before and after Curing
% of Initial Dry Weight Primed Stalk-cured Connecticuta Pennsylvania b Before After Before After curing curing curing curing 1.8 2.7 2.9 2.0 Oxalic 4.8 6.8 3.6 5.8 Malic 3.4 2.9 4.7 0.6 Citric Unknown (as malic) 2.5 0.5 0 Calculated from data of Vickery and Pucher (40) b Calculated from data of Haskins (19).
equivalent to decreased protein nitrogen. Presumably a considerable fraction of the protein nitrogen becomes the amide nitrogen of asparagine and glutamine. The increase of ammonia nitrogen occurs in quantities greater than can be accounted for by the deamination of amino acids. Thus some ammonia probably is formed from undetermined compounds ~ 4 4 ) . The disappearance of a variable amount of nicotine is observed during air-curing. I n some crops this loss is extremely small (44), whereas in others the loss may amount to 25y0 of the initial amount (17). As a rule, a small loss of nicotine occurs during natural curing. Since the results quoted here are from older work, the term nicotine is synonymous with “steam volatile alkaloids.” A survey of certain constituents of the B-vitamin complex in Pennsylvania cigar leaf ( 5 ) has shown that an increase in pantothenic acid and thiamine takes place during air-curing (Table VI). No change was found in the nicotinic acid content and a significant decrease occurred in riboflavin. The values reported for nicotinic acid are in fair agreement with those of Frankenburg and Gottscho (11). Crude Fiber, Pentosans, and Pectic Substances. Results of analyses of cured and uncured cigar tobacco have shown no change in crude fiber content during curing (17, 39). Pentosan content of leaves attached to or removed from the stalk does not change during natural curing, according to the results of Garner, Bacon, and Foubert ( 1 7 ) . The pectic substances of cured cigar leaf have been reported as 8 to 9% of the dry matter (16). The midribs contained about twice as much pectin as the leaf web. A moderate loss of pectin is reported to take place during aircuring (8). Starch. Pennsylvania cigar tobacco of the Swarr-Hibshman atrain contains starch with an amylose content of 23% and an
Vol. 44, No. 2
amylopectin content of 77y0(48). Thus this leaf starch is quite similar to cornstarch in its ratio of amylose to amylopectin. Periodate oxidation studies have shown that tobacco leaf amylopectin contains 26 glucose residues per nonreducing end group (89), a value which is similar to the values obtained with amylopectin from storage organs of plants. The estimated chain length of tobacco leaf amylose was about 40 to 47 anhydroglucose units (89). After curing no starch could be separated by calcium chloride extraction methods for amylose and amylopectin determinations (48). Ward (45) has shown that at the half-cured stage Canadian stalk-cured cigar tobacco had lost about 90% of its starch content. During this same period there was an increase of dextrins, although the actual amount, after curing, was less than the amount of starch. ‘ Usually, however, starch has been found to be absent in fully cured cigar tobacco (16, 48). Sugars. The total amount of sugars in freshly harvested cigar tobacco varies with the strain, soil type, fertilization, crop year, maturity, and the analytical methods employed (8). Although Ward (46) found 2.6% of reducing sugars to be present after 2 weeks of stalk-curing, the fully cured cigar leaf may contain only a few tenths of 1% of reducing sugars (16, 17, 45). No sucrose was found in cured cigar tobacco by Garner, Bacon, and Bowling (16). Vickery, Pucher, Wakeman, and Leavenworth (44) followed the changes in carbohydrates that were extracted from leaves as a result of boiling for 1 hour in water a t p H 4. Compounds soluble under these conditions were subdivided into fermentable and unfermentable carbohydrates. The composition of this extract is shown a t four time intervals after harvest (Table VII). Carbohydrates soluble at p H 4 will probably include dextrins and starch as well as sugars. I n these particular samples only 3.7% of the dry weight of mature leaves was found in the soluble fraction, whereas the analysis of a previous crop (S9) had yielded 5.2% of soluble carbohydrates. That a considerable part of this carbohydrate was either glucose, fructose, or mannose was demonstrated by the isolation as phenylglucosazone of 48% of the sugar in the extract (39). Complete disappearance of either the unfermentable or fermentable fraction did not take place. Organic Acids. Extensive investigations of the organic acids of cigar tobacco have been made by Vickery, Pucher, and coworkers ( S I , 38, 40, 42). They have made significant improvements in the determinations of nonvolatile acids in biological materials (32-36). The most important known organic acids that are present in tobacco are oxalic, citric, and malic acids. The amounts of these acids that have been found in typical samples of cigar tobacco are shown in Table VIII. The values presented in Table VI11 have been calculated from the data of Vickery and Pucher (.bo),secured from the analysis of primed wrappers, and from the data of Haskins ( 1 9 ) , secured from the analysis of stalk-cured filler tobacco. Oxalic acid undergoes little change in either primed or stalkattached leaves during curing (19, 31, 40). However i t is characteristic of the air-curing process that malic acid decreases and citric acid increases; the increase of citric acid is roughly proportional t o the decrease of malic. This process probably takes place by transformations of the tricarboxylic acid cycle (30). Ether-Soluble Compounds. The ether extract of tobacco leaves contains a number of unrelated compounds. The principal components of the extract are paraffin hydrocarbons, terpenes, alcohols, fatty acids, phenolic acids, waxes, fats and oils, volatile oils, resins, sterols, and plant pigments. Although small amounts of free’acids, such as malic and citric, and free alkaloids are removed by ether, these substances do not form a large fraction of the extract (8). The volatile oils of tobacco leaves, found in the ‘‘gum” which covers the surface of the leaves, are believed to form an important fraction of the compounds that produce the characteristic odor of
INDUSTRIAL AND ENGINEERING CHEMISTRY
burning tobacco (8). The effect of natural curing on the volatile oil content of American cigar tobacco has not been reported. The less volatile compounds of the ether extract are called tobacco resins. Cured cigar tobacco contains about 5 to 10% of etherextractable substances (16,41) of which the greater part is resin (16). Total ether-soluble substances decrease during air-curing of cigar leaf, the decrease being about 20% of the initial amount in primed tobacco (39)and about 50% in stalk-cured leaves (14). Plant Pigments. The disappearance of chlorophyll when tobacco leaf is being cured can be observed as the green color of the mature leaf changes t o yellow and finally t o brown. The chlorophyll content of green leaves may vary from 0.6 to 4% (IO). Dunlap reported that the amount of chlorophyll in harvested Connecticut cigar leaves was about 1.4% of the dry weight (6). During curing this rapidly decreases. Vickery and Pucher (@) found that the destruction of chlorophyll was practically complete after 12 days of curing primed leaves. Wheq abnormally rapid drying takes place, much of the chlorophyll will be,unchanged and the cured leaf will have a green color. The total amount of the yellow pigments, carotene, and xanthophyll is about one fifth to one third that of chlorophyll in mature tobacco leaves (6). The p-carotene content of samples of the 1950 crop of Pennsylvania cigar tobacco was about 0.014% of the dry weight before curing. After stalk curing p-carotene had decreased to 0.005% of the initial dry weight, a loss of over 60% of the pigment (11). Evidently 8carotene constitutes only a small fraction of the yellow pigments. Phenols, Polyphenols, and Tannins. The yellow rompounds, visible during the second stage of curing, are later masked by the brown pigments t h a t develop aa curing proceeds. The brown colors are believed t o be formed partly from substances of the phenol, polyphenol, and tannin classes (8). One of the constituents of this group of compounds is the phenol glucoside, rutin. Although green tobacco leaves have been found t o contain about 1% of rutin (.27),the air-cured leaf has been shown b y Couch and Krewson t o contain no rutin ( 4 ) . The product of r u t h oxidation is dark brown and probably comprises an important fraction of the brown compounds t h a t appear in the final stages of natural curing (16). The determination of phenols in%obacco has been reported b y Naghski, Beinhart, and Couch (15). Their method consisted of distilling the phenols with steam from acidified samples, treating the distillate with the Folin phenol reagent (7), and comparing the light absorption at 7000 A. with a standard. T h e results were regarded as reasonably accurate but not precise. B y this method cured Pennsylvania cigar leaf contained 0.038% phenols, calculated as catechol. No values were found for the phenol content of uncured leaves. The method for the determination of the heterogeneous group of substances called polyphenols and tannins is quite uncertain. In a typical method total reducing substances are determined and a correction applied for reducing sugars. The remainder is called polyphenols and tannins (8). Reports of changes in total polyphenol and tannin content during curing of American cigar tobacco have not been found.
Enzymes Harvested tobacco leaves continue to exhibit some of the characteristics of living material during the curing period. One of these characteristics is the utilization of enzymes for the control of chemical changes. Certainly enzymes play the principal role in determining the changes t h a t occur in the early stages of curing and are probably important at all stages. Tobacco enzymes and their importance in the curing process have been described and thoroughly discussed by Frankenburg (8). A review of the subject is beyond the scope of this paper. However, our information on the role of enzymes in curing cigar tobacco is elementary when considered from the viewpoint of the newer knowledge of enzymology.
Literature Cited Anderson, P. J., Conn. Agr. Expt. Sta., Bull. 517 (1948). Bucher, F. S., and Street, 0. E., Penna. Agr. Expt. Sta., Leaflet 71 (1940). Chapman, 0. H., Conn. Agr. Exp. Sta., Tobacco Sub-station, BUZZ.3 (1922). Couch, J. F., and Krewson, C. F., U. S. Dept. Agr., Eastern Regional Research Lab., Rept. (July 1944). Dohner, J. F., M.S. Thesis, The Pennsylvania State College (1951). Dunlap, A. A., Phytopathology, 18, 697 (1928). Folin, O.,and Ciocalteu, V., J. Biol. Chem., 73, 627 (1927). Frankenburg, W. G., Advances in Enzymol., 6, 309 (1946). Frankenburg, W.G., Arch. Biochem., 14, 157 (1947). Frankenburg, W.G.,Sozlthern%hemist, p. 315 (March 1949). Frankenburg, W. G.,.and Gottscho, A. M., Arch. Biochem., 21, 247 (1949). Frdar, *W., and Hibahman, E. K., U. S. Dept. Agr., Farmer's Bull. 416 (1917). Garner, W. W., U. S. Dept. of Am., Bur. Plant Ind., Bull. 143 (1909). Garner, W. W., Ibid., Farmer's Bull. 523 (1913) [Revised by J. E. McMurtrey, Jr., and C. W. Bacon (1947)l. Garner, W. W., "Production of Tobacco." pp. 155-78 and 398-408,Philadelphia, Blakiston, 1946. Garner, W. W.,Bacon, C. W., and Bowling, J. D., IND. ENO. CHEM.,26, 970 (1934). Garner, W. W., Bacon, C. W., and Foubert, C. L., U. S. Dept. Agr., Bull. 79 (1914). Haley, D. E., and Haskins, A. L.,The Pennsylvanis, State College, unpublished results. Haskine, A. L., M.S. Thesis, The Pennsylvania State College (1944). Jeffrey, R. N.,Ky. Agr. Expt. Sta., Bull. 496 (1946). Jensen, C. O., and Looker, J. B., unpublished resulta. Johnson, J., and Ogden, W. B., Wis. Agr. Expt. Sta., Research Bull. 110 (1931). Johnson, S. W., Conn. Agr. Expt. Sta., 16th Ann. Rept., p. 31 (1893). M o b , E. C. J., Landw. V e r s J t a . , 59,253 (1903). Naghdci, J., Beinhart, E. G., and Couch, J. F., IND.ENO. CHEW.,36, 556 (1944). Neuberg, C., and Kobel, M., Enzymologia, 1, 177 (1936). Neuberg, C., and Kobel, M., 2. Unterszcch. L e b e m . , 72, 113 (1936). Olson, O.,and Haley, D. E., Penna. Agr. Expt. Sta., Bull. 240 (1929). Palmer, J. K.,M.S. Thesis, The Pennsylvania State College (1950). Pucher. G. W., and Vickery, H. B., J . Bwl. Chem., 178, 557 (1949). Pucher, G. W.,and Vickery, H. B., Proc. Natt. Acad. Sci., 19, 623 (1933). Pucher, G. W.,Vickery, H. B., and Leavenworth, C. S., IND. ENQ.'CHEW.,6, 190 (1934). Pucher, G. W., Vickery, H. B., and Wakeman, A. J., IND. ENG. CEEM.,ANAL. ED., 6, 140 (1934). Ibid., p. 288. Pucher, G. W., Vickery, H. B., and Wakeman, A. J., J. Biol. Cham., 97,605 (1932). Pucher, G. W., Wakeman, A. J., and Vickery, H. B., IND. ENQ. CEEM.,ANAL.ED., 13.244 (1941). Street, 0. E., U. 8. Dept. Agr., Farmer's Bull. 200 (1948). Vickery, H. B., and Abrahame, M. D.. J . Biol. Cham., 180, 37 (1949). Vickery, H.B., and Pucher, G. W., Conn. Agr. Expt. Sta., Bull. 324 (1931). Vickery, H. B., and Pucher, G. W., Ibid.,352 (1933). Ibid., 374 (1933). Vickery, H. B., and Pucher, G. W., J. Biol. Chem., 90, 637 (1931). Vickery, H.B.,and Pucher, G. W., Science, 73, 397 (1931). Vickery, H. B., Pucher, G. W., Wakeman, A. J., and Laavenworth, C. S., "Chemical Investigations of the Tobacco Plant," Washington, D. C., Carnegie Inst. Washington (1933). Ward. G. M.. Can. DeDt. Am.. Tech. Bull. 37 (1942). Whitney, M.; U. S. Dept. A&.: Farmer's Bull. 60 (1902). (47)Whitney, M.,U. 8. Dept. Agr., Rept. 63 (1900). (48) Zelitch, I., M.S. Thesis, The Pennsylvania State College (1948). RaralPIvln August 8 , 1951. Authorized for publication on Ootober 19, 1951, No. 1692 in the Journal Seriea of the Pennsylvania Agricultural Experiment Station.