SEED HEADSCOVERED WITH PAPERBAGS TO PREVENTCROSSPOLLINATION ON A TOBACCO SEEDFARM
E
Flue-Cured Cigarette
QA
LL TOBACCOS, whether air-cured, suncured, fire-cured, or flue-cured, are not suited for consumption in manufactured tobacco products immediately after curing. Depending upon the type of tobacco, method of aging, and local terminology, such tobaccos are referred to as “raw,” “green,” “unfermented,” or “unaged.” These terms refer to the presence of irritating properties, the lack of aroma, and to differences in the burning qualities of the tobaccos a t this stage. After curing, tobaccos are subjected t o fermentation and aging or aging according to the methods found peculiarly fitted to the particular type, These methods vary, from the very wet (40 per cent moisture content) pile sweats of cigar tobaccos in which high temperatures are reached (49’ C.) where there is considerable pyrogenesis (22” C.) and an abundance of carbon dioxide and ammonia is evolved, to the mild aging that flue-cured cigarette tobaccos undergo. The latter proceeds a t low temperatures (3” to 26” C.) and at low moisture content (8.5to 10.5per cent), with very little (if any)
Tobaccos
L. F. DIXON, F. R. DARKIS, F. A. WOLF, J. A. HALL, E. P. JONES, AND P. M. GROSS Duke University, Durham, N. C.
1
A previous paper in this aerie8 appeared in 1935 (6)
180
FEBRUARY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
self-heating, very little evolution of carbon dioxide, and the evolution of only traces of ammonia. I n all types of fermentation or aging there are appreciable changes in chemical composition, physical properties, color, aroma, burning quality, taste, and smoking quality. The most pronounced changes occur in the fermentation of cigar leaf, whereas the changes in flue-cured cigarette tobaccos during the protracted aging, although just as essential and perceptible, are not so pronounced. Following Nessler (SO), numerous workers have attacked the problem of tobacco fermentation from many angles. Adequate reviews of these investigations are available (6, 17, 20,21 , 2 4 , 3 6 , @ ) . All the literature deals with the fermentation of air-cured tobaccos (largely cigar types) that have not been subjected to thermal curing processes and therefore could differ in chemical composition ( l l ) , in bacterial and and enzyme content, from fungous flora and population (M), flue-cured tobaccos that are cured at high temperatures (maximum, 93" C. for 12 to 18 hours) (10, 12) and subsequently redried a t moderately high temperatures (66' to 82°C. for 25 minutes). Furthermore these earlier studies were made with tobaccos of high moisture content (20 to 52 per cent) which showed considerable degree of pyrogenesis and appreciable evolution of carbon dioxide. The aging of flue-cured tobaccos is the extreme type of lowmoisture-content aging and contrasts markedly with fermentation studies described in the literature. The behavior of air-dried tobacco under such conditions has been observed by many workers to be quite different from the usual cigar type fermentations (8, 9, 17, 90, 30). Here it is hoped that the study of the aging of flue-cured tobacco, with its associated high thermal processes prior to aging, may yield information on fermentation studies in general as well as provide specific information relative to this stage in the handling of one of our most important tobacco crops.
Material
181
reconditioned, graded, tied into bundles, and marketed. This period ranges from a few weeks to 4 months, depending upon the tobacco type and the marketing conditions. Flue-cured tobacco is marketed a t a moisture content of 15 to 25 per cent. Probably by reason of the high sugar content of these tobaccos, spoilage occurs quite readily in the upper range of moistures, and necessitates a thorough drying and reconditioning to a proper moisture content by the purchaser. This latter process is known as redrying, and is carried 0u.t by the use of tobacco-redrying machines which are modified textile driers. Since no data for the redrying process are available in the literature, curves showing the ordinary conditions of drying and reordering are given (Figure 1) so that cognizance may be taken of this process, as well as the fluecuring conditions, when considering the aging behavior of this tobacco.
Sampling The samples for this work were commercial grades of flue-cured cigarette tobacco. Initial samplings for analysis and observations were made immediately after prizing the redried hogsheads. These hogsheads are 48 inches in diameter, 54 inches high, and 56.5 cubic feet in capacity, and contain approximately 1000 pounds of leaf tobacco. Immediately after the initial sampling, the hogsheads were stored in an unheated well-ventilated tobacco storage (approximately 75 X 125 X 15 feet). The conditions of storage were those for the commercial a ing of this type of tobacco in every particular. Samples were taten at 6-month intervals (April and October). Six hogsheads of each type were sampled in each crop year, at each of the intervals mentioned for a period of 30 months. The samples for chemical analyses were taken in the same manner as described in a previous article (6). Samples for the determination of fungous and bacterial flora were taken with a 1-inch (2.5-cm.) sterilized auger; the tobacco particles were allowed to fall from the auger into a tared, sterilized, screw-top jar, whose top was momentarily removed to allow the tobacco t o enter. The samples were carried immediately to the laboratory and weighed, and the contents poured into a flask containing 200 cc. of sterile water to prepare the suspension. Samples of the air from about the middle of the hogsheads were obtained at the same time for the determination of carbon dioxide concentration. This was accomplished by means of a long capillary steel tube attached to a 250-CC.filter flask equipped with a stopcock. The flask had previously been evacuated to 0.1 mm. of mercury.
The present studies deal entirely with flue-cured cigarette tobacco which in chemical composition (6, I l ) , method of curing (10, 12), method of aging, and criterion of quality is vastly different from the cigar types. These differences are such as to effect marked differences in fermentation behavior and necessitate some description of the material and its pre-aging treatment, The aging, under industrial conditions, of the six main In flue-curing the harvested leaves are types of flue-cured tobacco for three successive crops was suspended from sticks in rather tight barns studied for 30 months. over sheet iron heating flues which rest upon Environmental conditions in these tobaccos were not the dirt floor of the barns. The method confavorable for the activity of bacteria and molds, and sists of an initial starvation process lasting 36 to 48 hours a t a temperature of 32" to 41 O C. only small amounts of them were found in the tobacco during which time the tobacco loses its at any time. I t is believed that these agencies played a chlorophyll and becomes yellow. The temvery minor part in the aging process. Little self-heating perature is then raised to 43-49' C. for about (only at the beginning of the period of aging) took place. 12 hours to "set" the color. The temperaSmall amounts of carbon dioxide, acetic acid, formic ture is then increased to 52-60" C. and maintained for approximately 24 hours until all acid, and ammonia were evolved during aging. parts of the leaf except the midrib are dry. A chemical study, covering changes taking place over Next the temperatures are gradually raised a 30-month period, of several groups of constituents to 82-93" C. and maintained from 12 to 18 shows an increase in moisture and a decrease in sugar, hours, or until such time as the midribs betotal nitrogen, water-soluble nitrogen, amino nitrogen, come extremely dry and brittle. This] cured dry leaf is then allowed to nicotine, total acids, and pH. I t is believed that the hang in the barn until sufficient moisture aging of flue-cured tobacco is essentially a chemical prochas been absorbed to permit handling withess, the main reaction being that between sugars and out breakage. It is then packed in piles in amino compounds with the formation of melanoidins storage barns with a moisture content of and carbon dioxide. 10 to 15 per cent, where it remains until
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TOBACCO
TEMPERATURE
TOBACCO
MOISTURE C O N T E N
a t the aging intervals noted in Table I. The aging period was considered as beginning a t the redrying date. The redrying dates ranged over a period of 6 weeks. I n that no significant difference was observed between different types and grades, it seems unnecessary to present the detailed data; therefore only the arithmetical average of the results a t each aging interval is given. Microorganisms were present in all samples throughout the aging period. Bacteria were much more abundant than molds in all cases. Although no attempt was made to identify the specific species of bacteria, a total of twenty-five distinct species appeared on the plates, such as are commonly found in soil and air. These included such forms as Sarcina subjlava, Flabobacterium Jlavum, Bacillus subtilis, and Bacillus mycoides. There was no evidence of the predominance of any species a t any period of the year.
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18 1
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12 1 8 20 2 4 28 32 38 40 4 4 48 5 2 3 8 60 84 E L A P S E D PROCESS TIME IN MINUTES
I
TABLE I. AVERAGE FUNGI AND BACTERIA COUNTSAND CARBON DIOXIDECONTENT OF GASESOF FLUE-CURED CIGARETTE TOBACCOS DURING ONEYEAROF AGING
FIGURE 1. CONDITIONS EXISTING DURING THE REDRYING OF FLUE-CURED TOBACCOS The steel tube (such as is used in boring rifles) was
6/le
inch
(0.8 om.) outside diameter with a '/*-inch (0.32-cm.) hole throughout its length (36 inches, or 91.4 cm.). Through this tube passed
a stiff steel wire to which was soldered a metal cone which closed the capillary opening during the insertion of the tube into the tobacco. In practice the steel rod was pushed into the center of the end of the hogshead to a depth of 28 inches (71.1 om.) and then slightly withdrawn, and the wire was pushed down to open the end of the tube which was closed by the cone during insertion. The evacuated flask was then attached to the exposed end of the steel tube by rubber tubing, the stopcock opened, and the flask filled with gases from the interior of the tobacco mass. Air samples collected in this manner were immediately taken to the laboratory and analyzed for carbon dioxide. The conditions of this test, which were purposely chosen to be as near those of actual aging practice as possible, precluded the possibility of determining oxygen absorption. Such studies have been carried out in the fermentation of tobaccos of the air-cured type. In the flue-cured samples under consideration, tests for oxygen absorption would probably have been of minor significance since the plant material dealt with had been killed by a drastic thermal treatment.
Methods The suspension for counts of organisms was obtained by dropping the weighed tobacco (approximately 5 grams) from the sterilized sampling jars into 200 cc. of sterilized water, shaking vigorously, and allowing to stand for 3 minutes. Immediately 0.5-cc. portions of this suspension were pipetted into sterilized Petri dishes. To each Petri dish approximately 10 cc. of melted, cooled, nutrient agar was added; the dish was rotated to mix thoroughly the agar and suspension and was then incubated 48 hours at room temperature. Counts were made of both fungi and bacteria and computed to number of organisms per gram of tobacco. The nutrient agar was standard Bacto-agar to which lactose and dextrose were added. For enzyme tests the crude enzyme-containing matrix was prepared as follows: Samples of finely ground tobacco were soaked in distilled water for 2 hours and filtered, and 95 per cent alcohol was added to the filtrate. This produced a brown jelly-like precipitate which was collected on a filter paper, washed through several changes with 95 per cent alcohol, and allowed to dry (24 hours) to a semi-solid mass. The tests for various enzymes were made according to the methods described by Oosthuizen and Shedd (SI). The carbon dioxide content of the air from within the hogshead of tobacco was determined on a modified Anderson apparatus as described by Jones (18)and expressed in parts of carbon dioxide per 10,000 parts of air. The chemical methods were those described in a previous paper (6).
Bacteria and Fungi Counts of bacteria and fungi were obtained on all flue-cured cigarette types and included the entire cigarette-grade range
(Carbon dioxide content of air in storages, 3 to 5 parts per 10,000) Parts COZ Elapsed Av. No. No. per Time HogaMoisFungi Bacteria 10,000 of Approx. from Re- head ture Dry per Gram per Gram Hogshead B&s Tobacco Tobacco Gases Date drying Temp. Days ' C. P e r cent 20 0 43 9.6 75,200 10-30 1017 79 46 4 1 300 0.5 10-30 650 76 46:400 1 46 10-31 890 68 .. 29,540 45 1.5 10-31 193 63 .. 2 26 640 11-1 43 482 66 42 .. 2.5 32:800 297 11-1 43 41 31,400 11-2 3 462 17 31 8 11-7 13 .... 29 9 11-8 3 9:7 26,000 36 8 2i47 12-5 4 4 . . 19,600 80 880 1-18 6 10,500 6 125 440 3-4 6 17 1 4-13 1835 10: 18 900 165 7 200 19 14:740 6-18 670 8 2,150 235 23 6-22 276 7 7 430 265 26 7-22 264 7 10:3 6'130 280 26 390 8-6 5 178 1:540 300 24 8-26 4 55 550 10: 5 15 10-20 365
.... ..
..
..
....
....
In like manner the specific identification of fungi was not attempted; however, such cosmopolitan genera as Penicillium, Aspergillus, Mucor, Rhizopus, Alternaria, Cladosporium, Oospora, and Sterigmatocystiswere always found. No species appeared to be predominant a t any sampling interval. The highest number of organisms was found immediately after the tobacco was packed into the hogshead and prized. The number a t this point was considered low (10 to 60 per cent of that found by some workers on cigar leaf, 5, 7, 19,23, 29, QO), especially when it is considered that the tobacco was packed by hand. The number of organisms decreased to practically 50 per cent of their initial value during the first 12 hours, followed by a gradual decline in numbers throughout the aging period. There was no marked tendency during any period of storage for the number of organisms to increase above that of former periods.
Carbon Dioxide Content of Gases from the Tobacco Mass The production of carbon dioxide in fermenting tobacco masses has long been considered a criterion in fermentative action and the results of oxidation processes (6, 20, 56, 38). In this investigation the gases were withdrawn from the interior of the aging tobacco mass, simultaneously with sampling for organism counts, a t progressive aging intervals. The average carbon dioxide content is expressed in parts per 10,000 and given in Table I to allow convenient comparison with
FEBRUARY. 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
numbers of organisms, hogshead temperatures, and date of sampling. Immediately after redrying, the carbon dioxide content of the prized tobacco rose from an average of 20 parts to 79 parts within 12 hours. This was the highest concentration reached a t any time during the subsequent aging and was maintained for approximately 24 hours. One individual case was found where the concentration increased to 125 parts per 10,000 during this period, Following this the concentration diminished as the temperature of the tobacco fell, until the tobacco had reached storage temperatures. This usually requited between 14 and 21 days, and a t this time the carbon dioxide concentration coincided with or was slightly above that of the outside air (3 to 5 parts per 10,000). A concentration of 3 to 8 parts per 10,000 was maintained throughout the remainder of the aging period, with no noticeable change which could be attributed to changes of seasons, moisture content, or temperature. The highest content in any individual sample during this period was 12 parts per 10,000. A smaller number of determinations were made over a period of 30 months (not shown in Table I ) and showed a range of 2 to 7 parts per 10,000 with no apparent correlation with seasonal changes. This indicates that the period immediately after redrying (7 days), when hogshead temperatures are 32" to 46" C.,is the only time in the aging period when the changes taking place proceed a t such rate as to raise the carbon dioxide content of the tobacco atmosphere much above the content of the storage air. Nothing is known of the diffusion rate or of the total quantity2 evolved during this period. It is known, however, that a t the end of this period no aging effects can be detected in the tobacco, and it is believed that the decrease in carbon dioxide content over this period is indicative of the rate of loss by diffusion of the gas formed within the first 12 hours. The heads of the bundles (representing 14.5 per cent of the total weight) are tied with a tobacco leaf into a compact mass which dries very little during the drying process but retains its heat and moisture which it transfers to the remainder of the bundle after prizing in the hogshead. It is known that this equalization of heat accounts for a portion of the rise in hogshead temperature observed, and it is probable that organism activity proceeds in these moist, warm stems during the first 24 to 36 hours and accounts for the carbon dioxide produced during this period. Subsequent equalization of moisture, combined with heat losses, inhibit this action; the organisms are subjected to very unfavorable conditions, and oxidation proceeds a t a very low rate.
Temperature Phenomena In that pyrogenesis is associated so closely with the fermentation of air-cured tobaccos, repeated efforts have been made to demonstrate its presence during the natural aging of normally dried flue-cured tobaccos. In a few cases in which the tobacco was insufficiently dried and prized a t high moisture content, the entire tobacco mass self-heated and charred. Other instances are known where tobaccos were dried in the manner described and placed a t high temperatures (49' C.), and then showed self-heating to a marked degree. With the exception of the very slight (2.0' C.) heating effects observed during the first 24 to 36 hours after redrying, there was no evidence of any heating effect of normally dried and stored flue-cured tobacco. Temperatures of tobacco in hogsheads illustrating the thera Some work has been done by this laboratory on the quantity of carbon dioxide evolved from flue-cured tobacco when held at 40' C. and 50 per cent relative humidity. These results show from 0,0900 to 0.0143 gram to be liberated per kg. of tobacco per 24 hours. This is an average of 6 per cent of the quantity found by Schloesing (56) when air-dried tobaccos were used. He reports finding 0.258 to 1.367 grams per kg. of tobacco per 24 hours.
183
mal behavior of aging tobacco for one year after reaching storage temperature are given for information (Figure 2). These temperatures were obtained a t daily intervals and compared with maximum and minimum storage temperatures. Since the storages were extremely well ventilated, they vary with and are very close to outside temperatures. By this comparison no evidence of self-heating was found. Because of the extremely irregular nature of the storage temperatures, they were not included in Figure 2. When the temperature increases occurring immediately after redrying (Table I) were first noted, the entire increase was attributed to pyrogenesis. However, since the bundle heads were very compact, they lost their heat less readily than the fluffy tails, both in the cooling chamber of the drier and during packing into the hogshead. As a result an armored thermometer inserted between the bundle layers, in contact largely with tails, recorded a temperature below the actual mean temperature. Because of this phenomenon the temperatures of heads and tails were investigated with thermocouples placed within these portions of the bundle and prized in the usual manner. It was found as an average that initially the heads were 2.3O C. higher in temperature than the tails and that the weighted mean temperature of the tobacco mass could be calculated as 44.1" C. The armored thermometer denoted a temperature of 43" C. Twelve hours later an increase of 1.5" C. was shown in the heads and 1.8" C. in the tails, with a calculated mean temperature of 45.9" C. The armored thermometer registered 46" C. a t this point. On the basis of these data it might be concluded that the self-heating was shown during this period to be approximately 2" C. After due allowance is made for the possibility of variations in temperature within the 1000-pound tobacco mass, these data seem to point a t least to the possibility that a very slight selfheating effect may exist during the first 12 to 24 hours after redrying.
Enzymes Flue-cured tobaccos, owing to repeated high-temperature treatments offer an opportunity to observe the practical application of the enzymic theories advanced by Loew (25, 26, 27) and supported by Vriens (41), Behrens ( S ) , Johnson (17), Kolenev (2@, Barta (Z), Jouravsky (20), Fodor and Reifenberg (Q),Neuberg and Kobe1 (31). Loew (25) found that 65-66" C. for 3 minutes destroys oxidase, and that 8788" C. for 3 minutes destroys peroxidase. Behrens (3) was of the opinion that oxidase can withstand 80" C. and peroxidase 90" C. Vriens (41) states that 100" C. for 5 minutes destroys peroxidase, 100" C. for 10 minutes destroys zymogen, and 100" C. for 15 minutes destroys peroxidase and zymogen. Johnson (17) finds that 80" C. for 24 hours is required to destroy the peroxidase. Since flue-cured tobacco is subjected to a temperature of 82" to 93" C. for 12 to 18 hours and remains above 52' C. for about 40 hours in the curing process,
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FIGURE2. TEMPERATURE OF FLUE-CURED TOBACCO STORED IN HOQSHEADS UNDER NATURAL AQINGCONDITIONS
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Tobacco Type
Crop
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0
7
Georgia
1928 1929 1930 Av.
South Carolina
Av. EasternNorthCarolina Av. Durham Av. Daiiville
AY. Winston Av. Grand av. % change
Georgia
1928 1929 1930 1928 1929 1930 1928 1929 1930 1928 1929 1930 1928 1929 1930
10.31 8.90 7.34 8.85 8.25 8.11 7.63 8.00 8 28 8.62 8 60 8.50 9.41 8.55 8.13 8.70 10.20 9.11 9.42 9.58 9.42 9.03 8.49 8.98 8.77 0
1928 21.61 1929 23.59 1930 25.12 AV. 23.44 South Carolina 1928 20.38 1929 22.16 1930 21.22 Av. 21.25 Eastern NorthCarolina 1928 18.85 1929 18.38 1930 18.27 Av. 18.50 Durham 1928 15.52 1929 19.84 1930 18.39 Av. 17.92 Danville 1928 14.70 1929 1 7 . 9 4 1930 1 6 . 6 1 Av. 16.42 Winston 1928 14.96 1929 1 7 . 1 7 1930 1 7 . 0 2 Av. 16.38 Grand av. 18.99 % change 0
VOL. 28, NO. 2
TABLE11. CHEMICAL CHANGES OF GROUPS OF ORGANICCONSTITUENT Time of Sampling, Months: 6 12 18 24 30 Moisture. ~. 7. " 10.39 10.36 10.33 10.71 10.73 9.21 9.35 9.93 10.04 10.09 8.14 9.01 9.29 9.61 9.61 9.24 9.57 9.85 10.12 10.14 8.60 9.39 9.60 10.11 10.05 8.20 8.84 9.20 9.68 9.35 8.62 8.91 8.81 9.46 9.61 9.05 9.20 9.75 9.67 8.47 9.25 9.46 9.98 10.30 10.95 9.18 9.44 9.19 9.27 8.62 10.18 10.09 9.39 9.44 9.66 10.16 9.56 9.89 9.18 9.35 10.35 10.50, 10.09 10.35 9.79 9.90 9.93 9.92 9.31 9.39 10.36 9.70 10.11 9.70 9.89 10.25 9.99 10.13 9.60 9.79 11.01 11.61 11.72 11.31 11.40 9.44 9.62 10.15 9.89 9.92 10.53 10.61 10.52 10.83 10.86 10.68 10.73 10.61 10.80 10.36 9.92 10.32 10.58 10.91 10.56 9.52 9.67 .10.47 10.22 10.33 9.49 10.01 9.62 9.87 10.21 10.22 10.33 10.37 9.64 10.00 10.22 9.94 10.15 9.42 9.73 $7.41 4-10.95 -I-13.45 4-15.74 4-16.53
21.29 22.71 25.23 23.08 19.49 20.80 20.80 20.36 18.39 17.25 18.10 17.91 15.08 19.29 17.73 17.37 14.57 17.40 15.94 15.97 14.14 17.19 16.20 15.84 18.42 -3.00
Total Sugar, % 20.75 20.40 22.15 22.18 25.30 25 00 22.73 22.53 19.20 18.86 20.27 20.47 20.73 20.51 20.07 19.95 18.09 17.96 17.06 17.01 17.18 17.11 17.44 17.36 14.91 14.47 18.38 18.45 17.52 16.95 16.94 16.62 13.97 13.41 16.40 16.48 15.83 15.37 15.40 15.09 13.85 13.62 16.56 16.50 16.34 16.23 15.53 15.51 18.02 17.84 -5.11 -6.06
19.87 21.93 24.65 22.15 18.46 20.42 20.55 19.81 17.58 16.69 16.75 17.01 14.55 18.04 16.86 16.48 13.58 16.05 15.62 15.08 12.80 16.24 16.14 15.06 17.60 -7.32
and since this treatment is followed by a redrying process where tobacco temperatures are maintained from 66" to 80' C . for 25 minutes, we would expect these conditions to approach those required for inactivation of these enzymes. Tests were made for enzymes along the lines outlined by Oosthuizen and Shedd (3.9). Diastase, catalase, and invertase were either absent or present in small amounts in flue-cured tobacco. Proteolytic enzymes were not found at any time during the aging of flue-cured tobacco. Apparently some samples of flue-cured tobacco had been heated to such an extent as to eliminate enzymes entirely. The enzyme content of the samples tested appeared to decrease with increasing age. I n air-dried tobaccos, diastase, catalase, and invertase were found to be present in significant amounts when tested for by the same procedures. These differences are indicated by the results for the catalase test which is the one most capable of quantitative evaluation. The average volume of oxygen evolved from a 2-gram sample of flue-cured tobacco was 0.7 cc. as compared with 11 cc. collected from samples of aircured tobacco. When it is recalled that air-dried tobaccos have not undergone the drastic heat treatment to which fluecured tobaccos are subjected, it seems logical to conclude that the heat treatment has inactivated the enzymes in the latter. This comparison of the enzyme content of flue-cured and air-cured tobaccos seems valid, since the moisture content of
20.21 21.86 24.23 22.10 18.48 19.66 19.93 19.36 17.53 17.01 16.85 17.13 14.30 18.12 16.73 16.38 13.38 16.30 15.49 15.06 13.06 16.24 16.24 15.18 17.54 -7.64
1.72 1.65 1.51 1.63 1:70 1.76 1.78 1.75 1.73 1.91 2.12 1.92 2.08 1.87 1.99 1.98 2.01 1.76 2.07 1.95 2.25 2.08 2.17 2.17 1.90
0
1.73 1.64 1.49 1.62 1.66 1.73 1.72 1.70 1.70 1.89 1.93 1.84 1.91 1.80 1.84 1.85 1.92 1.70 1.91 1.84 2.12 2.01 2.06 2.06 1.82 -4.17
Time of Sampling, Months: 12 18 24 Total Nitrogen, % 1.66 1.69 1.66 1.63 1.63 1.55 1.50 1.45 1.47 1.60 1.59 1.56 1.65 1.61 1.64 1.73 1.73 1.72 1.74 1.71 1.69 1.71 1.68 1.68 1.70 1.68 1.68 1.88 1.88 1.83 1.93 1.86 1.86 1.84 1.81 1.79 1.91 1.88 1.93 1.79 1.83 1.83 1.82 1.82 1.80 1.84 1.84 1.85 1.93 1.85 1.93 1.70 1.71 1.68 1.83 1.79 1.81 1.82 1.78 1.80 2.00 2.00 2.06 2.00 2.00 2.00 1.96 1.92 1.96 1.99 1.97 2.01 1.80 1.78 1.78 -5.26 -6.32 -6.32
1.11 1.04 0.97 1.04 1.11 1.10 1.16 1.12 1.14 1.12 1.35 1.20 1.37 1.13 1.31 1.29 1.34 1.14 1.40 1.29 1.42 1.39 1.46 1.42 1.23 0
1.06 1.02 0.98 1.02 1.03 1.07 1.16 1.09 1.06 1.09 1.33 1.16 1.20 1.08 1.35 1.21 1.22 1.11 1.38 1.24 1.35 1.34 1.44 1.38 1.18 -4.07
Water-Soluble Nitrogen, % 1.04 1.01 1.01 1.01 1.03 1.02 0.95 0.93 0.94 1.00 0.99 0.99 1.03 0.97 0.98 1.07 1.07 1.06 1.12 1.08 1.10 1.07 1.05 1.04 1.01 1.03 1.03 1.09 1.10 1.09 1.27 1.24 1.23 1.12 1.12 1.12 1.16 1.16 1.18 1.09 1.09 1.08 1.33 1.29 1.27 1.19 1.17 1.18 1.23 1.19 1.20 1.10 1.08 1.10 1.30 1.30 1.31 1.21 1.19 1.20 1.33 1.33 1.34 1.34 1.34 1.32 1.39 1.40 1.42 1.35 1.36 1.36 1.16 1.15 1.15 -5.69 -6.50 -6.50
0
6
30
1.67 1.66 1.43 1.59 1.61 1.69 1.68 1.66 1.69 1.79 1.87 1.78 1.85 1.78 1.78 1.80 1.93 1.66 1.80 1.80 2.03 1.95 1.96 1.98 1.77 -6.84
.
1.00 1.00 0.95 0.99 1.00 1.04 1.11 1.05 1.01 1.os 1.28 1.12 1.18 1.08 1.28 1.18 1.20 1.04 1.32 1.10 1.34 1.28 1.41 1.34 1.15
-6.50
both tobaccos is approximately the same during aging and under the conditions of the experimental tests. Although this does not prove the absence of highly specialized enzymes for which reliable tests are not available, it seems reasonable to assume that the conditions of temperature in the flue-curing process would destroy these enzymes also. In view of the fact that the greater portion of the enzyme content of flue-cured tobaccos has been destroyed and that quite different over-all aging effects are observed in air-cured tobaccos, the inference seems justified that the contribution of enzymes to the aging of flue-cured tobacco is a minor one.
Changes in Aroma and Color during Aging It has been previously stated that aging produces considerable change in color, aroma, and smoking quality. The tobaccos used in this study were evaluated for aroma, color, and general character, a t each sampling, by an experienced judge of tobaccos.3 Odor progresses from a green hay-like a The accuracy of such empirical judgment of aroma is i n some meaaure attested b y the .results of Powell (88) with tobacco beetles (Lasioderma serricornefabr.) for their reaction toward tobacco a t different shgea of aging. H e found i n a statistical study that the beetles were attracted i n the order of increasing age and aroma of tobacco as follows: 6-month-old tobacco, 61 per cent; 18-month, 75: aO-month, 89. This was in effect a partial check, by what amounted to a biological assay, of the validity of such empirical judgment.
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FEBRUARY, 1936
FLUE-CURED TOBACCOS DURING VARIOUSSTAGES OF AGIXG
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c Time of Sampling, Months: Time of Sampling, Months:0 6 12 18 24 30 6 12 18 24 30 a-Amino Nitrogen, % ’ 7 -Total Nonvolatile Aciditya0.116 0 . 119 0.113 0.114 0,121 0,142 11.48 11.71 11.53 11.71 11.83 1928 12.36 Georgia 0,126 0.123 0,137 0,147 0,154 0,169 12.70 * 12.58 12.83 12.58 12.72 1929 12.99 0.136 0.131 0,148 0.140 0,157 0,170 12.17 12.73 12.55 12.73 12.17 1930 12.49 0.126 0.12 0.130 0.136 0,144 0,160 12.12 12.30 12.12 12.34 12.24 12.61 Av. 0.150 0,152 0.151 0 , 1 5 9 0 , 1 5 9 0.179 10.73 1 1 . 4 4 10.82 11.44 11.50 1928 11.64 South Carolina 0 . 1 5 6 0.158 0 , 1 6 2 0,166 0 , 1 9 1 0.178 11.49 11.71 11.36 11.71 11.70 1929 11.87 0,158 0.162 0.167 0,170 0,192 0,195 11.93 12.15 11.98 11.81 12.15 1930 12.04 0.155 0.157 0.160 0,165 0.176 0.188 11.38 11.77 11.39 11.38 11.67 11.85 Av. 0.158 0.160 0.166 0.163 0.169 0,201 EasternNorthCarolina 1928 12.95 12.41 12.31 12.31 11.80 11.65 0.183 0.178 0.195 0.187 0,196 0,226 1929 12.94 12.61 12 0 3 12.03 12.18 12.06 0.179 0.181 0.190 0,188 0.220 0.226 1930 12.86 12.41 12.44 12.44 12.56 12.56 0.173 0.179 0.173 0.184 0,195 0,218 12.05 12.09 12.18 12.26 12.48 12.92 AV. 0,181 0.168 0.192 0.192 0,197 0.223 11.18 10.57 11.35 11.35 11.61 Durham 1928 11.97 0.164 0.158 0.163 0 . 1 6 0 0.187 0,194 12.48 12.49 12.60 12.60 12.58 1929 13.13 0.172 0.171 0.183 0.177 0.217 0,225 13 73 14.27 13.49 13.49 13.62 1930 14.02 0 , 1 7 2 0.166 0.176 0.179 0.200 0.214 12.32 12 44 12.48 12.46 12.60 13.04 Av. 0.163 0.163 0.161 0.163 0,198 0,180 Danville 1928 14.01 13.57 13.05 13.05 12.84 12.14 0.130 0.136 0.126 0.140 0.162 0.188 1929 13.94 13.28 13.26 13.26 13.30 13.02 0 .150 0 , 1 7 0 0 . 1 5 1 0.169 0.208 0.208 1930 15.00 14.58 14.54 14.54 14.69 14.68 0.148 0.156 0.146 0.157 0,198 0.183 13.54 13.28 13.61 13.62 13.81 14.32 AV. 0.211 0,226 0.213 0,235 0.230 0,263 12.87 12.90 12.92 12.92 13.28 Winston 1928 13.90 0.193 0.217 0.192 0,235 0.235 0,242 14.00 14.05 14.11 14.11 14.12 1929 1 4 . 2 9 0.186 0.186 0.197 0.195 0.234 0,239 15.16 15.49 15.03 15.03 15.57 1930 1 5 . 6 1 0.197 0.197 0.235 0.221 0.213 0,248 13.99 14.15 14.02 14.01 14.32 14.60 Av. 0.161 0,169 0.162 0,174 0,189 0,204 12.57 12.58 12.66 12.75 12.85 13.22 Grand av. -21.08 -17.16 -20.59 -7.35 -14.71 0 -4.92 -4.84 -4.24 -3.55 -2.80 0 yo change Nicotine, %H-Ion Concn. Expressed as p H 2.18 2.11 2.16 2.21 2.27 2.24 Georgia 1928 5.17 5.12 5.07’ 4.92 4.91 4.82 2.24 2.08 2.13 2.21 2.29 2.33 1929 5.30 5.21 5.03 4.98 4.90 4.87 2.03 2.02 2.06 2.02 2.23 2.28 1930 5.51 5.21 5.11 5.07 5.04 5.05 2.lO 2.09 2.15 2.15 2.25 2.29 Av. 5.326 5.18 5.07 4.98 4.95 4.91 1.91 1.93 1.91 1.91 1.97 4.85 4.81 4.91 4.97 5.02 South Carolina 1928 5.21 1 . 9 0 1.92 1.94 2.00 2.03 4.85 4.85 4.95 4.98 5.26 5.32 1929 2.35 2.36 2.30 2.31 2.55 4.81 4.84 4.84 4.88 4.89 1930 5.11 2.05 2.07 2.05 2.07 2.18 4.84 4.83 4.90 4.94 5.03 Av . 5.21b 1.92 1.90 1.91 1.93 1.95 2.09 EasternNorthCarolins 1928 5.15 5.02 4.98 4.88 4.81 4.74 1.93 2.04 1.94 2.09 2.11 2.20 1929 5.27 5.19 5.02 4.89 4.80 4.73 2 .59 2.81 2.62 2.79 3.00 3.01 1930 5.18 4.89 4.87 4.82 4.76 4.78 2.27 2.15 2.25 2.16 2.35 2.43 4.79 4.75 4.86 5.03 4.95 Av. 5.19b 2.57 2.55 2.67 2.60 2.67 3.07 4.66 4.67 4.84 4.94 4.94 Durham 1928 5.20 2.18 2.24 2.21 2.25 2.26 2.46 4.75 4.78 4.94 4.96 5.16 1929 5.29 2.99 2.93 3.25 3.23 3.26 3.41 4.80 4.85 4.82 4.85 5.04 1930 5.24 2.71 2.58 2.57 2.69 2.98 2.73 4.76 4.76 4.91 4.86 5.03 5.25b Av. 2.98 2.96 3.08 3.12 3.41 3.10 Danville 1928 5.36 5.07 5.00 4.93 4.79 4.80 2.83 2.87 2.73 2.88 2.88 3.13 1929 5.36 5.19 4.90 4.90 4.79 4.82 3.52 3.50 3.73 3.76 3.82 3.90 1930 5.13 4.95 4.81 4.82 4.80 4.78 3.12 3.06 3.21 3.25 3.27 3.48 4.79 4.80 4.88 4.90 5.06 AY 5.27b 2.51 2.58 2.60 2.58 2.66 2.90 4.83 4.73 4.87 5.05 5.28 5.14 Winston 1928 2.37 2.29 2.23 2.37 2.38 2.54 4.78 4.77 4.99 5.02 5.18 1929 5.30 3.54 3.38 3.36 3.77 3.52 3.79 4.80 4.81 4.86 4.81 5.02 5.29 1930 2.73 2.72 2.84 2.83 3.08 2.93 4.77 4.90 4.80 4.94 5.11 Av. 5.29b 2.46 2.44 2.54 2.54 2.74 2.62 4.80 4.90 4.82 4.95 5.07 5 25b Grand av. .10.22 -10.95 .7.30 -.7.30 0 -4.38 -8.19 -8.57 -3.43 -5.71 -6.67 0 4, ,” chnnnp u . a Milliliters of 0.1 N alkali required to neutralize the acid in 1 gram ,of tobacco b Since p H values, which are logarithmic unlts, may n o t be averaged i n the same manner as ordinary numbers, these averages were obtained as follows: The p H values t o be averaged were converted to moles per hter; these values i n moles per llter were averaged, and the result was then converted to p H units.
Crou
Tobacco TvDe
7
0
7
~
7 -
~~
-
~~
-
odor through a rather horehound-like odor (rancid in the case of primings) carrying green odor characteristics to the fruity, spicy, mellow aroma of fully aged tobacco. The rate of this progression varies with tobaccos of different character and differing aging rates. At times some tobaccos become quite aromatic within a year. Others require 18 months to lose the green hay-like odor and acquire aroma, and still others require 2 or 2.5 years. In general, the characteristic odors described (horehound or rancid) will precede by 6 months the mellow aroma. The process of acquiring aroma is gradual. The green hay-like odor develops traces of fruitiness or spiciness and finally gives way to full aroma. In general, a t the end of a year there is considerable volume of the green hay-like odor with detectable amounts of the fruity spicy character. At the end of 18 months the horehound or rancid odor is noticeable, developing into true aroma and maximum volume a t the end of 2 years. Then there is a gradual loss of volume of aroma and an acquirement of mellowness. This progresses slowly for years (tobaccos 15 years old have been observed) with considerable modification of quality and decrease of volume of aroma. Unaged flue-cured tobacco has a certain “flash” or liveliness of color in all the yellow or orange shades which progressively gives way to darker shades and a more dull or “dead” character of shade. This change is concurrent with the aroma development. I n other words, B light orange shows some
-
dnrkening as an accompaniment of the first slight aroma development, yet it exhibits some of the flash or liveliness of color associated with unaged tobaccos. As aging advances, the color continues to darken slightly and becomes more dull in character as the hay-like odor gives way to full aroma. This phenomenon is very marked and so characteristic that judges of tobacco consider that very flashy yellow tobaccos of light “body” (6) require more time for aging and are incapable of developing the maximum aroma. Handlers of tobacco also associate large loss of color during aging with high moisture content of the tobacco after redrying. “Reddening” of color is associated with the maintenance of high temperatures in tobaccos of relatively low moisture content.
Changes in Constituents during Aging The chemical phase of this investigation differs from the majority of fermentative studies in that an attempt has been made to follow the changes in certain groups of organic constituents during aging under exact commercial conditions, rather than to follow changes in small bulks under controlled or abnormal conditions. This has necessitated following a comparatively great number of samples, representative of all types and grades of flue-cured cigarette tobaccos in three successive crops. Six hogsheads of various grades were selected from each type in each of three crops. These hogs-
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INDUSTRIAL AND ENGINEERING CHEMISTRY
VOL. 28, NO. 2
month period for these constituents may readily be calculated from data given in Table 11. The percentage change in some tobacco constituents is shown in Figure 3. One of the most interesting features of these curves is the regular increase in moisture content over the aging period studied, irrespective of seasonal conditions. It might be 4 expected that the moisture content changes would originate from and vary during extended periods of greatly differing INTER 6 S U M M E R 12 W I N T E R I B S U M M E R 2 4 W I N T C R 30 relative humidities and temperatures in the storages whose air conditions vary with outside weather conditions. It should be remembered, however, that the tobacco is stored in large compact masses not readily affected by the surrounding atmosphere. This is borne out by the relatively slight increase observed in moisture content (usually less than 0.1 per cent) of such tobaccos in hogsheads when held from 7 to 10 days in an atmosphere a t about 43O C. and 85 per cent relative \\ humidity. Therefore the major portion of the increase in PO moisture content must be attributed to changes taking place LAMINO-N. 22 within the tobacco and not to external moisture variations. A C E IN MONTHS The increase in moisture content, due mainly to changes FIGURE 3. PERCENTAQE CHANGEOF GROUPS OF within the tobacco during aging, is shown by the curve in ORGANIC CONSTITUENTS DURING NATURAL AGING Figure 3. This increase is noticeable within 30 days after OF FLUE-CURED TOBACCOS redrying and generally continues for 2 to 2.5 years. The length of this period is conditioned by the initial moisture content and the rate of aging. A peak in moisture content occurs heads were initially sampled immediately after redrying, in a t about the time of maximum aroma development, after the fall of the crop year and each succeeding April and October which there is a gradual decrease in moisture-holding capacity for 30 months. This sampling has resulted in running 1296 and moisture content. This is in line with the observations individual analyses for each constituent tabulated, and has of other investigators (80) and explains the behavior of very made it necessary to consider over 10,000 individual deterold flue-cured tobaccos which exhibit a decided loss in hygrominations. In addition, there have been available approxiscopicity and which become very dry and friable. The loss mately 3000 analyses of these same tobaccos sampled a t in water-holding capacity is shown (Figure 4) in an exagshort intervals during the summer and winter, which have gerated form by an experiment in which tobacco samples not been included in these data. were held a t 39" C. and 50 per cent relative humidity for 10 The volume of data has necessitated tabulation as averages. months. This loss has also been demonstrated in naturally These averages have been compiled (Table 11) by constituaged tobaccos and is indicated in these data in the flattening ents in such manner that the average content is shown for of the curve after the thirtieth month. each type a t crop and successive sampling intervals. FurThe tobacco is practically in equilibrium with the air in the thermore, all types have been averaged into a grand average, and the percentage change from the original content has been tobacco mass after the first 2 weeks following redrying. Thus, by knowing the moisture content and the relative humidity a t noted for each aging period. This percentage change for equilibrium, the relative humidity of the air in the tobacco flue-cured tobaccos as a whole is plotted by constituents mass may be ascertained for consideration in respect to either (Figure 3). outside conditions or bacterial activity. A curve showing the Sampling dates were set a t the first week in April and the moisture content of average flue-cured tobacco in equilibrium first week in October (tobacco temperature, approximately with various relative humidities at 30" C. is given in Figure 5. 16" C . ) because of the prevalent idea among tobacco men of Therefore, we would expect the relative humidity of the air the existence of the so-called May and September sweats. within the tobacco mass to be from 45 to 56 per cent. Thus samples would be taken just prior and subsequent to The initial steep rise in moisture content is attributed to an maximum aging activity. Additional samples were obtained accelerated change liberating water during this period, which a t monthly intervals during this period, but, since no evidence might be due to organisms but which is more probably the was found of marked changes in chemical composition a t result of chemical reaction. This rise may be slightly augthese short intervals, the data are not presented. Some differences in aging behavior are also exhibited by different crops within a given type and between different types, but it is not within the scope of this article to discuss this phase of the problem. All determinations are expressed in terms of weight of dry tobacco. It is realized that some loss of dry matter is experienced during this type of aging, but, since this loss hardly reaches 2.5 per cent even during protracted aging, it has been disregarded in the calculation of these data. I n actual aging practice, the weighing of the total contents (approximately 1000 pounds per hogshead) of a large number of hogsheads before and after aging has shown that the loss in dry weight (after due allowance for moisture changes) averages below I I 2 3 4 5 6 7 B 8 ?o 2.5 per cent. This figure is in good agreement with the sum ELAPSED T I M E I N MONTHS (2.31 per cent) of the losses of sugars (1.45 per cent), nitrogen F I G U R4.~ DECREASE IN WATER-HOLDING CAPACITY (0.13 per cent), nicotine (0.30 per cent), and total acids OB FLUE-CURED TOBACCO MAINTAINEDAT 39" c. AND 50 PER CENT RELATIVE HUMIDITY (calculated as malic, 0.43 per cent). The losses over the 30-
FEBRUARY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
mented by small amounts of moisture obtained from infiltered air. After the loss of heat during this first period, the reaction proceeds a t a lowered rate and the rate of increase of moisture content decreases. An increasing moisture content is conducive to accelerated aging$ but, on the other hand, increased aging results in a lowered water-holding capacity of the leaf. Thus the curve continues t o flatten and finally reverses its direction. This point corresponds in general with the inflections in the curves for other constituents. Thus it appears that a proper amount of moisture is required before adequate aging results, and that the initial moisture content influences the rate of aging. Moist tobaccos become aromatic more quickly and show a flattening of the moisture curve a t an earlier date. These facts are significant in that the rate of aging and resultant loss in color are conditioned to some extent by the initial moisture content and can be of practical henefit to the manufacturer in obtaining the desired end result. The decrease in total sugar averages 1.45 per cent of the dry weight of tobacco. This decrease occurs quite regularly over the aging period a t a continually decreasing rate and represents the greatest actual loss of any group of constituents. This decrease is probably largely the result of a reaction with some of the nitrogen constituents to be discussed later in the paper but appears to be accompanied by an oxidation process in which there is liberation of acetic acid, formic acid, acetaldehyde, ammonia, carbon dioxide, and water. These substances have been identified in the air aspirated from aging tobacco masses over prolonged periods (60 days). The increase in moisture content of the tobacco may be considered as some evidence of the chemical splitting off of water. It is also possible to regard this increase as the chemical result of the slow action of a small quantity of bacteria, fungi, or enzymes over a long period. The small organism and enzyme content, combined with the unfavorable conditions to which they are exposed and their evident decrease in numbers with time, leads, however, to the conclusion that any activity they possess is expended during the first 2 or 3 weeks of aging and that subsequent changes are purely chemical in nature. The percentage changes in constituents at consecutive aging intervals are shown in Table I1 and plotted in Figure 3. Undoubtedly the losses given are real. There may be some question as to the influence of changes in dry weight, but this factor would tend to increase the differences shown. It is believed that the relationships shown may be considered as very closely related to those actually occurring, in view of the large number of samples studied. In the nitrogen fractions the greatest rate of change occurs from the time of redrying until the end of the first summer (12-month sample), after which the rate decreases. There is also a decided decrease in rate after the second summer (24-month sample). It is clear from the behavior exhibited by the sugar, nicotine, and amino nitrogen that there is a definite increase in activity during the summer months when tobacco temperatures are highest (26' C.) This period of increased temperature would be conducive to increased organism or chemical activity. The following over-all changes in content of the various nitrogen fractions may be obtained in part from the data of Table I1 and are shown in condensed form in Table 111. The evidence available (Table 111) indicates that the losses in amino, nicotine, amide, and ammonia nitrogen comprise practically the entire nitrogen loss. Thus their sum (0.111 per cent) comprises 85.4 per cent of the total nitrogen loss. The amino nitrogen and nicotine are the most active of the nitrogen fractions and account for 72.3 per cent of the loss in total nitrogen. The sum (0.076 per cent) of the losses of the water-soluble
187
90
BC
7c
d 4
so
+
0
I
5c
$
40
3c
za I C
C
I
I
i
5
IO
15
MOISTURE
20 CONTENT
25
%
30
35
40
FIGURE5. AVERAGE EQUILIBRIUM MOISTURE OF FLUE-CURED TOBACCOS AT RELATIONSHIPS 30" C.
nitrogen constituents, made up of the amino nitrogen, watersoluble nicotine nitrogen, and amide and ammonia nitrogen, is the equivalent, within the accuracy of the methods, of the determined water-soluble nitrogen loss of 0.080 per cent. This loss in water-soluble nitrogen represents 61.5 per cent of the loss in total nitrogen. Presumably any nicotine lost must be by vaporization from the free form. Any free nicotine would be water-soluble and appear in the water-soluble nitrogen. For this reason the average amount of free nicotine nitrogen has been included with amino, amide, and ammonia nitrogen in the watersoluble nitrogen. This makes the distribution of the losses in water-soluble nitrogen approximately as follows : amino nitrogen, 56.6 per cent; free nicotine nitrogen, 22.4; amide and ammonia nitrogen, 21.0. TABLE111. AVERAGH LOSSESIN PERCENTOF DRYTOBACCO 1. a-Amino N obsvd. 0.043 2 . Free nicotine N obavd.= 0.017 3. Amide and NHa N 0bsvd.b 4. Water-sol. N calcd. (1 2 3) 0.076 5 . Obsvd. water-sol. N 0.080 6. Bound nicotine N obsvd. 0.035 7. Total N calcd. (4 6) 0.111 8. Obsvd. total N 0.130 a Although the determination of free nicoti.ne was not made on all the Sam les a coiisiderable number of determinations were made on the types grad&, hnd ages of tobacco represented. These ranged between 0.0 and 0.; per cent. A value of 0.1 per, cent was used here. b Amide and ammonia nitrogen determinations were made on all the samples studied b u t were not included in Table I1 because of the questionable nature of the method. Though a p roximrtte, the total changes in theae constituents were included here as an aid% considering the nitrogen fractions.
+ +
0.018
+
The change in the average pH value amounts to 0.45 pH unit (5.25 to 4.80 pH). The decreases appear to be affected by the summer season and show the same general character of curve as that shown by the amino nitrogen, nicotine, and sugar. It is conceivable that these changes could result from loss of nicotine or ammonia. The total nonvolatile acids decrease from 13.22 to 12.58 (expressed in cc. of 0.1 N alkali per gram of tobacco) or 0.64. The greater portion of this loss occurs during the first year. The loss is very small after this time and practically nothing after 18 months of aging.
Suggested Mechanism of Aging Maillard (as), Ruckdeschel (34), and Ambler (1) studied the reaction between sugars and amino acids resulting in the
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INDUSTRIAL AND ENGINEERING CHEMISTRY
liberation of carbon dioxide and the formation of melanoidins (highly colored aromatic compounds). This reaction proceeds to varying degrees of completion under different conditions of temperature, pressure, and concentration of reacting constituents and products. While the conditions in the aging tobacco mass are not favorable to rapid progress of this type of reaction, it is conceivable that it could take place to a limited extent over the period of aging. This would explain the dull orange tinge of color produced in flue-cured tobacco during the period of aging and could account, a t least in part, for the aroma development. Laboratory experiments following the procedure of Ambler and using dextrose, alanine, and glycine as reactants, produced melanoidins whose color and aroma were believed by an experienced judge of tobacco to be closely akin to those of well-aged tobaccos. This resemblance was particularly striking in the case of alanine. The aging tobacco loses its flash or brightness of color and acquires a duller darker shade and maximum aroma a t the time of maximum moisture content, which is coincident with the end of the time required for beneficial aging. The change in color is gradual and concurrent with aroma development. The time required for best aging results is from 24 to 30 months for most flue-cured tobaccos as shown by Figure 3. If subjected to further aging, tobaccos gradually become less hygroscopic, develop very dull dead shades of color, and lose some aroma. This behavior is also suggestive of the melanoidins which lose aroma and color on drying (84). In view of this parallelism and of the fact that the greatest chemical losses in aging tobacco are found to be in the sugar and amino nitrogen content, it seems plausible to attribute a t least a part of the changes in color and aroma in aging to the formation of melanoidins by the reaction between amino compounds and sugars.
Discussion of Results It appears that the various hypotheses concerning the causes of the fermentation of tobacco have had their origin, a t least in part, in the differentiated methods of curing and fermenting used for the various types. Undoubtedly all tobaccos in the uncured condition are subject to change due to bacterial, fungous, enzymic, and chemical activity which would continue until checked by the subsequent alteration of conditions. A number of investigations which have been attempted to explain the various kinds of tobacco fermentation on the basis of the behavior of any particular type, or to ascribe it to any one of the described causal agencies, have failed to produce evidence that is applicable to all fermentations (3,4, 24, 26, 26, 36, 39, 42). It appears unwarranted, a t present, to consider that the predominating activity exhibi€ed in any specific type of fermentation should be applicable to fermentation or aging in general. I n practically all the previous studies, outstanding exceptions have been attributed to curing conditions and environmental factors during fermentation. Numerous investigators have recognized the possibility that all or any portion of the causal agencies named might affect to some degree the results of aging (18-17, 20, W1,37) and have considered that the prefermentation treatment and the fermentation conditions determine the type and degree of fermentation found. For instance, Johnson (17), after adding considerable support to both microbial and enzymic hypotheses for the course of certain cigar tobacco types of fermentation, stated that in the case of redried tobaccos “it is quite conceivable that a slow oxidation, entirely independent of microorganisms, may proceed in such tobacco.” Jensen (16, 16) concluded that fermentations proceeding a t a moisture content of 20 per cent or below differ greatly from those carried out a t above
VOL. 28, NO. 2
20 per cent moisture. Kissling (21) and Smirnov (37) were of this general opinion also. The present study, although not disproving the possibility of some bacterial, fungous, or enzymic action, indicates that they do not play a dominant role in the process. The relatively small extent of the changes in major chemical constituents observed during the period of natural aging, together with the negligible amount of self-heating and the unfavorable conditions for organism or enzymic action, leads to the conclusion that the aging of flue-cured tobacco is in large measure a purely chemical process. In some respects this might be considered analogous to “autolysis”-a general term used by Smirnov (37) in describing the nature of the fermentation of Russian tobacco of low moisture content. The length of time required is conditioned by the low thermal and moisture levels, both of which would limit and inhibit the interdiffusion of and the reaction between constituents. I n considering the relation of the findings of this paper to the more general aspects of this problem, we may say that properly aged flue-cured tobacco, from the commercial standpoint, must have the following features: (a) It should be devoid of the green hay-like odor, and irritatkg and disagreeable smoking qualities possessed by unaged tobaccos. (b) It should possess R rich full aroma that will materially enhance the value of the finished product. ( c ) It should have a color in line with trade requirements. (d) The tobacco should retain sufficient elasticity to prevent undue losses by breakage during handling. The proper treatment with reference to each of these requisites is necessmily qualified by varying preferences. Smokers of Great Britian value highly the bright flashy color, and raw taste and odor possessed by unaged tobacco. This requirement results in the purchase of bright tobacco of low aging capacity (67, which is further drastically dried and stored a t a low moisture content to inhibit aging and color loss. Proper aging as used in this paper is based on the American preference which calls for a mild smoking taste without the rawness characteristic of unaged tobacco, a full fruity and spicy aroma devoid of green or hay-like odors, and dull shades of orange color rather than the flashy lively shades of unaged tobacco. Evidence of the validity of American aging practices is indicated by the changes in chemical composition shown by the curves in Figure 3. These changes reach maxima or minima after times approximately coincident with those used empirically for aging. More detailed study of the data of Table I11 shows that the initial moisture content of a tobacco can be used to influence the rate of aging and the time of attainment of maximum moisture content. Thus different tobaccos age at various rates dependent upon the initial composition, the initial moisture content, and the temperature a t which they are maintained. Some tobaccos are well aged after 1 year of storage. Most tobaccos are well aged within 2 years. A few tobaccos require 30 months and even then do not lose all rawness or develop full aroma. This information provides a means for regulating the end point by the control of initial moisture content, length of storage, and storage temperature. It is possible t o follow changes during aging by chemical analysis (such as total sugar and amino nitrogen), by moisture content, or by pH. However, since color changes and aroma development are concurrent with aging progress, it is more practical to follow the changes in these properties which are more adaptable to commercial use. The chemical methods are more adapted to the formulation of procedures for efficiently aging tobaccos of various types and character. The results presented here have laid the groundwork for future investigation by giving a chemical survey of the course of the natural aging of the most valuable type of cigarette tobacco. It is hoped that they will provide a basis for more
FEBRUARY, 1936
INDUSTRIAL AND ENGINEERING CHEMISTRY
detailed investigation of methods for shortening, improving, or evaluating aging technic.
Literature Cited (1) Ambler, J. A., IND. EXG.CHEM.,21, 47 (1929). (2) Barta, L., Biochem. Z . , 257, 406-10 (1933). (3) Behrens, J., Centr. Bakt. Parasitenk., 11, Abt. 2, 514-27, 540-45 (1896). (4) Boekhout, F. W. J., and Vries, J. J. 0. de, Ibid., 11, Abt. 24, 496-511 (1909). (5) Cohen, N. H., Meded. Proejstat. Vorstenland. Tabak., 12, 1-21 (1914). (6) Darkis, F. R . , Dixon, L. F., and Gross, P., IND. ENQ.CHEM.,27, 1152 (1935). (7) Davalos, J. W., “Cronica MBdico-Quirurgica. Habana (1892), No. 15,” abstract in Centr. Bakt. Parasitenk., I, Abt. 13, 390-2 (1893). (8) Faitelowitz, A,, Biochem. J . , 21, 262-4 (1927). (9) Fodor, A., and Reifenberg, A., 2. physiol. Chem., 162, 1-40 (1926). (10) Garner, W. W., U. S. Dept. Agr., Farmer’s Bull. 523 (1913); rev. 1922. (11) Garner, W. W., Bacon, C . W., and Bowling, J. D., IXD.ENQ. CHEY.,26,9 7 0 4 (1934). (12) Garner, W. W., Bacon, C . W., and Foubert, C. L., U. S. Dept. Agr., Bull. 79 (1914). (13) Hirmke, K., Fachl. M i t t . oster. Tabaksregie, 10, 41 (1910). (14) Izvoshtshikov, V. P . , State Inst. Tobacco Investigations, Krasnodar, U. S. S.R., Bull. 56 (1929). (16) Jensen, H., Centr. Bakt. Parasitenk., 11, Abt. 21, 469-83 (1908). (16) Jensen, H., Meded. Proefstat. Vorstenland. Tabak., 12, 22-38 (1915). (17) Johnson, J., J. Agr. Research, 49, 137-60 (1934). (18) Jones, E. P., IND. ENG.CHEN.,Anal. Ed., 2, 195-6 (1930). (19) Jorgensen, A. P. C., “Micro-Organisms and Fermentation,” 5th ed. rev., 1925. (20) Jouravsky, G. J., State Inst. Tobacco Investigations, Krasnodar, TJ. S. S.R., Bull. 58 (1929).
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