March 1950
INDUSTRIAL AND ENGINEERING CHEMISTRY
reaction, at different rates. If only one bond breaks, a free radical or an ion will result which could react with iron to form an iron mercaptide. The iron mercaptide could then decompose to ferrous sulfide and an organic product. If the reaction does not involve dissociation fragments it may be a bimolecular reaction between the iron atom (or ion electrons) and the sulfur containing molecule, and the intermediate complex may decompose to ferrous sulfide or to iron mercaptide, Speculations as to the course and mechanisms of the reaction illustrate lack of knowledge of the actual reaction steps. Investigation of the changes through which the organic portions of a number of organic sulfur compounds pass on reaction with iron will probably be necessary before the functional differences cited can be explained on a logical chemical basis.
+
519
LITERATURE CITED
(1) Greenhill, E. B., J.Inst. PetroEeum, 34,659 (1948). (2) Hagerman, D. B.,Anal. Chem., 19,381 (1947) (3) Holt, L. C.,U. S. Patent 2,443,823,Example 1 (June 22,1948). (4) Merchant, M. E., preprint, American Society of Lubrication Eneineers. Annual Meetinn. Buffalo. N. Y. (1948). (5) Prutton, C. F., Turnbull, D.,and Dlouhy, G., IND. ENG.CHEM., 37,1092(1945). (6) Prutton, C.F.,Turnbull, D., and Dlouhy, G . , J . Inst. Petroleum, 32,90 (1946)., (7) 8.A.E. Journal, 39,23-4(1936). (8) Westlake, H.E., Chem. Rev., 39,219 (1946);Farmer, H.E.,and Shipley, F. W., J. Polymer Sci., 1,293(1946). (9) Wolf, H. R.,and Mougey, H. C., Proc. Am. Petroleum Inst., 1932, pp. 118-30. RECEIVED April 7, 1949. Presented as a part of the Symposium on Organic Sulfur Compounds before the Division of Petroleum Chemistry at the 115th CHPJMICAL SOCIETY,San Francisco, Calif. Meeting of the AMERICAN
Fermentation of Cigar-Type Tobacco C. 0. JENSEN' AND H. B. PARMELE, P. LoriZZard Company, Jersey City, N . J .
T
-
Bacteria were found to be the agents which initiate the H E present study deals same as those which function with the fermentation in bulk fermentation (4, 6, fermentation process in bullcs of cigar leaf tobacco. Enor sweating of leaf tobacco IO), although a similar result zymes of the tobacco plant, fungi, and chemical reactions preparatory to the manufacmay be accomplished. Forced unaided by microorganisms are discussed as possible causature of cigars and of scrap tive agents. Actively fermenting bulks were found to sweating at 50" or above (1, 1.9, 21) may also be an enchewing tobacco. It aims to have 700 million bacteria per gram at some stages. The explain the primary reaction rise in temperature of bulks followed an increase of bactirely different process. which takes place when such terial numbers. Seven species of bacteria were isolated Invariably heat, carbon leaf is piled in bulks of about from fermenting Wisconsin cigar leaf tobacco. Four of dioxide, and ammonia are these species produced heat when grown on sterilized to2000 to 40,000 pounds (1000 g e n e r a t e d in f e r m e n t i n g to 20,000 kg.) at 30 to 40% bacco; -threedid not. tobacco (IO, l 7 , 1 8 ) . Aloss of moisture. dry matter takes place; this The procedure followed in includes nicotine and other the bulk sweating of tobacco has remained essentially unnitrogen compounds, crude fiber, and ether-soluble and waterchanged for many- vears. In preparation for bulk sweating soluble subst&es (6-9,13). Frankenburg has shown that there enough water is added to the dry bales to prevent breakage is an increase in the protein nitrogen fraction (6) and an increase in nonalkaloid pyridine compounds during the process ( 7 ) . of the leayes when bands of the latter are removed. The hands of filler tobacco, which includes all tobacco except that Reviews of previous work have been made by Johnson (IO), used for cigar binders and wrappers, are usually untied and, in Reid, McKinstry, and Haley ( I @ , and others (4, 1.9,l a ) . Sevsome cases, the midribs are removed from the leaves before fereral theories have been advanced to explain tobacco fermentamentation. In any event the whole leavw, stemmed strips, or tion. This process has been said to be caused by the enzymes of hands are adjusted to the necessary moisture content and placed the leaf, by bacteria, by fungi, and by chemical changes occurin piles. Bulks of cigar wrappers may contain 4000 pounds ring without the aid of either microorganisms or enzymes. Most (2000 kg.) of tobacco or less, 28 to 32% water, and be allowed to published studies have supported either the enzyme or microorganism theories of fermentation as applied to the preparation of ferment until a temperature of 50' C. is reached. For binders and fillers the bulks may contain from 20,000 to 40,000 pounds (10,000 leaf for cigar-making purposes. The work of Johnson (10) lends to 20,000 kg.) of tobacco, 32 to 40% water, and be allowed to support to both of these theories, with particular emphasis on the reach a temperature of 55' to 70' C. High moisture contents and probable importance of fungi. Reid, McKinstry, and Haley (16, high temperatures are permitted when dark leaf is not objection16) support the bacterial hypothesis. It is the purpose of the able, whereas low moisture contents and low temperatures are work described in this paper to indicate more definitely the fundaemployed when light colored leaf is desired, as is the case with mental cause of cigar leaf fermentation. high grade cigar wrappers. Sour wine or vinegar is sometimes CHEMICAL THEORY OF FERMENTATION added along with the necessary water before the leaf is bulked. Such treatment is said to assist in the development of an atThe rate and extent of fermentation can be measured by protractive odor or aroma. duction of carbon dioxide and ammonia, changes in pH, and rise The process of bulk fermentation is often designated as rein temperature. Changes in odor, taste, and burning qualities sweating, when the tobacco subjected to it has been stored for a can also be used as criteria of fermentation. By using any or all year or more, and allowed to age or undergo natural seasonal of these indexes, it can be shown that sterilized tobacco, adjusted sweating in bales or cases. The agenh which initiate aging procto the optimum moisture content with sterilized water, remains esses in cigar leaf and in cigaret tobacco are not necessarily the entirely inactive. The agents required in fermentation are destroyed by heating, which means that the process is biological and 1 Present address, The Pennsylvania State College, State College, Pa.
520
INDUSTRIAL AND ENGINEERING CHEMISTRY
depends on the presence of living matter or enzymes. Evidence supporting this statement will be found in subsequent tables. Without a donbt the theory that tobacco fermentation is purely chemical can be discarded. It should be borne in mind that this conclusion refers to bulk fermentation of cigar-type tobacco at a relatively high moisture content and not to the aging of cigaret types. The latter age at a low moisture content, and it may be possible that under these conditions slow chemical changes occur without the aid of microorganisms or enzymes. ROLE OF FUNGI IN FERMENTATION
It is well known in commercial practice that visible mold growth does not occur on the inside of actively sweating piles of tobacco. This inhibition of mold development may be due to relatively low concentrations of oxygen, to high concentrations of carbon dioxide, or to the presence of other products of bacterial metabolism. However, when the temperature of the tobacco has reached a maximum and begins to decline, the bulks must be disassembled or molding will occur. Tobacco so molded is characterized by a musty odor and is considered unfit for use. When samples of leaf were removed from a fermenting bulk of tobacco, ground, extracted with sterilized water, and a portion of such extract plated a i t h nutrient agar, only a few molds were found to develop. In addition to nutrient agar, Czapek's sodium nitrate sucrose medium, and wort agar, each a t several different acidities were also tried. Again molds were found to be present in small numbers. These low mold counts substantiate the findings of Reid, McKinstry, and Haley (16), but are contrary to the results of Johnson (IO). However, in the latter instance, samples were taken from very small lots of tobacco, which were undergoing what might be called a laboratory fermentation. Experience with certain small lots of sterilized tobacco in the authors' laboratory has shown that accidental contamination leads to a vigorous visible growth of mold with production of carbon dioxide, heat, and a musty odor. Microorganism counts made on such tobacco have shown a large number of fungi and few bacteria. An entirely different flora exists when samples are obtained from a commercial bulk. ENZYMES VERSUS BACTERIA
The foregoing discussion has indicated that bulk fermentation of cigar-type tobacco is not a purely chemical process, but is dependent upon the presence of living material. Likewise, the idea that fungi play a part in this process does not seem credible. I t seem more likely that the fermentation in question depends on the activity of one or both of the remaining probable agents-i.e., plant enzymes or bacteria. The following experimental n-ork ~ v m performed with the aim of establishing whether plant enzymes or bacteria are essential agents in the bulk fermentation of cigar-type tobacco. Johnson (IO) reported that sterilized tobacco strips in Dewar flasks treated with filter-sterile extracts of unsterilized tobacco showed none of the usual signs of sweating. He does not report the effect of additions of unfiltered extracts to sterilized tobacco.
CARBOW DIOXIDEPRODUCTION, A ~ ~ ~ M OPRODUCTION, NIA AND pH CHANGES.One hundred-gram samples of Ohio cigar leaf tobacco at 7% moisture, ground to pass a 20-mesh sieve, were placed in 1-liter Erlenmeyer flasks. With the exception of controls, these were sterilized in an autoclave at 15 pounds' pressure for 20 minutes. Sufficient sterilized water or an equal volume of water extract of tobacco iws added aseptically to each sample to bring the moisture content to 37%. Water extracts of unfermented and unsterilized Ohio tobacco were made by grinding the leaf with sand and water in a mortar and pestle. The extract was separated from the !esidue by filtering through cheesecloth and filter paper, Approxlmately 50 ml. of extract were prepared from 100 grams of leaf. Filter-sterile extracts were made by passing the above liquid through a sterile Berkefeld filter or through a sterile Seitz filter and tested by plating out samples on nutrlent agar. The flasks of tobacco, prepared and treated m described above,
Vol. 42, No. 3
were placed in a constant temperature bath at 37.5" C., and connected with a compressed air line and absorption train by sterilized stop ers and tubing. Air forced through sterilized cotton plugs, sofa lime, and concentrated sulfuric acid was passed through each flask. The effluent gas was passed through a series of tubes which contained dilute hydrochloric acid, concentrated sulfuric acid, soda lime, and calcium chloride. Carbon dioxide was determined by gain in weight of the soda lime and calcium chloride tubes. Ammonia was determined in the hydrochloric acid tube by nesslerization, according to the method of Folin and Bell ( 5 ) ,as modified by F'ickery and Pucher (20). The final pH of the ground tobacco was determined by the method of Bodnar and Barta ( 3 ) . Preliminary trials indicated that the nature of the results was the same after 4 days of incubation as after 15 days. Consequently, the results given in Table I are the averages of 3 determinations on samples which were incubated for the shorter period of time. The results indicate that certain essential agents responsible for the production of carbon dioxide, ammonia, and the accompanying change in pH are present in unfiltered tobacco extracts and are removed from the extract by bacterial filtcra.
TABLEI. EFFECTOF FILTER-STERILE AND UNFILTERED EXTRACTS o s 100-GRAMSAMPLES OF OHIO TOBACCO (Incubated for 4 days) COe. G. Sterile tobacco 0.12 Control 1.04 Sterile tobacco plus filter-sterile extract 0.11 Sterile tobacco plus unfiltered extract 0.90
SHB, Mg.
0.10 1.09 0.15 1.83
Final pH 6.7
7.5 6.8 7.8
THERMOGENESIS. Four large-mouth, 1-gallon Dewar flasks were placed in an incubator which was maintained at 34" * 0.5" C. In each flask was placed a 2-quart, cylindrical, cardboard ice cream container filled with coarsely ground Ohio cigar tobacco. Small holes were made in the bottom of each container and plugged Tith cotton, t o allow the escape of carbon dioxide, which was absorbed by soda lime placed in the bottom of each flask. A hole was made in the top of the container directly in line with a hole in the cork stopper of the Dewar flask, in order to accommodate a thermometer. One container was filled with tobacco, sterilized by autoclaving at 15 pounds' pressure for 20 minutes, and treated with sterilized water. The second container was filled with tobacco, sterilized, and treated with an extract of unsterilized tobacco. The third contained sterilized tobacco to which a filter-sterile extract was added, whereas the fourth acted as a control, and contained unsterilized tobacco and water. A11 tobacco samples were brought to a moisture content of 4Oajo0. Thermogenesis began almost iinniediately in 2 of these flasks and reached a maximum in 48 hours. Temperatures attained after 24 and 48 hours are recorded in Table 11.
TABLE11. THER~IOGENESIS I N 500-GRAM TOBACCO 7 -
SAMPLES O F
OHIO
-Temperature Attained, -C . Sterilized Tobscoo Plus
- .."". WiltP,.
Time, Hr. 24 48
Control 39 41
Sterile water 34 34
sterile extract 34 34
Unfiltered extract 36 40
The results indicate that essential agents responsible for the production of heat are removed from the extract by bacterial filters. Although an unfiltered extract is capable of initiating fermentation as shown by heat, carbon dioxide, and ammonia production, and significant changes in pH, a filter-sterile extract is not able to do so. Some enzymes pass through bacterial filters readily, whereas others do not; probably most of them will be found in the filtrate in low concentrations. However, the absence of an essential enzyme in the complex system undoubtedly required for sweating could prevent the filtered extract from carrying on the fermentation process. Enzymes do not reproduce or multiply in nonliving cells in contrast to microorganisms. If the active agents of an unfiltered extract can be carried from one lot of sterilized tobacco to another through a sequence of several transfers, without loss of fermentative ability, these agents are bacteria and not enzymes.
C t.
521
INDUSTRIAL AND ENGINEERING CHEMISTRY
March 1950
BAC T E RIA
2
4
6
8
IO
20
12 14 16 I8 AGE OF B U L K - D A Y S
24
22
26
28
Figure 1. Relation of Temperature Rise to Number of Bacteria in a Bulk of Ohio Cigar Leaf Tobacco
ThermogeneTRANSFER OF AGENTS CAUSING FERMENTATION. sis in two series of samples was measured by the Dewar flask method, The first flask contained sterilized tobacco to which sterilized water was added, the second contained unsterilized tobacco brought to the proper moisture content with ordinary tap water. At the end of 48 hours a hole was aseptically cut in both ends of each container and sterilized water was allowed to percolate through the tobacco. In this manner an extract was obtained from the sterile sample and from the control flask. These extracts were collected in sterilized receptacles, made up to the necessary volume with sterilized water, and added to separate lots of sterilized tobacco. After 48 hours extracts were prepared from flasks 3 and 4 and added to sterilized samples 5 and 6, respectively. This rocedure was repeated for the treatment of samples 7 and 8. $he data acquired are recorded in Table 111 The data in Table I11 show that the agent which causes'fermentation can be transferred through a series of sterilized tobacco samples. The results obtained from the procedure employed indicate that fermentation is not caused by plant enzymes but is initiated by bacteria.
bulks of ferme?ting Wisconsin tobacco. The presence of cytochrome oxidase was determined by the method of Keilin (11). Peroxidase waa determined by the method of Loew (14),whereas catalase WM estimated by the method of Appleman (,%). The results of these tests are included in Table IV. BACTERIAL SPECIESFOUND I N FERMENTING. BULKS. Seven species of bacteria were isolated from the samples of fermenting Wisconsin tobacco. These seven species were tentatively identified as Micrococcus sensibilis Zettnow, Micrococcus bicolor, Bacillus sphaericus Neide, Phytomonas mellea, Bacillus mycoides,
TABLE 111. TRANSFER OF HEAT-PRODUCINQ AQENTTHROUGH Flask NO.
COMMERCIAL BULKS
The number and kind of bacteria occurring in 30,000-pound bulks of fermenting Wisconsin cigar leaf were determined. Samples were obtained from bulks which were 5, 11, 18,23, 27, and 30 days old. Each sample was taken from as near the center of the bulk as possible and prepared for bacterial counts by drying and grinding to pass a 20-mesh screen. The importance of grinding the leaf has been pointed out by Reid, McKinstry, and Haley (16). Nutrient-agar plates prepared from these samples contained few molds, but large numbers of bacterial colonies. The number of bacteria, shown in Table IV, increased rapidly for the first 5 days, reaching a high of 110 million. A decrease then occurred, until in a 23-day-old bulk only 170,000 organisms per gram were present. I n preliminary tests, cytochrome oxidase, peroxidase, and catalase were found to be active in samples of fermented tobacco. These enzymes were lacking, or present in small amounts, in dry unfermented tobacco taken directly from storage. Qualitative determinations were made of the activity of these enzymes in the
A
SERIESOF STERILESAMPLES
1
2 3 4 5 6 7 8
Description Sterile tobacco plus sterile water Unsterile tobacco plus tap water Sterile tobacco plus extract of flask 1 Sterile tobacco plus extract of flask 2 Sterile tobacco DIUS extract of flask 3 Sterile tobacco plus extract of flask 4 Sterile tobacco plus extract of flask 5 Sterile tobacco plus extract of flask 6
Temp. a t 48 Hr.,
c.
34 41 34 39
34 ..
40 34 38.5
TABLEIV. BACTERIAA N D ENZYMES EXISTINGIN BULKSOF FERMENTINQ WISCONSIN TOBACCO Temp. Description of of Bulk, Sample O C. Stored in warehouse ..,, 3 days in ordering room Davs in bulk
....
-0 5 11
18 23 27 30
.... .
I
.
.
32.2 48.9 52.2 55.0
68.3
No. of CytoBacteria chrome per Gram Oxidase 21,500 Absent
Peroxidase Absent
Catalase Trace
110,000 Absent
Absent
Trace
Absent Present Present Present Present Present Present
Absent Present Present Present Present Present Present
Trace Present Present Present Present Trace Trace
240,000 110,000,000 25,000,000 560,000 170,000
