STUDIES ON THE FERMENTATION OF TOBACCO 1
`
`By JAMES JOHNSON
`Horticulturist, Wisconsin Agricultural Experiment Station, and Agent, Division
`of Tobacco and Plant Nutrition, Bureau of Plant Industry, United States
`Department of Agriculture
`
`INTRODUCTION
`
`After curing, cigar-leaf types of tobacco in particular are allowed to
`undergo one or more fairly definite periods of fermentation or "sweat(cid:173)
`ing." This process is characterized chiefly by an exchange of gases,
`the generation of heat, and a modification of the flavor and aroma of
`the leaf. The aging process in tobacco is not in all cases clearly dis(cid:173)
`tinguishable from fermentation, except that the rate of activity in the
`latter is more rapid and results in an appreciable liberation and
`accumulation of heat and gases. Although the subject of fermentation
`has received considerable attention in the past no satisfactory technic
`for measuring improvement in quality has been devised, and estimates
`of the progress of fermentation are dependent largely upon the opinion
`of those experienced in judging tobaccos.
`As there is little exact knowledge concerning the nature of the pro(cid:173)
`cess of fermentation, comprehensive investigations from several
`different angles will be necessary to establish the facts. The present
`investigations, in which the Dewar-flask method was used, are pri(cid:173)
`marily concerned with the possible relationship between micro(cid:173)
`organisms and the changes involved in the fermentation of cigar-leaf
`types of tobacco.
`
`EARLIER STUDIES
`
`The chemical changes occurring in tobacco during fermentation
`have been given particular attention by several investigators, but
`need not be reviewed here. It should be recalled, however, that tobacco
`fermentation is generally believed to be an oxidation process, and the
`close relation of air to the results secured has been generally recognized.
`Analyses show significant decreases in nitrogen compounds, including
`nicotine, and other organic substances, accounting for a loss of solid
`matter sometimes exceeding 5 percent, during fermentation. The
`total loss of weight, including that of free water, may be considerably
`higher during the process. On the other hand there is a marked libera(cid:173)
`tion of ammonia and carbon dioxide gases as a consequence of this
`activity. The improvement of the aroma, flavor, burn, and other
`qualities is to be regarded as of major importance even though not
`chemically determinable. Separating the essential and desirable
`changes from incidental changes constitutes one of the chief difficulties
`of the chemical aspects of the problem.
`The previous investigations which are of most interest in relation to
`the results secured in the present investigations are those dealing with
`the possible relationship of enzymes and micro-organisms to the
`fermentation process. There have be~n certain claims (17) 2 and
`1 Received for publication Mar. 12. 1934; issued August 1934.
`• Reference is made by number (italic) to Literature Cited, p. 1.59.
`
`Journal of Agricultural Research,
`Washington, D.C.
`
`(137)
`
`Vol. 49, no. 2
`July 15, 1934
`Key no. Wis -61
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`
`there is some evidence for the contention that, given the proper con(cid:173)
`ditions, fermentative changes may occur in the absence of any enzymic
`or microbial activity, but this theory has not received much support.
`The problem seems rather to involve the relative importance of the
`enzymic and the microbial activities during normal fermentation,
`even though these may be regarded only as agencies speeding up the
`rate of oxidation and other chemical changes.
`As early as 1858 an analogy between tobacco fermentation and
`alcoholic fermentation was implied by Koller (10), who added yeast
`with the purpose of increasing the rate of tobacco fermentation.
`Later, tobacco fermentation was compared with that of silage (4),
`"brown" hay (1), manures (19), etc. The bacterial theory of tobacco
`fermentation was most definitely brought forward in 1891 by Suehs(cid:173)
`land (21), who isolated bacteria from sweating tobacco, prepared pure
`cultures, and inoculated these back into tobacco. His claim that the
`flavor and odor of a specific type of tobacco could be developed in
`another type through the use of bacterial cultures has not, however,
`been substantiated. Miciol (15), Davalos (3), Vernhout (22),
`Behrens (1), Koning (11), Jorgensen (9), and others soon afterward
`isolated organisms from tobacco and in general supported Suchsland's
`hypothesis. These workers showed that a variety of organisms were
`present, in what were considered large numbers, i.e., as high as
`112,500 bacteria and 12,500 fungus spores on 100 cm2 of freshly
`fermented leaf.
`Suchsland's bacterial theory soon fell into disrepute under a
`vigorous though not convincing attack by Loew (12) in 1899. Loew
`not only claimed that bacteria were not present in sufficient numbers to
`influence fermentation, but that sufficient moisture was not normally
`present for their development, and that even if sufficient moisture
`were present, bacteria do not find tobacco a congenial medium for
`growth.
`An even stronger argument against the bacterial hypothesis, how(cid:173)
`ever, was the enzymic theory of tobacco fermentation developed by
`Loew and treated in further detail in two succeeding papers (13, 14).
`This theory ascribes tobacco fermentation to oxidizing enzymes nor(cid:173)
`mally present in all living material, as oxidase, peroxidase, and cata(cid:173)
`lase, the latter being present in dried, cured, and fermented tobacco,
`even after several years. The chief role in fermentation was first
`ascribed to peroxidase, an enzyme readily identified by its reaction
`with tincture of guaiacum in the presence of hydrogen peroxide.
`Loew's theory has since been supported mainly by Boeckhout and Ott
`de Vries (2) and Jensen (5), but very little new evidence to support or
`controvert the enzyme theory has appeared in the literature. Jensen
`(6) in 1915 used the Dewar-flask method of study and concluded that
`fermentation of leaf tobacco cannot be inhibited through the addition
`of chemicals detrimental to micro-organisms. The data presented are
`not clear on this point, however, and the variability of the tempera(cid:173)
`tures in the incubator used was such as to render the data of doubtful
`value. On the other hand, Jensen suggests the existence of two types
`of fermentation, namely, one which proceeds at a moisture content of
`20 percent or below and another which requires a higher moisture
`content. Recent investigations in Russia, discussed in considerable
`detail by Smirnov (20), evidently are in general agreement with
`Jensen's conclusions.
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`J uly 15, rn:H
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`St·udies on the Fermentation of Tobacco
`
`139
`
`It is evident from the literature that the determination of the
`nature of tobacco fermentation is complicated by the problem of what
`consti tutes true fermentation and by the variation in th e practical
`requirements of different types of tobacco .
`Studies of a related nature have been conducted by many investiga(cid:173)
`tors on the spontaneous generation of heat in hay, straw, silage, ma(cid:173)
`nures, etc. The purely
`chemical, enzymic, and
`microbial explanations
`have all had stanch
`supporters, butitisin(cid:173)
`teresting to note tha t
`investigations
`recent
`support the microbial
`theory (16, 18), at least
`under te mp er a tu r c
`and moisture condi(cid:173)
`tions und er which or(cid:173)
`ganisms will multiply ,
`and even discount the
`c ooper a tion of e n (cid:173)
`zymes (16).
`
`MATERIALS AND
`METHODS
`At firs t the present
`s tudi es w e r e c on (cid:173)
`ducted with the ordi(cid:173)
`nary narrow-mouth 1-
`quart thermos bot tles.
`Later, wid e - m ou t h
`flasks were s ecur e d
`which were easier to
`fill and permitted of
`handling the tobacco
`under fairly satisfac(cid:173)
`tory aseptic and pure(cid:173)
`culture condition s
`when desired. Thi s
`was accomplished by
`first placing the tobac(cid:173)
`co in moisture-proof
`cellophane containers,
`s tcrilizing it with heat,
`and inoculating it with
`water suspensions of
`cultures of organisms
`by means of u Luer syringe inserted through the cellophane at one or
`more points. The cellophane containers were at first made th e desired
`shape and size before being filled with tobacco ; inoculations were then
`made by injections at u large number of points (fig. 1).
`However, more even distribution of inoculum could be secured by
`preparing larger sealed cellophane bags in which the tobacco was held
`
`FIGURE 1.- A simple method for preparing tobacco samples for fer(cid:173)
`mentation studies with pure cultures. T he sterilfzed tobacco in
`the cellophane roll (A) may be inoculated at any desired points by
`Inserting the syringe (B) through the cellophane before the roll is
`placed in the wide-mouth Dewar flask (C).
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`loosely and into which the inoculum was injected with a syringe and
`mixed with the tobacco by turning and agitating it. The tobacco
`was then pressed into one end of the bag, which was rolled into form
`to fit the wide-mouth Dewar flask (fig. 2).
`It was found that the tobacco could be adequately sterilized in these
`containers without apparent physical injury to the leaf or any appre(cid:173)
`ciable change in moisture content by placing the roll or bag in a scaled
`copper container which was placed in an ordinary steamer. Forty(cid:173)
`five minutes of steaming, during which the tobacco reached a tem(cid:173)
`perature of 80° C., was sufficient to prevent subsequent thermogcnesis,
`and plating out showed that no organisms were present.
`In practice,
`
`}·wuRE 2.-The cellophane-bag (A . B , C) method permits of a uniform application of inoculum under
`aseptic conditions; the tobacco ln the cellophane hag is sterilized in a copper, rubber-tube-sealed contain er
`(D), without undergoing any change in percentage of moisture.
`
`however, the steaming was allowed to continue for 1 hour, during
`which time the tobacco reached a temperature of at least 85°. At
`these temperatures peroxidase was destroyed.
`In other tests peroxi(cid:173)
`dase was found to be destroyed in water extract if heated for 10 min(cid:173)
`utes at 83°, and Loew (12 ) reports that the enzyme is destroyed at
`87° in 3 minutes.
`A limited amount of aeration was made available to the tobacco by
`various means.
`In the case of the narrow-mouth bottles a pliable
`In the ease
`perforated lead tube extended to the bottom of the flask.
`of the cellophane containers, aeration was only provided by puncturing
`the celloph ime at several points with the syringe or a hot needle and
`by not, sealing the mouth of the flasks tightly. No other provisions for
`aeration were made, but since, as a rule, the tobacco was only loosely
`packed and the experiments continued for only 10 days, it is safe to
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`Studies on the Fermentation of Tobacco
`
`141
`
`say that the amount of oxygen available was equal to that in normal
`fermentation in large boxes or bulks. At least, preliminary trials
`with air aspirated through the bot tles did not appreciably inereasc
`thermogenesis, whereas the replaeement of the air with nitrogen,
`followed by sealing, greatly reduced the thermogenie power.
`The tobacco used in the tests was largely of the local variety known
`as Havana No. 142, grown on the Wisconsin Experiment Station form.
`This tobacco contained approximately 30 percent moisture, which is
`close to normal for 'Wisconsin tobaceo in the bale soon after stripping.
`The leaves were first stemmed and eut into strips about one-fourth
`ineh in width; this made the pack more uniform and easier to
`handle. One hundred and fifty grams (about 5% ounces) of this
`
`l•01GURE 3.- 0ne of the constant-temperature incubators and the thermos bottles used in the earlier
`experiments.
`
`tobaeeo was usually used in the I-quart thermos bottles. The
`moisture eon tent was usually raised to 35 pereent or more by atomi7.ing
`In most
`the tobaeeo with the desired quantity of distilled water.
`instanees the ehemicals were applied with the water.
`As soon as the bottles were fill ed they were plaeed in automatieally
`regulated eonstant-tcmperature ehambers. Most of the experiments
`were run in a large 30° C. ineubator in which the temperature nor(cid:173)
`mally varied less than 1 ° (fig. 3). More signifieant results would 110
`doubt have been seeured in some eases by mcubating at a somewhat
`lower
`temperature. Readings were
`taken at 24-hour intervals
`(i.e., at 9 a.m. ), usually over a period of 10 days. The temperature
`inereases shown in the tables were then based on the average of the
`incubator readings subtraeted from the average of the flask readings
`over the 10-day period. The maximum increases, which usually
`occurred about the fifth day, were considerably higher than the
`average.
`In most eases the incubator temperature reeorded was
`75317- 34- -4
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`Journal of Agricultural Research
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`that of a thermometer inserted in a corresponding empty thermos
`bottle.
`After the completion of the test in the thermos bottles, rough esti(cid:173)
`mations of ammonia and carbon dioxide were usually made by draw(cid:173)
`ing air from the aeration tube over concentrated hydrochloric acid
`and through limewater. These estimations of gaseous products are
`to be largely regarded as confirmatory information as to the direction
`and degree of activity exhibited. Exact quantitative determinations,
`while desirable, did not appear to be justified in the present connec(cid:173)
`tion, and would probably not have influenced the conclusions reached.
`The tobacco was then withdrawn from the fl.ask, and a random sample
`of approximately one-half gram, or 10 square inches of leaf was
`introduced into about 5 cc of sterile water, agitated, and allowed to
`stand for about 1 hour before being plated out in nutrient agar or
`potato agar. Notes were also made at this time on the color of the
`
`~341----+---+-----+---+----~-+----+---+----t-----l
`
`t
`w
`.. a:
`a:
`~ 33 1----+-+-+---+--+---+--+-" ...... -+--+---+--1
`.J .. :::,
`
`w
`Q.
`:le
`~ 32 l----t--1---t----t---t----t---+----+---+----+--~
`
`t;
`< 31 1----fl---t----+---+----+---+----+---+----t-----l
`
`DAYS
`FIGURE 4.-Thermogenic behavior of tobacco in a thermos bottle held for JO days at an incubator tempera(cid:173)
`ture of between 30° and 31° C.
`
`water extract, and the odor, as well as on tests for peroxidase with
`tincture of guaiacum and hydrogen peroxide. The entire lot of
`tobacco was again weighed and then placed in an oven for determina(cid:173)
`tion of dry weight.
`
`EXPERIMENTAL RESULTS
`
`In the present investigation about 350 separate fermentation trials
`were made in Dewar fl.asks. The presentation of all the data secured
`seems unwarranted, and consequently only portions will be used to
`illustrate the conclusions drawn. The trials were rarely run in
`duplicate, preference being given to repeated successive trials, which
`were feasible since practically all environmental factors were under
`control and the data were based on average temperature readings.
`Control flasks, i.e., fl.asks containing untreated tobacco, were run,
`however, in connection with practically all trials.
`The typical thermal behavior of such a control may be noted from
`the graph shown in figure 4. The tobacco being placed in the fl.ask
`at room temperature and the fl.ask then placed in an incubator averag(cid:173)
`ing 30.5° C. (87° F.) 1 the temperature rose very rapidly during the .
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`Studies on the Fermentation of Tobacco
`
`143
`
`first 24 hours as a consequence of both the heat from without and the
`thermogenesis from within, which in the presence of sufficient moisture
`usually gets under way the first day. The temperature normally
`continues on the upgrade in the flasks for 3 to 5 days, after which time
`it usually declines gradually. This decline is in part due to a reduc(cid:173)
`tion of the potential heat-producing constituents of the tobacco,
`since if this same lot of tobacco is removed and replaced in the flask
`with the original moisture content, the temperature curve is lowered
`and shortened. However, a gradual reduction in available oxygen
`and an accumulation of gases such as ammonia and carbon dioxide
`injurious to thermogenesis may also play a role in this decline.
`
`EFFECT OF MOISTURE
`
`The percentage of moisture in Wisconsin tobacco at the time it is
`baled on the farm averages approximately 30 percent,3 the variation
`being generally between 25 and 40 percent. This tobacco may be
`fermented in several different ways. If the leaf is sorted, it is packed
`tightly in boxes or placed in bulks, where it goes through one or more
`sweats before being packed in boxes. If the leaf is stemming tobacco,
`the bales are stacked in large bulks, where they undergo fermentation.
`Following a period of aging, usually accompanied by natural drying,
`this leaf is moistened heavily before being stemmed, and the strips
`are again fermented in large bulks at a moisture content of about
`40 percent. The results secured under the various methods of
`handling stemming tobacco may naturally be expected to be quite
`different, and the results in this type as a whole are apparently not
`comparable with those obtained in tobacco types stored at moisture
`contents of 20 percent or below, where little or no thermogenic
`activity occurs.
`In large bulks or under high pressure, tobacco may evidently go
`through a normal fermentation process at a relatively low moisture
`In thermos bottles with only 150 g of
`content (22 to 28 percent).
`tobacco, loosely packed, no significant thermogenic activity was
`obtained at a moisture content of below 30 percent. As the moisture
`content is increased above 30 percent, the thermogenic power was
`found to increase proportionately (table 1). The rate of this increase
`was distinctly raised by increasing the pressure, i.e., placing 300 g
`of tobacco in the same volume (fig. 5). However, significant results
`by the thermos-bottle method are dependent upon having a moisture
`percentage of 35 to 40, which, though comparable with the amount of
`moisture present during the practical process of fermenting stemming
`tobacco, is not comparable with the usual percentage of moisture in
`sorted tobacco. It seems reasonable to assume, however, that within
`certain limits higher moisture contents in the thermos bottles for a
`time at least only serve to hasten activity of the same character as
`proceeds more slowly at lower moisture contents.
`In order that the
`generation of heat may be measurable in the thermos bottles, it must
`naturally accumulate at a more rapid rate than that at which it is
`lost through the insulating power of the flask. Furthermore, moisture
`is actually liberated during normal fermentation, as is suggested by
`the term "sweating", which no doubt renders the leaf and the environ(cid:173)
`ing air increasingly favorable for additional forms of activity. Taking
`
`a The moisture percentages as given throughout this paper are computed on the basis of total weight.
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`Vol. 49, no. 2
`
`all factors into consideration, limited generalizations with respect
`to the similarity of fermentation activity at ranges of from 25 to 40
`percent are permissible. The situation in tobacco stored at moisture
`contents below approximately 20 percent may be expected to be of
`
`5
`
`r;
`/?
`'--'
`LIJ 4
`0:
`:::,
`I-
`<!
`0:
`LIJ
`a.
`::!:
`LIJ
`I-
`~
`LIJ
`(/)
`<!
`uJ
`0:
`0
`==
`LIJ
`"'
`
`3
`
`2
`
`I
`
`<!
`0:
`LIJ
`>
`<!
`
`,' Tl GHT PACK
`I
`f-----1----------1,,-----t------1------1-----'-I
`I
`I
`I
`✓✓--+-~~f----+-----t------i
`
`;
`I
`I
`f-----+----t-/'--,,----lf-----+----t-----+-------t
`
`0
`
`0
`
`25
`
`30
`
`40
`35
`MOISTURE ( PERCENT)
`FIGURE 5.-Relation of percentage of moisture and tightness of packing to the thermogenic activity of
`tobacco in thermos bottles held at an average temperature of 30.2° C.
`
`45
`
`50
`
`55
`
`quite another nature, as are those in tobacco maintained at excessively
`high moisture contents.
`
`TABLE 1.-Relation of percentage of moisture in tobacco to its fermentation in
`Dewar flasks 1
`
`- -
`
`Average temperature
`
`Moisture
`(percent)
`
`Ammonia Carbon
`dioxide
`
`Odor
`
`Color of
`extract
`
`Increase
`Incubator Flask
`- - - - - - - - - - - - - - - -
`0 c.
`o C.
`o C.
`0 Raw ______
`+
`+
`30.4
`30. 3
`31.0
`0. 7
`+ ___ do _______
`++
`++
`32. 9
`30. 3
`31. 3
`1.0
`+ Mild ______
`++
`++
`34. 2
`30. 3
`31. 9
`1. 6
`++ __ _do _______ +++
`+++
`37. 9
`30. 3
`32. 9
`2. 6
`+++ +++ Good ______ +++
`41.5
`30. 3
`32. 7
`2. 4
`+++
`+++ Strong ____ +++
`50.0
`30. 3
`34.8
`4. 5
`+++ +++ --AO------- +++
`54. 3
`30. 3
`35.0
`4. 7
`t In this and succeeding tables, plus signs indicate relative amounts, i.e., + small, ++ medium, +++
`large.
`EFFECT OF TEMPERATURE
`
`Under favorable conditions cigar-leaf tobacco is ready for normal
`fermentation as soon as it is properly cured.
`In practice, some
`months may elapse between the completion of the curing and the
`beginning of fermentation. While this interim is partly a result of
`the time required for marketing, sorting, packing, and storage, fer-
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`Studies on the Fermentation of Tobacco
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`145
`
`mentation does not normally get under way until favorable natural
`or artificial temperatures are presented.
`Little is definitely known about the most favorable temperature
`for fermentation, or regarding the lowest or highest temperatures at
`which it occurs. The solution of this problem presents many diffi(cid:173)
`culties. An investigation of the problem by the Dewar-flask method
`suggests, however, many points of interest. The results of a series
`of trials are shown in table 2. The various temperature control
`chambers used could not be maintained regularly at a variation of
`less than 1 ° C.; consequently small temperature increases at the ex(cid:173)
`tremes were difficult to detect. Evidently, however, little or no
`activity occurred at temperatures below 10° C. (50° F.). At about
`16° C. (61 ° F.) fairly evident thermogenesis began, reaching a maxi(cid:173)
`In other trials the indications
`mum at 20° C. (68° F.) in this series.
`were that the maximum of heat production lies closer to 25° C.
`(77° F.) (fig. 6). At any rate, spontaneous generation of heat begins
`
`5
`
`,..,
`
`0
`
`f------+------+-----+------+--=--
`
`~--+--------1----+----e----------a
`
`\s3 PER.CENT MOISTURE
`
`I
`
`"' a: 4
`
`<(
`
`0
`
`;j
`I-
`<(
`a:
`"' a.
`;; 3
`"' I-
`"=
`"' "' 2
`"' a:
`"=
`"' I
`C>
`<(
`a:
`"' ::.
`
`0
`
`0
`
`5
`
`10
`
`15
`
`30
`25
`20
`TEMPERATURE ('C.)
`
`35
`
`40
`
`45
`
`50
`
`FIGURE 6.-Relation of temperature of incubation to the thermogenic activity of tobacco at two different
`moisture contents.
`
`to drop off at 30° C. (86° F.) and is apparently entirely eliminated
`before a temperature of 50° C. (122° F.) is reached. Temperatures
`as high as 50° C. are rarely, if ever, reached in fermenting boxes of
`leaf tobacco, though in bulk fermentation considerably higher tem(cid:173)
`peratures may be attained (23), whereas in stemming tobacco the
`bulk may be allowed to reach 60° C. (140° F.). For cigar wrapper
`and binder tobacco, however, it is now generally believed that tem(cid:173)
`peratures above 50° C. are injurious to the quality of the leaf. Nor(cid:173)
`mally the bulk is therefore turned or the leaf transferred to boxes
`before this temperature is reached. The curve of the temperature
`rise in such a bulk is typically such as is shown in figure 7, in which
`it will be seen that following a rapid rise of temperature, the rate is
`diminished considerably beyond 43° C. (110° F.) and, in the fourth,
`fifth, and sixth rebulkings, drops off at increasingly lower tempera(cid:173)
`tures, the primary reason perhaps being the lowered moisture content.
`In practice, bulks are now usually turned only once or twice before
`being boxed.
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`TABLE 2.-Relation of temperature to the fermentation of tobacco in Dewar flasks 1
`
`Average temperature
`
`Moisture
`{percent)
`
`39.3
`38.3
`39.1
`37.8
`37.0
`37.3
`37.0
`38.0
`
`Ammonia Carbon
`dioxide
`Incubator Flask
`Increase
`- - - - - - - - - - - - - - - - - -
`o C.
`o C.
`o C.
`2-0.5
`9.8
`9.3
`16. 5
`17.8
`1.3
`20.0
`23. 7
`3. 7
`25.4
`28.0
`2.6
`27.0
`28.4
`1.4
`38.4
`1.8
`36. 6
`44.9
`44.1
`.8
`48. 5
`48.3
`'-.2
`
`Odor
`
`Color of Peroxl-
`extract
`dase
`
`- - -
`++
`++
`0 Raw .......
`0
`++
`++ +++
`++ ..... do ..•.. -
`++ +++ Good _______ +++ +++
`+++ +++ Strong _____ +++ +++
`+++ +++ -·---do ______ +++ +++
`+++ +++ -----do.-•• -• +++ +++
`0 Mild _______ +++ +++
`+
`0 -----do ______ +++
`+
`0
`
`1 See footnote, table 1.
`
`'Decrease.
`
`Judging from the results obtained with the thermos bottles, fer(cid:173)
`mentation apparently does not proceed much below 18° C. (65° F.)
`
`110
`
`..:
`!, 100
`1'J
`0::
`
`0::
`
`:, ... <t
`l:'. 90 t - · I+ - - ' 1--+·H----+-•·--1-- +---+- -+--!-----+--~
`::I:
`1'J ...
`
`40
`DAYS
`FIGURE 7.-Generation of heat in a large bulk of cigar-leaf tobacco following repeated rebulking.
`
`or above 45° C. (113° F.). Several trials with tobacco "incubated"
`in a moist condition at temperatures between 45°-55° C. (113°-131 °
`F.) for 20 days yielded little or no evidence of normal fermentative
`changes. On the other hand, there is some chemical evidence that
`heating tobacco directly may bring about changes resembling certain
`of those due to true fermentation, although evidently such heating
`cannot replace fermentation. Apparently, it is not the actual tem(cid:173)
`perature reached in fermentation which is significant, but the actual
`increase in temperature above the surrounding atmosphere, since
`this increase represents the intensity of the process. Taking all
`facts into consideration, it will be seen that problems of considerable
`practical importance arise in this connection. Apparently fermenting
`rooms should preferably be maintained at approximately 22° C.
`(72° F.), and temperatures occurring in the fermenting leaf above
`
`RJRV EX 1018
`
`

`

`July 15, 1934
`
`Studies on the Fermentation of Tobacco
`
`147
`
`45° C. are less likely to be effective than are lower temperatures. A
`30-degree increase in temperature between 75° and 105° F. should,
`for example, be considerably more effective than a 30-degree rise in
`temperature between 100° and 130° F. Hence, bulkings which result
`in excessive accumulation of heat may actually retard true fermenta(cid:173)
`tive changes.
`
`EFFECT OF HEAT AND ANTISEPTICS
`
`If tobacco is heated to a sufficiently high temperature in dry or moist
`heat, both the microbial flora and the enzymes present are destroyed.
`If such heated tobacco is placed in a thermos bottle in a moist con(cid:173)
`dition, heat generation will still occur, though after some delay. This
`is true microbial thermogenesis, resulting from reinfestation with
`If, however, the technic is modified so as to insure
`micro-organisms.
`the introduction of heat-sterile tobacco into the container and the
`maintenance of aseptic conditions,
`thermogenesis is completely
`prevented, and the leaf is essentially preserved and unchanged
`(table 3). However, it is evidently not possible by the use of heat to
`destroy the microbial flora without destroying or injuring the plant
`enzymes or other constituents of the plant cells which may be con(cid:173)
`cerned with normal fermentation, since the thermal inactivation points
`of most forms of living matter often too closely overlap.
`It seemed quite conceivable, however, that certam chemicals,
`especially those of an antiseptic nature, might exert a selective action
`between microbial activity and the activity inherent in plant cells.
`Johnson and Murwin (8), for example, found in testing various dis(cid:173)
`infectants for tobacco seed, that while corrosive sublimate under
`certain conditions completely checked germination, silver nitrate
`under similar conditions was not injurious to the germination of the
`seed, a function evidently dependent upon enzymic activity. Both
`chemicals, however, were about equally destructive to micro(cid:173)
`organisms. Chloroform, toluene, and acetone are usually conceded
`to be effective germicides, but with the additional property of not
`being particularly destructive to enzymes or harmful to their activity
`at low concentrations.
`
`TABLE 3.-Ejfect of heat applied to moist tobacco in air-tight copper containers on
`subsequent behavior in Dewar flasks when rein/ estation with micro-organisms is
`prevented 1
`
`Heat treatment of 150 g of
`tobacco
`
`Average
`tempera- Ammonia Carbon Peroxi- Bacteria
`dioxide
`ture in-
`dase
`crease
`~ - - - - - - - - - - -
`
`Fungi
`
`None _________ - -_ - -- - --- --- --
`20 minutes, to 45° c __________
`::JO 1ninutes, to 67 ° c _________
`40 minutes, to 74° c __________
`4/i minutes, to 80° c __________
`60 minutes, to 85° c __________
`
`o C.
`4.3
`2.8
`2. 0
`. 2
`
`,_ . I
`
`'-.2
`
`+++
`+++
`+
`+
`0
`0
`
`++ +++ +++
`++
`++ +++
`+
`+
`++
`+
`0
`0
`0
`0
`0
`0
`0
`0
`
`++
`++
`+
`+
`0
`0
`
`1 See footnote 1 table 1.
`
`2 Decrease.
`
`According to Loew (14), many salts and bases are not harmful to
`catalase activity, and some may be beneficial. Mercuric chloride
`however, was found to be distinctly harmful, as was formaldehyde in
`high concentrations (1 to 5). Chloroform was not found to be
`
`RJRV EX 1018
`
`

`

`148
`
`Journal of Agricultural Research
`
`Vol. 49, no. 2
`
`destructive. While the data on this subject are not entirely clear
`in differentiating between destruction and inactivation, nevertheless
`the report by Jensen (5) that mercuric chloride, formol (formalde(cid:173)
`hyde) and chloroform do not prevent tobacco fermentation has been
`regarded by some as strong evidence in support of Loew's theory of
`enzymic fermentation, and good evidence against the microbial theory.
`The results of the experiments here reported, in which the thermos(cid:173)
`bottle method was used, have been quite contradictory to those of
`Jensen. Not only do mercuric chloride and chloroform effectively
`prevent thermogenesis, but acetone, toluene, beta-napthol, and other
`antiseptics at low concentrations also prevent thermogenic activity
`(table 4). The data secured with formaldehyde were less certain.
`Under the conditions of the experiments, both enzymes and microbes
`were apparently inactivated but not necessarily destroyed by many
`antiseptics. Chemicals other than antiseptics, such as ethylene
`chlorhydrin, acids, and bases, were tested with the purpose of dis(cid:173)
`covering possible stimulatory action on thermogenic activity, but no
`conclusive evidence in this direction was secured.
`
`TABLE 4.-Effect of antiseptics and other chemicals on tobacco fermentation in
`Dewar flasks 1
`
`Average temperature
`
`I
`
`Treatment of 150 g tobacco
`
`Mois-
`ture
`
`Per-
`cent
`37. 0
`None ______ --------------------
`Acetone, 5 cc __________________
`37.0
`Toluene, 5 cc __________________
`37. 0
`Chloroform, 1 cc _______________
`37. 8
`Formalin, 2 cc _________________
`51. 1
`Mercuric chloride, 1 g _________
`37.1
`Silver nitrate, 1 g ______________
`37. 1
`Silver nitrate, 3 g ______________
`41. 3
`Ethylene chlorhydrin, 1 cc ____
`43.8
`None _____ --- __________________ 43.8
`Potassium hydroxide, 2.5 g ____
`38. 5
`Oxalic acid, 0.5 g _______________
`41. 0
`
`C (,,'.
`30.3
`30. 3
`30. 3
`30.3
`30.0
`30. 5
`30.2
`30. 2
`27. 7
`27. 7
`30.5
`30. 3
`
`Car-
`Am-
`bon
`monia diox-
`ide
`
`Odor
`
`Perox-
`idase
`
`Staphylo-
`cocci to
`the
`square
`inch•
`- - - - -
`
`In-
`Incu- Flask
`crease
`bator
`- -- - - -
`oc.
`oc.
`Number
`3.4 +++ +++ Mild ____ +++
`33. 7
`100,000
`0 raw _____ +++
`30. 7
`.4
`0
`120
`0 ___ do ____ +++
`.4
`30. 7
`0
`0
`+ __ _do ____ +++ ----------
`+
`30.5
`.2
`1. 7 +++ ++ ___ do ____ +++
`31. 7
`500
`0 __ _do ____ ++
`30.8
`. 3
`0
`0
`+ +++ ___ do ____ +++
`1. 5
`31. 7
`0
`+ +++ __ _do ____ +++
`31. 6
`1. 4
`0
`2. 6 +++ +++ Strong __ +++
`30. 3
`20,000
`4. 7 +++ +++ __ _do ____ ++
`32.4
`50,000
`2.9 +++ +++ __ _do ____ ++
`33.4
`100,000
`4.1 +++ ++ __ _do ____ +++
`34. 4
`10. 000
`
`t See footnote 1, table 1.
`
`2 Estimated.
`
`The results secured with silver nitrate are of particular interest as
`indicative of a differential effect on microbial and enzymic activity.
`Under the conditions of the experiments, silver nitrate reduces ther(cid:173)
`mogenesis to about one-half that of the untreated controls. Th