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U. S. DEPARTMENT OF AGRICULTURE.
Report 3STo. 59.
CURING AND FERMENTATION
CIGAR LEAF TOBACCO.
OSCAR LOEW,
Division of Vegetable Physiology and Pathology.
"WASHINGTON:
GOVERNMENT PRINTING OFFICE.
1899.
I
i.
LETTER OF TRANSMITTAL
U. S. Department of Agriculture,
Division of Soils,
Washington, D. C, February 19, 1899.
Sir: In accordance with the recommendation in my annual report
for 1898, approved by you, and the authorization of Congress, a com-
prehensive line of tobacco investigations, to extend and supplement
the tobacco soil investigations of the Division of Soils, has been under-
taken. The work includes the mapping of soil areas, studies in fer-
mentation, improvements in breeding and selection, investigations of
the conditions of growth and manipulation in foreign countries, and
the question of supplying tobacco to foreign markets.
In examining and classifying the soils of the principal tobacco districts
of the United States certain facts developed in regard to the commercial
value of the crop from certain soils which could not be clearly under-
stood or explained without a further investigation of the methods of
curing, fermenting, and handling of the tobacco, and possibly also of
breeding new varieties. Only in this way could the full value of the
soil work of this Division be shown. As soon as it was definitely
determined that the work could be undertaken, I requested the Chief
of the Division of Vegetable Physiology and Pathology to detail an
expert to investigate the curing and fermentation of tobacco, this work
naturally pertaining to his Division. In accordance with this request
Dr. Oscar Lo6w was detailed to carry on the investigations, and at
once went to Quincy, Fla., where he spent some time during the fer-
mentation season.
Other Divisions have also been asked to cooperate in a similar manner
in other phases of the comprehensive investigation. In view of this
extensive cooperation it is proposed to issue a series of reports on
tobacco investigations, to which all the Divisions of the Department
may contribute matter pertaining to the subject.
Dr. Loew's discovery of the real cause of the fermentation of cigar
tobacco, as remarked by Mr. Galloway in submitting this report, can
not fail to prove of great scientific interest and economic value, and
will unquestionably modify the methods of curing and fermenting
when the investigation has been carried further and the conditions
and principles of the process are better understood.
3
216439
This treatise, which is more or less technical, will be followed by a
more popular one giving the substance of Dr. Loew's investigations in
connection with some temperature studies which have been made in
the fermenting piles of tobacco in Florida and Connecticut. I respect-
fully recommend that the manuscript herewith submitted be published
as Keport No. 59 of the Department.
Bespectfully,
Milton Whitney,
Chief of Division,
In Charge of Tobacco Investigations.
Hon. James Wilson,
Secretary of Agriculture.
LETTER OF SUBMITTAL
U. S. Department of Agriculture,
Division of Vegetable Physiology and Pathology,
Washington, D. C, February 19, 1899. '
Sir: I respectfully submit herewith the manuscript of a bulletin
prepared by Dr. Oscar Loew, of this Division, on The Curing and
Fermentation of Cigar Leaf Tobacco. The work on tobacco has been
carried on in accordance with the plan of cooperation recommended in
your report to the honorable Secretary of Agriculture for 1898. The
investigations have involved bacteriological, chemical, and chemico-
physiological studies, and the interesting results obtained will, it is
believed, open the way for further work along important lines.
The chemical work has been carried on in the laboratory of the
Division of Chemistry, and we are greatly indebted to Dr. H. W. Wiley,
the Chief of that Division, for facilities furnished. At Quincy, Fla.,
Dr. Loew received much information and valuable assistance from
Mr. Henry Storm and Mr. W. M. Corry, second vice-president and
general manager, respectively, of the Owl Commercial Company^ which
has a very large tobacco plantation at that place and which has done
more than any other agency in developing the tobacco industry in
Florida. We wish to express our thanks to these gentlemen for their
kindness.
Respectfully, B. T. Galloway,
Chief of Division.
Prof. Milton Whitney,
Chief, Division of Soils,
In Charge of Tobacco Investigations.
\
CONTENTS.
Page.
Introduction 9
The curing 10
Decrease of protein 10
Regulation of heat and moisture 11
Flavor 12
Color 12
Ammonia 13
The sweating or fermentation process 13
Rise of temperature 14
Oxidation 14
Losses ■- 15
Development of gases 16
Starch 16
Sugar 16
Tannin 17
Fiber 17
Ashes 17
Nitrate 17
The cold sweat, aging, or after- fermentation 18
The petuning of the tobacco 19
The bacterial fermentation theory of Suchsland 20
The oxidizing agency in the fermenting tobacco leaf 23
Views on the physiological functions of the oxidizing enzyms 26
The tobacco oxidase and peroxidase 27
Summary 33
Recent foreign literature 34
7
j j j j j j ,* J ,
CURING AND FERMENTATION OF CIGAR LEAF
TOBACCO.
INTRODUCTION.
The production of tobacco adapted to the different market demands
has become a prominent factor in national economy. Of particular
importance is the production of superior cigar leaf tobacco. The filler
leaf of a cigar must above all things have a good flavor, good aroma,
and good burn. In the wrapper leaf, however, still other qualities come
in, such as elasticity, pliability, size, shape, color, size of the veins,
the fineness and peculiar grain of the Havana type^ and the smooth
silkiness of the Sumatra.
Little is known of the chemical properties of the leaf, especially of
those which contribute to the flavor and aroma. It is probable that the
actual amount of nicotine is relatively unimportant, and it is certain
that the excellence of the leaf and its adaptation to market demands
is not dependent, except in a very general way, upon the amount of
nicotine. It has long been known that certain of the potassium salts,
especially potassium chlorid, can not be used at all for the production
of high types of cigar tobacco, as they give the leaf a poor burn. It is
furthermore an old experience of tobacco growers that excessive nitrog-
enous manuring tends to produce a large leaf, of inferior quality, con-
taining an increased amount of nicotine. If the prime object of tobacco
culture were the production of nicotine, as the prime object in raising
sugar beets is the production of sugar, then the rational procedure
would be to furnish an excess of nitrogenous manures, but nicotine
alone does not make a good cigar tobacco any more than alcohol
alone would make a good wine. The substances producing the flavor
and aroma, therefore, although probably present in minute quantities,
are much more important than the actual percentage of nicotine found
in the cured leaf.
Whitney 1 has shown that tobacco suited to our domestic cigars is
grown only upon certain soils and under certain climatic conditions. It
appears, therefore, that the leaf capable of being converted into a cigar
leaf through the ordinary processes of curing and fermentation must
1 Bull. No. 11, Division of Soils, U. S. Department of Agriculture.
9
10
possess certain characters. A fresh leaf has no specific taste, nor has
it any specific odor, but the finished leaf has a sharp, saline taste and
a characteristic odor.
From the time the tobacco leaf is gathered in the field until the
manufacture of the cigar and even afterwards a series of highly inter-
esting changes take place in the leaf, as a result of which the charac-
ters of the finished leaf are developed and fixed. There are three
stages in these changes, viz, (1) the curing process; (2) the sweating,
or fermentation; and (3) the cold sweat, after-fermentation, or aging, as
it is variously called.
THE CURING.
There are two periods in the curing process: The first period, in
which the cells of the leaves are still alive and induce processes of
metabolism; and the second period, in which the cells have died and
the chemical changes have therefore no connection with the living
protoplasm. In the former period, which may last only a few days
(longer with the ribs), the starch content is dissolved and the sugar
formed is partly consumed by an increased respiration 1 and partly
transported to the ribs, where, as Miiller-Thurgau has shown, starch
may be formed again. In the latter period the enzyms alone are active.
Decrease of protein. — With the consumption of a large amount of the
sugar a state of inanition or starvation sets in, and the reserve protein
is attacked by an enzym, trypsin-like in character, the action of which
will continue after the death of the cells. A cold-prepared aqueous
extract of a fresh leaf will show albumin on the addition of nitric acid
and warming, while the cured leaf does not give this reaction. The
reserve protein and a certain albuminous portion of the nucleo-proteids
of the protoplasm will thus finally be split and transformed into amido
compounds and bases, only the remaining nucleins resisting, hence the
decrease of protein matter in the curing and fermentation process will
stop at a certain point. Such proteolytic processes proceed not only
in plants exposed to darkness, which means their starvation or inani-
tion, but also in all cases where reserve protein must be dissolved to
enable further development, as in germination or development from
bulbs.
It is in full accordance with physiological principles that when cells
are in want of nourishment they produce a larger amount of enzyms
than when well nourished. This explains why tobacco leaves killed
immediately after being gathered will show imperfections when after
having been moistened they are subjected to the curing process. The
enzyms that have been produced during the inanition state of the
1 The respiration of a pile of such fresh leaves may soon lead to a considerable and
even injurious rise of temperature, as in the respiration of germinating barley on the
malting floor. A moderate t rise is often intentionally brought on, as it hastens
tbft curing. Sometimes; tfrib *ise of temperature is called sweating, although the
"•use here is a' different' one 'from the true sweating, or fermentation, following
r caring.
11
cells, however, will naturally remain active after the death of the pro-
toplasm from starvation has set in. 1
Considerable variation has been found in the total nitrogen content
of the fresh leaves, as well as in the amido nitrogen content of the
cured leaves. The amount of the former may vary in American, Greek,
and German tobaccos from 2, 3, or 4 per cent to 8 per cent of the dry
leaf. One-third of this and even more can turn into amido compounds
in the curing process.
Regulation of heat and moisture. — Further changes, relating to color
and flavor, set in with the death of the cells. However, it requires a
most judicious regulation of the moisture, temperature, and ventila-
tion of the barn where the tobacco leaves are hung up to obtain those
changes which characterize cured tobacco of a superior quality. This
curing process may last four weeks or even much longer. 2 When the
weather is too dry all the chemical changes in the loaves come to a pre-
mature stop, but on the other hand when it is too moist the danger of
mold development arises. In the former case the barns must be
eventually kept closed and water introduced, while in the latter case
careful application of heat may be resorted to.
An interesting experiment in curing by artificial heat has been
described by E. H. Jenkins. 3 Some farmers have tried the burning of
sulphur with the intention of killing the mold spores by sulphurous
acid, but this requires the utmost precaution, as the leaves themselves
might easily be injured and even all further action in them stopped.
Sometimes mold fungi will develop unnoticed in the stems, appearing
distinctly later on, when the sweating operation has begun. All dis-
eased leaves must be discarded before fermentation begins in order to
avoid further damage by the spreading of the fungi. Tobacco growers
in Florida recognize the white mold, the yellow mold, the blue mold,
and the stem-rot mold, the latter being the worst and causing much
damage. Sturgis has described a bacterium causing pole burn of
tobacco, 4 and further determined the fungus causing the stem rot to be
Botrytis longibranchiata. 5 Jenkins reports that the pole burn disease
"may destroy a portion or even the whole of the harvested crop within
forty-eight hours after the time when the trouble is first noticed."
1 It is somewhat difficult to prove the presence of diastase in healthy normal
leaves, as very small quantities may resist extraction.
2 The drying, or curing, for good cigar tobacco requires about as much time in
America as it does in Europe. Tscherwatscheff, a Russian, has described the Ameri-
can method as requiring but four days with applications of artificial heat (Landw.
Jahrb., 1875). What he had seen, however, was nothing but the preparation of
light-colored cigarette tobacco as practiced in North Carolina, Virginia, and Ken-
tucky. In curing cigar tobacco fire is resorted to only when damp, foggy weather
prevails for a long time.
3 Conn. Agr. Expt. Sta. Ann. Rept., 1897; Ibid., 1892, p. 38.
4 Conn. Agr. Expt. Sta. Ann. Rept., 1891.
6 Ibid., Sturgis's list of tobacco diseases.
12
Flavor. — The development of the flavor of cured tobacco has not yet
been explained. At first a decided flavor of cucumbers 1 is generated,
which later on is entirely replaced by the rank and common straw
smell of cured tobacco, giving rise finally to the superior tobacco flavor
developed by the sweating or fermentation process.
Color. — As jregards the brown color of cured and fermented tobacco,
there can hardly be any doubt that not only one but several compounds
contribute by their chemical changes to its development. Of course
the first supposition would be that the tannin, by being changed into
a phlobaphene (a brown product), is the principal cause. 2 Thus, for
example, in the autumn, when the leaves of oaks and of various other
trees containing tannin die off, a brown coloration sets in. But the
intensity of the brown color of the fermented tobacco leaf does not run
parallel to the different concentration of the tannin in the cell systems
of the leaf. A healthy tobacco leaf was placed with its base in a dilute
solution of ferrous sulphate (about 1 per cent) for from twelve to fifteen
hours, at the end of which time this reagent had risen to the tip of the
leaf, thereby partly killing it. A reaction in the form of a black color
appeared, principally in the epidermis and to some extent also in the
mesophyll, but not at all in the vascular bundles. This black coloration
seemed to be restricted to the chloroplasts.
The epidermis of cured leaves, however, contains the least amount of
coloring matter and is sometimes entirely devoid of it, with the excep-
tion of the gland hairs, while the mesophyll cells always contain a brown
substance in irregular-shaped or rounded masses. The principal part
of the brown matter, however, is in the veins of the leaves and even the
most minute ramifications of the vascular bundles appear to be a much
darker brown than the neighboring mesophyll cells. The circumstance
that the veins contain less nicotine than the rest of the leaf also mili-
tates against the view that the coloring matter is principally due to
the oxidation of nicotine. However, there occurs in the veins a bitter
principle that does not seem to occur in the rest of the leaf, and perhaps
this may contribute to the color.
It is easy to show that several compounds contribute to the brown
coloration in well-cured leaves. In the first place, much brown matter
is extracted by cold water. Leaves thus exhausted will yield up another
portion of brown matter to warm, dilute sulphuric acid, and finally
still another portion 3 of a different chemical behavior is extracted by a
warm, dilute solution of potassium hydrate.
ir The expressed juice of a fresh tobacco leaf is at first without odor, but it gradu-
ally assumes that of fresh cucumbers, which later on is destroyed by putrefaction.
2 According to Savery, the tannin of tobacco is identical with that of coffee. There
exists, evidently, several kinds of phlobaphene, depending on the kind of tannin.
3 This latter portion is a mixture of several compounds, some colorless and pectose-
like, and one colored and phlobaphene-like. Twenty-five grams of fermented
tobacco from Florida yielded 0.51 grams of this product. The cell membranes of the
tobacco thus treated exhibit under the microscope a swollen appearance.
13
One author has assumed that the chlorophyll is first attacked in the
curing process and destroyed, but this is not correct. The green color
of the chlorophyll is in the beginning merely covered by the brown
substances. In the thin samples of fermented leaves of a light-brown
color green spots may frequently be noticed, and even dark-colored,
freshly fermented leaves may sometimes yield a greenish solution upon
extraction with strong alcohol. It is of some interest to note that the
brown matters are insoluble in absolute alcohol.
Ammonia. — An interesting feature in the curing and fermenting
process is the formation of a small amount of ammonia. As the green
leaves contain some asparagin, the formation of ammonia might be due
to a small extent to the decomposition of this amide, which readily
yields ammonia and aspartic acid. But in certain tobacco crops there
occur only minute quantities of asparagin. Certain amido compounds
formed by decomposition of proteids and also a part of thj3 nicotine in
decomposing probably yield the principal amount of ammonia. The
nicotine undergoes, in the fermentation process at least, a considerable
diminution, as explained below.
The opinion that the ammonia deteriorates the quality of the product
is certainly unfounded, as Fesca has correctly pointed out. It has been
demonstrated by Behrens that during the curing process a part of the
sulphur of the decomposed proteids is oxidized to sulphuric acid and
that the amount of compounds soluble in ether decreases. The latter
consist of a fatty substance and a volatile oil of disagreeable odor
derived principally from the gland hairs.
The total loss of dry matter in the curing process is subject to great
variation, depending mainly upon the amount of starch present at the
time of gathering, as above stated. The diminution of dry matter may
be as much as 40 per cent.
The principal changes in the curing process may be summed up as
follows :
(1) Disappearance of starch.
(2) Formation of sugar and its partial disappearance by respiration.
(3) Decomposition of protein with formation of amido compounds.
(4) Decrease of fatty matter.
(5) Decrease of tannin.
(6) Change of color and flavor.
THE SWEATING OB FERMENTATION PROCESS.
The so-called fermentation process develops in the tobacco leaves the
characteristic qualities of the commercial article. It is natural to sup-
pose that the same agency which finishes the curing process after the
death of the cells remains active during the so-called fermentation
process also. The fermentation follows immediately after the curing
when both are done by the grower, but where the cured tobacco is
bought up by manufacturers several months may pass before it is sab-
14
jected to the sweating process. This operation begins when the tobacco
is in the proper "order" or "case," being brought into this condition
naturally on a damp day, or by an exceedingly cautious moistening,
avoiding any visible water on the leaves. The amount of water applied
must just suffice to bring on moderate imbibition. The total amount
of water necessary to bring on a normal sweat is from 18 to 25 per cent
of the moistened leaf. A portion of this water (about one-fourth) is
again lost during the sweat.
The sweating of the Florida leaf in bulk requires from six to eight
weeks, the original crude and rank smell of the cured tobacco being
gradually changed to the proper aroma of the finished tobacco, and
the glossy appearance and the texture 1 being well brought out. Light-
colored wrappers require a slower and cooler fermentation than the
dark-colored leaves used as dark wrappers or fillers.
Rise of temperature. — When the cured tobacco is sold by the farmer
a large number of leaves are tied together at the base, forming "hands."
At the beginning of the sweat such "hands" are well shaken in order
to open all the foliage and admit air to every part. Then commences
the moistening, when necessary, which is done by exposing the " hands,"
under continuous shaking, to a current of steam issuing from a pipe; by
spraying with a fine spray; or by dipping, in which case the bases of
the "hands" are plunged into water and shaken, the adhering water
being soon drawn by capillary attraction into the leaf. These "hands"
are then packed, with the butts outside, in piles 4 to 5 feet wide and 12
to 15 feet long. The rooms, which contain a large number of such piles,
are kept warm, and steam passes freely from a number of pipes into the
air of these rooms to secure uniform moisture, as otherwise the warming
piles would soon become too dry. The temperature of these piles rises
in from one to two days considerably above the temperature of the fer-
menting room and may reach 52° C. (126° F.) or higher. Kepacking
becomes necessary in from three to four days in order to check the rise
in temperature and to shake out the leaves. The lower "hands" are
now placed on the top and the outer ones in the center in order to give
all leaves an equal chance to improve. The temperature now rises
more and more slowly, the next repacking not being necessary before
about seven or eight days. Altogether the piles are repacked from
five to eight times. When the temperature rises too high the color or
the aroma may be injured, hence frequent examinations are necessary.
These examinations are made by pushing the hand into the piles, a
decision being reached by the sense of feeling. 2
Oxidation. — Tobacco manufacturers are well aware of the fact that
a moderate quantity of air should gain access to the interior of the
1 The texture, or grain, of the leaf means to tobacco manufacturers small points
plainly visible on the extended leaf. It appears that these points are the bases of
the gland hairs, most of which break off in the curing and sweating processes.
2 For details relative to the treatment of the fermenting tobacco heaps the reader
is referred to Farmers' BuUetin No. 60 and to the next report on tobacco.
15
fermenting piles and that undue pressure must be avoided in order not
to diminish this access of air more than is necessary to insure an accu-
mulation of heat. Not only are numerous little channels left naturally
in the piles, but diffusion also will set in as soon as the air in the piles
becomes warmer than the surrounding atmosphere.
Repeated efforts have been made to replace the sweating or fermen-
tation process by a direct oxidation. Dr. Mew, of the Army Medical
Museum of this city, assures the writer that some experiments made by
him about twenty years ago to improve the cured tobacco leaf by direct
application of a dilute solution of permanganate of potassium resulted
in an essential improvement, the product being milder. Similar results
have been recently mentioned by Kiessling. 1 In Germany a patent
has been granted to the firm of Siemens & Halske for treating tobacco
with ozone. However, oxidation often takes quite an undesirable turn,
and the danger of destroying the aroma is quite as great, if not greater,
than the likelihood of developing it by artificial means.
Losses. — Jenkins has shown 2 that the losses in fermentation are
apparent in the nicotine, protein, amido compounds, nitrogen-free
extract, and also, to a much less extent, in the ether extract. The
loss of nicotine varies considerably in different samples and was
found by Jenkins to range from one-sixth to one-half in three samples
analyzed. Behrens observed in one sample a decrease of nicotine
from 1.46 per cent in the cured leaf to 1.07 per ceut in the fermented
leaf. Dambergis found in air-dry Greek tobaccos, having from 7 to 14
per cent of water, from 2.8 to 0.7 per cent of nicotine. 3
The question as to how much the loss of organic matter amounts to
during the sweating process can be answered only approximately and
by comparing parts of one and the same leaf, but a constant result
will never be reached, as the nature of the proceeding in fermentation
brings on differences in temperature, water content, and access of
oxygen, and thus leads to variations. In the fermenting heap thick
and thin leaves occur, often varying more than 20 per cent in weight
for an equal surface area. Leaves grown in the shade are thinner than
those exposed to direct sunlight, and in hot, dry summers the leaves are
thicker and coarser than in moist, rainy seasons. 4 These conditions of
course naturally influence the result.
Some tobacco manufacturers estimate the average loss during the
fermentation process to be 15 per cent (organic matter and water
together), while others estimate the loss of solid matter alone to be
1 Der Tabak, Berlin, 1893.
2 Conn. Agr. Expt. Sta. Ann. Rept., 1891.
3 Oesterreich. Chem. Zeitg., No. 16, 1898.
4 In the rainy season of 1891 Sumatra tobacco leaves weighed 52 grams per square
meter, while in the dry season of 1892 the leaves grown on the same spot weighed
80 to 90 grams per square meter. Behrens explains this difference by the larger
intramolecular spaces produced by excess of moisture (Landw. Vers. Stat., 1894,
Band 43, p. 272).
16
from 4 to 5 per cent. According to Jenkins, 1 the losses may be even
larger. He reports that "the upper leaves, short seconds, and first
wrappers lost, respectively, by fermentation 9.7, 12.3, and 9.1 per cent of
their total weight. But while three-fourths of the loss in the case of
the short seconds consisted of water, in the case of the upper leaves
almost three-fourths of the loss was dry matter. The first wrappers
lost a little less dry matter than water."
Development of gases. — The formation of ammonia can be noticed by
the characteristic odor in the fermenting rooms, but the amount is not
so high as one might naturally be led to suppose from the intensity of
the smell. About 3 liters of air from the interior of a fermenting pile
when drawn through 25 cc. of Nessler's reagent, produced a light yellow
color, indicating about 0.05 milligram of ammonia. No trace of hydro-
gen sulphide is given off. Test tubes containing filter paper moistened
with basic lead acetate remained perfectly colorless for twenty-four
hours in the fermenting heaps, hence it may be safely concluded that
no protein decomposition resembling putrefaction takes place. 2 The
amount of carbonic acid given off was also much smaller than would
naturally be expected from the apparent energy of the action.
Stareh. — Small quantities of starch are sometimes found in fer-
mented tobacco when the curing process has not been carried out prop-
erly in all parts of the leaf or in parts of leaves broken, or injured by
fungi, as observed by Miiller-Thurgau, but this occurrence of a small
percentage of starch interferes with the flavor just as little as does the
closely related cellulose. The well-prepared tobacco wrappers from
Florida examined by the writer did not show a trace of starch. The fact,
however, that in the curing process the solution of the starch is going
on with great energy forms a contrast to the observation that in cer-
tain cases remnants of starch remain unattacked during the fermenta-
tion process. This admits of hardly any doubt that the diastase 3 -is
gradually destroyed, perhaps by the proteolytic enzym.
Sugar. — As to the disappearance of the last remnant of sugar during
the sweating, amounting, according to Miiller-Thurgau, to from 1 to 3.3
per cent, some authors assume oxidation to carbon dioxide and water,
and others assume a partial transformation to acetic acid. When it is
taken into consideration that an alkaline medium can soon change
glucose into organic acids (gluconic, saccharinic, etc.), especially in
the presence of air, a more simple explanation would be at hand than
1 Conn. Agr. Expt. Sta., Ann. Kept. 1892, p. 28. The leaves used for comparison were
most carefully selected and were as nearly alike in color, size, and texture as pos-
sible.
sNessler's comparison of the sweating process to putrefaction is certainly not
admissible ; neither is his declaration that the formation of ammonia is not normal,
but simply a sign of true putrefaction.
3 Diastase is absolutely necessary to dissolve and saccharify the starch. The
dextrin and maltose thus formed may afterwards be transformed into glucose by the
living protoplasm itself, wherever this latter comes under consideration.
17
the assumption of a perfect combustion of the glucose. There are
organic acids present in the original tobacco leaf, such as citric, malic,
and oxalic acids, in the form of neutral salts. A part of these acids
may be changed and destroyed in the fermentation process, while other
acids may be formed by the changes the glucose undergoes. 1 The
nicotine is bound to organic acids and is not present in the free state;
besides, most of the ammonia formed is in combination with organic
acids, but a part of it is easily liberated by boiling the aqueous extract
of the fermented tobacco. These vapors have a strong alkaline reac-
tion and an ammoniacal odor, and are due either to the volatilization
of some ammonium carbonate or to the dissociation of a neutral ammo-
nium salt of a bibasic acid.
Tannin. — The amount of tannin, like that of nicotine, also decreases
in fermentation. It varies from 0.3 to 2.3 per cent in commercial
tobaccos. The Florida tobacco of 1898 contained only traces of tannin
after the fermentation was over. The amount of fatty matter, or, more
correctly speaking, of substances soluble in ether, was found by Jen-
kins 2 to decrease in fermentation from 3.5 to 2.8 per cent of the dry
matter. Behrens observed in one case a decrease from 9.14 per cent in
the cured to 8.34 per cent in the fermented leaf. The amount of such
fatty substances was found to vary in different samples from 1.8 to 10
per cent and in some cases even more. The decrease of fatty matter
during fermentation is probably due to the volatilization of a volatile
ethereal oil. It is certainly very improbable that some true fat was
oxidized to carbonic acid and water. Little attention has been given
thus far to the small amount of resins in tobacco.
Fiber. — In regard to the fiber, Jenkins determined its amount in
Connecticut tobacco as ranging from 13 to 14 per cent. Fesca and
Jmai found the range in Japanese tobacco to be from 13 to 15 per cent.
Only the ribs contained more — from 22 to 24 per cent. 3
Ashes. — The amount of mineral matter is subject to very great vari-
ation, namely, from 10 to 27 per cent.
Nitrate. — A question of special interest is the fate of the nitric acid
probably present exclusively as potassium nitrate in fresh leaves. Some
authors believe that nitrification goes on during the fermentation proc-
ess, which would lead to an increase of nitrate in the fermented leaf.
This, however, has never been proved by chemical analysis and is indeed
1 The precipitate obtained by copper acetate from a hot, aqueous extract of fer-
mented tobacco contains, among other things, some succinic acid. The writer did
not recognize butyric acid among the volatile acids in Florida tobacco, but acetic
acid was present.
3 Conn. Agr. Erpt. Sta., Ann. Kept. 1890. Correct comparison is, however, possible
only in calculating for a constant, e. g., cellulose.
3 There are still certain substances in the fermented tobacco which thus far could
not be characterized. Some analyses show from 1.7 to 18.9 per cent of nitrogenous
extractive matter and from 8.6 to 16.7 per cent of indefinite insoluble matter. There
exists great difficulty in isolating certain compounds from these mixtures.
15846 2
18
highly improbable. Other investigators, for instance, Jenkins, have
proved that the nitrate content undergoes only an insignificant decrease, 1
and still others, as Fesca and Behrens, assert that the nitric acid disap-
pears completely, probably by reduction. These contradicting state
ments may be due to the great variations occurring in the nitrate per-
centage. A small amount might disappear completely while a larger
amount decreases but little, although in both cases the absolute amount
disappearing may be the same.
The writer examined, qualitatively, both fresh and fermented leaves
from the same farm near Quiucy, Fla., and found a moderate amount of
nitrate in both. The samples of tobacco examined by Jenkins con-
tained from 1.89 to 2.59 per cent of nitric acid (N 2 5 ) at a water content
of 23.5 to 27.5 per cent, while Behrens's samples contained only 0.2 per
cent of this acid in the dry matter. From the disappearance of such a
small amount of nitric acid, it can not be inferred that larger quantities
would disappear entirely. In one of Jenkins's samples the amount of
nitric acid diminished from 2.59 to 2.35, that is, a diminution corre-
sponding to 0.4 per cent calculated for dry matter of the wrapper leaves.
Dambergis observed variations of nitric acid in Greek tobacco of com-
merce of from 0.5 to 3.37 per cent of the dry matter. Not only the mode
of manuring, but also the nature of the soil and the weather, influence
the nitrate content of the plants, hence large differences can not be a
matter of surprise.
The principal changes which take place during the sweating or fer-
mentation process, as found by various investigators, may be summed
up as follows:
(1) Decrease of nicotine.
(2) Increase of ammonia.
(3) Increase of alkaline reaction.
(4) Disappearance of sugar.
(5) Decrease of nitrate.
(6) Improvement of flavor and aroma. .
THE COLD SWEAT, AGING, OR AFTER-FERMENTATION.
The cold sweat which unfermented tobacco undergoes, and which
corresponds with the aging of wines, may be intentionally carried on
for as long as two years, where the main fermentation process has to be
shortened for any reason or is not thoroughly completed. Fully and
perfectly fermented leaves do not require this cold sweat, and the
manufacturers of a good product prevent after-fermentation by giving
such a degree of dryness in packing that further changes are stopped,
as after-fermentation might finally lead to great differences in the
product, which should be uniform. The interior of the piles or cases
would naturally become warmer, and the leaves would change more
iConn. Expt. Sta., Ann. Rept. 1892. To Jenkins belongs the credit of having first
compared the fermented with the unfermented leaf in regard to the chemical changes.
19
*
than those near the sides. In many cases, however, a further change
is found necessary, and slightly moistened sponges are placed in the
cases with the tobacco in order to maintain a certain degree of mois-
ture. After-fermentation carried on for too long a time might finally
destroy all good qualities by further oxidations.
THE PETUNING OF THE TOBACCO.
The petuning is an operation first practiced in Cuba, and consists in
spraying a liquid on the leaves during or after the sweating process.
The fillers only, and not the wrappers, are petuned, the intention being
to give them a darker color, an improved flavor, and the appearance and
character of a strong tobacco. The composition of the petuning liquid
used in Cuba is kept secret, and indeed each planter claims to have
something known only to himself. It is generally believed that one
method of preparing the petuning fluid is by pouring organic fluids
yielding ammonium carbonate over crushed tobacco stems, and letting
this mixture digest. This liquid is, of course, very liable to putrefy,
and consequently a most luxuriant growth of bacteria may be expected
within a few days in the warm Climate of Cuba. It is no wonder then
that on the surtacG of Havana tobacco various bacteria are found,
although it may be doubted whether they live long on these fermenting
leaves. The ammonium carbonate contained in the petuning liquids
increases the alkaline reaction already present in the fermenting leaf,
and thus supports the energy of the oxidizing process which brings on
the dark color frequently desired for the filler leaves.
One might naturally suppose that the ammonium carbonate would
dissolve some resinous matter from the stems which would lead to an
improvement of the aroma of the fillers if the hypothesis is correct
that this aroma depends to a great extent upon the resin content of
the tobacco plant, but this the writer holds is doubtful.
Petuning is practiced in some parts of the United States also, but
the opinion of tobacco manufacturers whom the writer has consulted
upon the subject is that the effect of the treatment is overrated. By
the most intelligent growers a hot solution of ammonium carbonate is
left to act upon the stems of Havana tobacco. This extract is pre
pared anew every day for use, which easily accounts for the fact that
the tobacco leaves thus treated do not show any bacterial flora on
their surface. The petuning liquid often has a different composition.
The tobacco stems are extracted with water containing rum, molasses,
or sour wine, consequently these liquids may swarm with bacteria after
they stand for a while. The molasses is supposed to disguise the bitter
taste derived from the stems.
Related to the petuning is the so-called "conditioning" of the
tobacco, consisting in the spraying with a 2 per cent solution of glyc-
erin. This operation is carried on only with chewing, plug, and
cigarette tobaccos, and is intended to keep these products moist and
pliable, as perfectly dry tobacco would easily crumble to a powder.
20
THE BACTERIAL FERMENTATION THEORY OF SUCHSLAND.
It has long been recognized that the main feature of the processes
going on in the sweating, or the so-called fermentation, of tobacco con-
sists in oxidations. These are accompanied by certain decompositions
liberating ammonia, and are the source of the striking development of
heat in the fermenting piles. Now, what is the cause of these power-
ful oxidations? Nessler, as well as Schlosing, asserts that it is merely
the common oxygen of the air that attacks certain compounds in the
cells with great ease, no other cause being required. Schlosing admits
bacterial action only 'for initiating the elevation of temperature, but
not for the main processes later on. On the other hand, Suchsland
attributes all the oxidations and the development of heat to the action
of certain bacteria, which are specific for different kinds of tobacco
and which impart to each of them a specific aroma.
Nessler's and Schlosing's views must assume substances of an unusual
affinity for oxygen, if the rather indifferent atmospheric ogygen could
exert such a powerful result without the intervention of any activifying
principle, hence Suchsland's view seemed more probable and soon found
many followers. He prepared pure cultures of microbes found upon
different kinds of tobacco, 1 and by transferring those obtained from
Havana tobacco to German tobacco he expected to develop the Havana
aroma in the German tobacco, but thus far no new developments have
startled tobacco growers. Davalos described mold fungi and microbes
occurring upon fermenting tobacco leaves in Havana, but without
proving their importance for the fermentation process (see Petuning).
Vernhout observed only one kind of bacterium upon fermented tobacco
leaves. This developed at 50° O. (122° F.) upon agar plates, and was a
thermophile kind of the group of Bacillus subtilis. 2 It developed also
in decoctions of tobacco and was capable of decomposing proteids with
development of ammonia. Vernhout, however, leaves it entirely unde-
cided as to whether this microbe plays any important part in the fer-
mentation process. Also Koning 3 % described several kinds of bacteria
from fermenting tobacco leaves which he found to be identical with
those occurring also on the green tobacco leaves. Besides the known
bacteria, B. mycoides and B. subtilis, he described five aerobic new
kinds, called B. tobacci Nos. I, II, III, IY, and V, of which B. tobacci
III seemed to have most influence on the aroma. These statements
may well be doubted, as a direct microscopical investigation of the
surface of the fermenting leaves is wanting.
!Ber. d. Deut. Bot. Ges., Vol. IX, 1891.
3 How some authors can assert that the fermentation is caused by anaerobic bacteria
when it is a known fact that the most important changes going on consist of oxida-
tions remains difficult to understand.
3 Zeitschr. fur Unters. der Nahrungs and Genussmittel, 1898, No. 3. It may also
be mentioned that this author claims to have discovered the bacteria causing the
mosaic disease of tobacco, while the most careful researches of Bejerinck have
proved that bacteria are not the cause of it.
21
The writer has repeatedly tried to scrape off bacteria from the sur-
face of freshly fermented Florida tobacco leaves, but has searched in
vain with the highest magnifying power for the millions of microbes
naturally to be expected if they really play a part in raising the tem-
perature of the fermenting heap and bringing on powerful chemical
chiyiges. These fermenting leaves are, however, exceedingly smooth
and clean, and the scrapings obtained from them consist almost exclu-
sively of particles of the epidermis. Only here and there, by applica-
tion of staining methods, some small globules become visible which
might represent spores or cocci. Certainly so few microbes could never
be held responsible for the action in the fermenting heap, but, on the
contrary, colonies of luxuriant growth, as seen spreading profusely
upon potatoes or agar, ought to be expected. It is very instructive
that Behrens in his attempt to isolate bacteria from the surface of
cured tobacco leaves, obtained two spore-forming microbes, Bacillus
subtilis and a Clostridium. One can not suppress the supposition that
both kinds have been present only as spores and as such would remain
inactive during the so-called fermentation process, as there is not
sufficient water to bring on their germination.
It is evident that for the proper examination of fermenting tobacco
leaves one must avoid petuned leaves, upon which all kinds of microbes
can be found when a putrefying petuning liquid is applied. But this
liquid is not at all essential for starting the fermentation process. The
fermenting Florida tobacco leaves the writer had under examination
were not petuned, and he most emphatically declares (1) that there are
no bacteria in the cells of the tobacco leaf, and (2) that the surface is
remarkably clean and is not covered by a bacterial coating. This
observation was made also by Mr. Albert F. Woods, of the Division of
Vegetable Physiology and Pathology, two years ago in his study of
spots on fermented leaves.
The chief object and pride of the tobacco manufacturer is to produce
a cigar leaf of faultless quality. This would be impossible were bac-
teria to develop their activity promiscuously on the surface, as their
first step would be to reach the nourishing material in the interior of
the cells, otherwise they would be incapable of multiplying except for a
short time. In gaining entrance to the cells the cellulose walls would
have to yield, or, in other words, the surface of the leaves would be
attacked.
We have here quite a different case from that of the fermentation of
sauerkraut, which contains over 92 per cent of water and a proportion
of cellulose to water as 1 to 62. In the fermenting tobacco leaf the
amount of water is generally below 25 per cent and the proportion of
cellulose to water is generally less than 1 to 1.5. In fact, the water
present merely suffices to impregnate the cellulose walls and contents
of the cells, and is entirely insufficient to bring organic matter from the
interior of the cells to the surface, where bacteria might feed upon it.
22
It is indeed a matter of interest to observe how the tobacco leaf
becomes less fit to support bacterial life after being cured and fer-
mented. While the expressed juice of the fresh tobacco leaf exposed
to the air at the ordinary temperature teems with myriads of- bac-
teria within twenty four hours, the equally concentrated extract of
cured or fermented leaves will remain perfectly clear for many days. 1
On the fresh tobacco leaf, as everywhere in nature, numerous kinds
of microbes occur, but these seem to die off when cured leaves are
fermented, as will be seen from the following experiment by the writer
with Florida tobacco leaves: Into about 15 cc. of sterilized beef broth,
contained in three test tubes closed with cotton plugs, were introduced,
with all necessary precautions, (1) a small scrap of fi esh leaf, (2) a small
scrap of cured leaf which had been packed two months waiting fermen-
tation, and (3) a scrap of fermented leaf. The tubes were kept at from
15° to 18° C. for several days. No. 1 was turbid after one day, when a
scum formed and the liquid became very turbid; the liquid swarmed
with bacteria, thick and thin rods and cocci being revealed by the
microscope. Kos. 2 and 3 remained perfectly clear, and after eight days
merely a trace of flocculi was seen at the bottom, in which a few cocci
(Sarcina ( ! ) ) could be recognized. Indeed, the juice of the fermented
tobacco leaf acts as an antiseptic upon the ordinary bacteria of putre-
faction. When a slice of meat is wrapped in a fresh tobacco leaf, and
another in a moistened, fermented tobacco leaf, it will be seen after a
few days that the former slice is rotten and the latter not. 2 This
property of course disappears upon considerable dilution of the juice,
as will be seen from the following experiments: Ten grams of fer-
mented and well-dried tobacco leaf were pulverized and extracted with
250 cc. of boiling water. A part of the filtrate received an addition
of sugar and another part an addition of peptone. The well-sterilized
flasks were infected with small chips of fermented tobacco leaf and
some of them kept at 50° 0. (122° F.) for three days, and some at from
18 to 20° C. (64.4° to 68° F.). There was more or less development in
all the flasks, but further tests, as the inoculation in peptone solution
or on potatoes, revealed as the only organism a bacillus resembling
B. subtilis. The development of the colonies, the mode of growth on
the surface of peptone solution and on potato, and the spore-forming
threads left no doubt on this point. This result is then in full accord-
ance with the observations of others — that is, that this bacillus can
be cultivated from fermented tobacco leaves. But as the most careful
searching of the surface of the fermented leaves for the bacillus itself
proved vain, it must be assumed that it exists on these leaves only in
the form of spores.
1 It may be mentioned, however, that a diluted (1 per cent) solution of a neutral
nicotine salt will permit a bacterial growth.
3 Southern manufacturers assert that the workmen in tobacco factories better
resist epidemics than those not so employed.
23
When by accident leaves are too much moistened before they are
subjected to the sweating, they will soon lose their coherence and
sbow spots and finally holes. The water content, the writer found, in
one such case amounted to 36 per cent. Here, then, is a true action of
bacteria, which can develop under these conditions, as the high per-
centage of water admits an abundant exit of organic compounds from
the interior of the cells to the surface and the formation of a diluted
solution. Now, it is the experience of every tobacco manufacturer that
the product will invariably spoil when the water content is increased
to such a point as to permit an exit of soluble organic compounds from
the cells. Here, then, begins the parallelism to the fermentation of
sauerkraut or ensilage, but not before. The objection that certain
kinds of thermophylic bacteria might be capable of developing on the
leaves in the presence of a smaller percentage of water can not be sus-
tained, as they require liquid food as well. 1 And how will they reach
the interior of the cells without eating through the cellular walls, that
is, without ruining the product? The claim that it is not the bacteria
but the enzyms they produce that enter can not hold good, as the
latter must be dissolved before they can migrate into the interior of
the cells, and hence a water increase is again required. The conclu-
sion that must invariably be reached, therefore, is that the bacteria
found upon the fermenting tobacco leaves do not participate in any
way in the fermentation process, but that they are accidentally pres-
ent and probably only in the form of spores.
THE OXIDIZING AGENCY IN THE FERMENTING TOBACCO LEAF.
After showing that the bacterial theory of Suchsland is erroneous,
as there exists no bacterial coating on the leaves, the question natu-
rally presents itself, what is the cause of the oxidizing action! The
assumption of Nessler and of Schlosing that the contact with the
atmospheric oxygen would suffice can not be correct for the following
reasons: (1) The substances undergoing oxidation (tannin, nicotine,
etc.) do not show such powerful affinities for oxygen as to account for
the considerable development of heat 5 and (2) neither curing nor fer-
mentation sets in when the fresh leaves are killed by direct application
of steam, although those organic matters which become oxidized in the
fermentation process are not changed at all thereby.
Neither the tannin nor the nicotine of the leaves can be energetically
oxidized by the molecular oxygen of the air without assistance or
stimulation of some sort. In the same way dilute alcohol can not be
oxidized into acetic acid by the common molecular oxygen of the air
except through the intervention of certain bacteria or platinum black. 2
1 Cohn made investigations on the growth of thermogenic micrococci in the refuse
from the cotton purifier. However, he had to add a fair percentage of water to
start the development.
2 MUller-Thurgau declares (1. c.,p. 508) that "In the beginning of the curing the
changes consist in an increased respiration, but later on, after the cells have died,
in other (' anderweitigen') oxidation processes," but he gives no explanation of the
cause of these latter ones.
24
The oxidations in the treatment of tobacco commence with the curing
process and are continued in the fermenting process. In the latter
case, but not in the former, the aid of bacteria has been invoked for
explanation. But when oxidations can go on in the curing without
bacterial aid, even after the death of the cells, then it might be sup-
posed that the same cause would also lead to oxidation later on during
the fermenting process. Now, what is the true cause of these phe-
nomena? There remains, in fact, as the only explanation the writer's
suggestion that an oxidizing enzym is the final cause of the energetic
oxidizing action after death of the cells as it is capable of instigating
certain compounds to take up the molecular oxygen of the air.
The formation of enzyms is a physiological necessity for every living
organism. Various enzyms come into action especially in the develop-
ment of shoots, as well as in the inanition state of the plants. Green
plants, as well as lower fungi, prepare enzyms, which may act on
protein, polyanhydrids of glucoses, glycosides, or fat, splitting or dis-
solving these bodies and thus making them more easily accessible
to the protoplasm. 1 The list of enzyms has been enlarged in recent
years by the oxidizing enzyms or the oxidases, which were brought to
our knowledge first by French savants, as Gabriel Bertrand, Bourque-
lot, Gouirand, Cazeneuve, and others. The most thorough investiga-
tions on this subject are those by Professor Bertrand, who has shown
their wide distribution through the vegetable kingdom. 2
The best reaction for oxidizing enzyms consists in the production of
a blue color with the tincture of guaiac, which reaction can be obtained
with various vegetable objects. This blue coloration is produced in
many cases only upon addition of peroxide of hydrogen, in which case
it was formerly considered as a reaction upon diastase. While the crude
diastase of malt gives this blue reaction in a very marked degree, the
diastase of certain fungi (Aspergillus oryzce) will, as the writer long
since ascertained, yield this reaction either only slightly or not at all,
although this diastase has very energetic qualities and produces glucose
from starch. Eaciborski and other authors also have proved that this
blue reaction is not characteristic of pure diastase, but only of an
admixture of an oxidase.
The brown, black, or reddish coloration of freshly prepared juices of
potatoes, turnips, etc., setting in when exposed to the air, the brown
color of the falling leaves in autumn, and similar phenomena are gen-
erally due to the action of the oxidases. The oxidation of tannin by
oxidases plays an important part in certain fruits ripening or overripe.
1 A trophic irritation is exerted when rapidly developing cells or cells in a state
of inanition require nourishment, and this stimulus leads to the production of
enzyms by the nuclei — an interesting caso of physiological adaptation. Well-
nourished cells killed in the full vigor of life often give only slight indications of
amylolytic and proteolytic enzyms.
2 Further contributions have been published by Grtiss and by Raciborski (Ber. d.
Deut. Bot. Ges., Vol. XVI, Nos. 3 and 5, 1898).
25
The blackening of bananas some time after they are gathered and the
brown color on the surface of a slice of apple may also be mentioned
as due to these agents.
Oxidizing enzyms also occur in animal organisms, as the investiga-
tions of Pieri, Abelous, Bougault, Salkowski, Yamagiva, Linossier,
Jaquet, and Sclimiedeberg have revealed. Such enzyms were found
in various organs, and are capable of easily oxidizing not only guaiac
tincture, 1 but also certain aldehydes, such as salicylic aldehyde. Spitzer
has determined the amount of oxygen liberated by different organs from
peroxide of hydrogen, and has observed that various poisons, such as
potassium cyanide, hydroxylamin, etc., small quantities of acids and
alkalies, and a temperature of about 70° C. (158° F.) destroy or
diminish the oxidizing action of this animal enzym. Water extracts
the enzym from the organs, and highly diluted acids, cautiously added,
precipitate it with all its original properties. (Abelous could, however,
prepare clear solutions only by application of potassium nitrate.) It
has the character of a nucleo-proteid and contains from 0.19 to 0.23 per
cent of iron. On the other hand, Bertrand and Villiers have found a
small amount of manganese in the vegetable oxidases.
That oxidations also can proceed in certain cases without the aid of
oxidizing enzyms is a well-known fact. But this is only the case with
substances of a specific kind showing a great chemical energy, and
even in such cases the presence of oxidizing enzyms will cause such a
powerful increase of intensity that the difference becomes most striking,
especially when chromogens consisting of certain derivatives of poly-
valent phenols are present. The colored product (brown, red, or black)
formed by oxidation will appear much sooner and in much greater
quantity in the presence of certain oxidizing enzyms than in their
absence. 2 On the other hand, oxidizing enzyms can bring on oxida-
tions with certain compounds, as, for example, ty rosin, which under
ordinary circumstances would not be oxidized at all by the indifferent
oxygen of the air.
Bertrand has characterized different oxidases. While the oxidase of
Rhus vernieiferaj the Japanese lac tree, oxidizes mainly benzene deriva-
tives containing at least two hydroxyl or two amido groups in ortho or
para position, another oxidase, isolated from certain green plants, as
well as from fungi, acts easily in tyrosin, which the former can not
affect, therefore he distinguishes the latter as tyrosinase from the
former as laccase. It is the laccase which acts upon the laccol in the
1 This blue reaction can be obtained not only by the action of oxidizing enzyms,
but also by that of powerful oxidizing chemicals. Such bodies (nitrous acid, free
chlorine, etc.) are usually absent when the test for oxidizing enzyms is made.
Any intelligent chemist will be able to decide at once by control experiments
whether he can trace the reaction rapidly setting in to oxidase's or not.
3 The attempt to explain such rapid oxidations by the assumption that certain salts
or ordinary albuminous matter would aetivify the oxygen, must be considered a
failure.
26
juice of the lac tree and converts it by oxidation into a black substance.
Laccase is killed at 75.5° C. (168° F.) and gives the guaiac reaction
without the aid of hydrogen peroxide. Like laccase, the oxidases of
Senecio vulgaris, Lactuca saliva, and Taraxacum dens leonis fail to attack
tyrosin. In certain objects, especially in fungi, however, laccase and
tyrosinase occur simultaneously.
The oxidation of polyvalent phenols by laccase leads not only to
organic acids, but even to the production of carbonic acid. Bertrand
observed in one case that for 23.3 cc. absorbed oxygen as much as 13.7
cc. of carbonic acid were produced.
Gouirand has observed in certain spoiled wines aa oxidase 1 which
oxidizes the coloring matter, the tannin, and the alcohol of the wine,
with production of carbonic acid. This oxidase is destroyed in plain
aqueous solution at 72.5° O. (162.5° F.), while in the wine a temperature
of 60° O. (140° F.) suffices. Very small doses of sulphurous acid will
also kill it. It is supposed to be derived from the fungus Botrytis
cinerea, which frequently grows upon ripening grapes, 2 while Martinand
and Tolomei observed an oxidase in ripe grapes. As to the Florida
tobacco leaf, the writer has demonstrated the presence of a relatively
large amount of oxidases in it.
These oxidizing enzyms belong, like other enzyms, to the protein
compounds, forming a special group of labile proteins, i. e., proteins
containing much chemical energy, which on the one hand is the cause
of their activity and on the other of their changeability to indifferent
proteids by heat, acids, and poisons. They are, as it is expressed,
easily killed. The labile, active atomic groups in the molecules change
thereby, the atoms migrating into more stable position.
Views on the physiological functions of the oxidizing enzyms. — As to
the physiological function of the oxidizing enzyms, no perfectly satis-
factory explanation has thus far been proposed. Some authors sup-
pose that they are important agencies in the respiration process and
that even respiration itself is caused by them when they are supported
by certain properties of the living protoplasm. This is, however,
improbable for several reasons: (1) Not every plant contains oxidizing
enzyms ; (2) many plants contain them only in certain stages; and (3)
carbohydrates and fat, the materials which by their combustion serve
for support of the respiration and for the production of energy, are not
attacked by the oxidizing enzyms, but are attacked very energetically
by the protoplasm.
Portier believes that the oxidase of the blood, of which he made a
special study, serves to augment the vitality of the leucocytes, which
prepare the oxidase and finally deliver it up to the blood when they
die. This hypothesis will certainly not find support. The suggestion
has also been advanced that the oxidizing enzyms play in plants the
'This (tMioxida.se is supposed by Bertrand to be identical with laccase.
2 See also Cazeneuve, Compt. rend., Vol. CXXIV.
27
same part that the haemoglobin does in animals, but neither is this
view justified, as the oxidizing enzyms are not carriers of molecular
oxygen, but simply instigators of oxidation.
The writer's view on this subject is that as the living protoplasm can
oxidize carbohydrates and fat, but does not attack or attacks only with
difficulty compounds of the benzene group, and, on the other hand, as
just the opposite takes place vith the oxidizing enzyms, it may be
inferred that there exists between the protoplasm and the oxidizing
enzyms a certain division of labor, the former oxidiziug the compounds
of the methan series and the latter those of the benzene series. The
former provides for the kinetic energy of the cells; the latter destroys
by partial oxidation noxious by-products. The oxidations in the former
case are generally complete, but in the latter only partial.
The oxidizing action of enzyms might be compared to that of plati-
num black. In both cases chemical energy is conveyed to certain
organic compounds, which are thus rendered capable of taking up the
oxygen directly from the air. The further inference might also be jus-
tified that just as platinum black brings on not only oxidations, but
also reductions under certain circumstances, the same may be possible
for the oxidases; for example, if platinum black is added to a mixture
of glucose and potassium nitrate in aqueous solution, a reduction of
nitrate to ammonia takes place by aid of hydrogen atoms in the sugar,
while the oxygen of the nitrate is thrown upon the glucose and organic
acids thereby formed. When the analogy of action of the oxidase to
platinum black is justified, there will be a simple explanation for the
disappearance of a certain portion of the nitrate and also of a certain
portion of the glucose during the fermentation process of tobacco. Pre-
liminary qualitative experiments by the writer have indeed proved the
formation of ammonia under these conditions. A full account of quan-
titative tests will follow in a later bulletin.
The oxidizing enzyms may occur in various parts of the plant — in
young and active as well as in dormant tissue. Griiss has observed that
there occurs frequently, but not always, a coincidence between the trans-
formation of starch and increase of oxidase. Whether the amount of
oxidase augments with the ripening of fruits has not been thoroughly
investigated. Tolomei observed that in olives it does increase during
the ripening process.
The juice of the fresh tobacco leaf soon turns dark upon exposure to
air and gradually forms a sediment, but if boiled this dark coloration
does not set in, the oxidase having been killed.
The tobacco oxidase and peroxidase. — There exist, evidently, two kinds
of oxidizing enzyms in the Florida tobacco leaf. The first kind oxidizes
guaiaconic acid (the characteristic reactive in the guaiac resin) to
guaiac blue without the aid of peroxide of hydrogen, but the second
kind oxidizes it only when this substance is present. Both kinds of
oxidizing enzyms, which may be distinguished as tobacco oxidase and
28
tobacco peroxidase, occur in the fresh as well as in the recently fer-
mented Florida tobacco leaf. 1 The former enzym is, however, much
more sensitive to heat than the latter, being killed at from 65° to 66°
O. (149° to 151° F.), while the latter is killed only at from 87° to 88°
C. (188.6° to 190.40 F.).
Dried tobacco leaf (not cured) was finely pulverized, and 1 gram of
the powder left with 20 grams of water for one hour at the ordinary
temperature. A part of the filtrate was heated for three minutes to
55° C. (131° F.). To about 2 cc. of this liquid were then added a few
drops of tincture of guaiac, 2 whereupon a blue coloration appeared in
a few minutes, exactly as in the control case. A second portion was
now heated to 65° 0. (149° F.) for three minutes, and the test applied
after cooling, but only a slight trace of a blue color was noticed after
ten minutes. Evidently most of the tobacco oxidase was destroyed at
that temperature.
The killing temperature of the tobacco peroxidase was determined in
a similar manner. However, here reaction is still obtained with great
intensity after the solution is heated for three minutes to 80° C.
(176o P.), but very feebly after heating for one minute to 87° 0. (188° P.).
Another reaction for oxidizing enzyms is the so-called indophenol
reaction, consisting in the production of a blue color when an alkaline
solution of ^naphthol with paraph eny lend iamine is acted upon by an oxi-
dase. This reaction must, however, quickly set in and with great inten-
sity, otherwise no reliable conclusion can be drawn. Cured and fer-
mented tobacco from Florida did not show this reaction in a marked
manner, 3 but it set in at once upon the addition of a little peroxide of
hydrogen. The latter alone will not produce this result in the absence
of an oxidizing enzym.
In the manner above mentioned the writer's investigations have
shown that dark tobacco two years old, from Quincy, Fla., yielded no
reaction for tobacco oxidase, but still a moderate one for tobacco perox-
idase, while a sample of light-colored tobacco four years old from the
same source yielded not the slightest reaction either for the oxidase or
the peroxidase. Evidently these enzyms themselves are gradually
changed. From these observations it may be inferred that the cold
sweat, or after-fermentation, might thus proceed for about two years
and end by the gradual dying off of the oxidase and peroxidase.
French savants were the first to call attention to this difference between the
oxidizing enzyms. The names oxidase and peroxidase, proposed by a French savant,
are not specific names, but group names. There may exist among various oxidases
and peroxidases as many differences as there are among protein bodies. Hence it is
entirely unjustifiable, at this stage of our knowledge, to introduce one specific name
for all peroxidases, as one author has done.
2 In all these cases freshly prepared guaiac tincture (1:50) was employed, as old
guaiac tincture is unreliable and with peroxide of hydrogen alone will sometimes
yield a greenish coloration.
3 Only a slow and weak reaction was thus developed.
29
When the fresh leaf of the tobacco is rapidly dried at about 60° 0,
(140 F.) and then moistened again and kept in a moist atmosphere, the
veins and their finest ramifications turn brown in about half an hour,
while the mesophyll and epidermal cells remain green even after a
week. Further investigations on this point will be made later. In
the fresh leaf, both oxidizing enzyios, the oxidase and the peroxidase
occur in the ribs and veins as well as in the parenchyma, the indica-
tions being that they are more abundant in the ribs than in the paren-
chyma. The bundle sheath and sieve tissue give the most intense
reaction on the oxidase, while the reaction on the peroxidase sets in
quickly and with about uniform intensity in all the cellular tissues.
The growing point and youngest leaves contain an especially large
quantity of the oxidase. A section through the stalk shows oxidase
only in the sieve tissue and bast parenchyma, while peroxidase also is
contained in the pith. 1 Both enzyms are found in the root, the former
more in the central and the latter in the peripheral parts and also in
the flower. The stigma of the pistil and the stigmatic fluid also show
strong reaction upon oxidase.
The two oxidizing enzyms are also contained in the young tobacco
plants. Several, dozen of these, measuring on an average not more
than 3 to 4 cm. from the tip of the root to the plumula, were rubbed in
a mortar with a little water aud some sand. The filtrate gave a very
intense reaction for oxidase, 2 and after this was destroyed by warming
to 70° C. an intense reaction for peroxidase also.
A colorless clear solution of the tobacco peroxidase can be obtained
in the following manner : A number of fresh tobacco leaves are well
crushed in a mortar, with the addition of sand and some dilute alcohol
of 30 per cent. This mixture is pressed and the turbid liquid directly
mixed with three times its bulk of strong alcohol. After standing two
hours the mixture is thrown upon a filter and the filter contents, after
being washed with some alcohol, extracted with about four to six times
its bulk of water at the ordinary temperature, heated for a minute to
70° C. (158° F.), and filtered. This clear, colorless filtrate gives no
indication of the oxidase, but a very intense reaction for peroxidase.
When this solution is compared with that of the juice of fresh tobacco
leaves it is easy to decide, what result is caused by the oxidase alone.
To determine whether the tobacco oxidase bears more resemblance
to the tyrosinase or the laccase a few drops of freshly expressed juice
of normal tobacco leaf were added to 2 cc. of a cold saturated tyrosin
solution, but even after four hours no characteristic darkening of the
'In order to observe the localization of the peroxidase small pieces of the tissue
are treated with strong, but not absolute, alcohol for three minutes at 70° C. (158° F-).
Thus the oxidase is killed and can not interfere with the tests for the peroxidase.
3 The indophenol reaction did not turn out satisfactorily, only a weak violet-blue
color resulting. On the addition of hydrogen peroxide, however, an intense blue
reaction was at once obtained.
30
mixture was observed. In this regard, therefore, that oxidase would
resemble laccase more than it would tyrosinase. 1
To extract oxidases from fermented or cured tobacco as completely
as possible it is necessary to thoroughly pulverize the samples and to
let the water act for some time at from 20° to 30° O. (68° to 86° F.)
before filtering. After complete drying, the samples can be easily pul-
verized very fine. The following experiment proves that when the tis-
sue is not pulverized the peroxidase is but very imperfectly extracted,
the passage through the cellular walls being quite slow. Fermented
tobacco leaves were three times soaked in water and the brown liquid
pressed out, the first soaking lasting half an hour and the second and
third soakings five minutes each. Although the sample was thus nearly
exhausted, it nevertheless yielded, when left with some alcohol of 30
per cent for one day, a light-colored liquid with a very intense reaction
for peroxidase.
It may safely be assumed that in the majority of instances the oxi-
dase will prove the more energetic of the two oxidizing enzyms. For
example, its action upon pyrocatequol and hydroquinone is much more
energetic than that of the peroxidase. On the other hand, however,
the former succumbs much more quickly to noxious influences, e. g., the
action of alcohol or rising temperature.
The fact that the peroxidase forms guaiac blue from guaiaconic acid
with the aid of hydrogen peroxide only does not indicate that its oxi-
dizing action in every case depends upon the presence of the latter. 2
The peroxidase can, on the contrary, also exert oxidizing action upon
various compounds without the assistance of hydrogen peroxide. Thus,
para-amidophenol is gradually changed by it to a dark brown sub-
stance.
Hydroquinone in dilute solution gradually assumes a reddish color in
the presence of the peroxidase, but in its absence there is scarcely a
trace of coloration within twenty-four hours.
Pyrocatechol is scarcely attacked by the peroxidase within twenty-
four hours, but on a further addition of a little hydrogen peroxide it
turns to a dark brown in five minutes. Hydrogen peroxide added alone
does not act thus.
Pyrogallol is slowly attacked by the peroxidase and turns brown in
twenty-four hours. The oxidase acts also in this case much more ener-
getically than the peroxidase.
Tannin solution shows in twenty-four hours a yellow color in the
presence of the peroxidase, but in the control solution merely a slight
1 The peroxidase of tobacco on the other hand bears resemblance to the peroxidase
of pus described by Linossier.
2 The hydrogen peroxide is decomposed by enzyms into water and oxygen, but
this oxygen in status nasceiis is charged with more chemical energy than the free
oxygen of the air, i. e., the two atoms constituting the molecule are for a time in
a more energetic motion than in the latter case, hence the action of the oxidizing
enzyms is facilitated by this nascent oxygen.
31
trace of coloration is perceptible. The addition of a little hydrogen
peroxide to both will increase the difference of coloration still more.
All these tests were made at from 18° to 20° 0. (64.4° to 68° F.). A
number of other compounds were also tested, such as arbutin, guaiacol,
and toluidine, but no decisive reaction was obtained within twenty-four
hours at the ordinary temperature.
There may exist great differences in the amount of tobacco oxidase
and tobacco peroxidase produced in different varieties of the tobacco
plant and under different conditions. The quantity of each may even
differ in the upper leaves fully exposed to the sun and the lower leaves
growing mostly in the shade. There may also be formed compounds
in certain varieties of tobacco that will more quickly destroy the
enzyms during curing, or fermentation, than in other varieties. Thus
considerable difference was goticed in comparing a sample of tobacco
from Connecticut with one from Florida. In the fermentation of the
former the tobaeco peroxidase was almost completely destroyed, while
in that of the latter a considerable part was still intact. Moreover,
neither the fermented nor the cured Connecticut leaf contained any
tobacco oxidase, although it was found in a greenhouse specimen of the
fresh leaf.
It is interesting to note that the best way of bringing the oxidizing
euzym to the fullest action possible is that practiced in the curing of
the Perique tobacco. 1 The rolls, or twists, of the tobacco leaves are
subjected to a pressure of about 7,000 pounds per square foot to bring
the juice from the interior of the cells to the surface. After twenty-
four hours the tobacco is taken out and aired a few minutes, which
causes a darkening to set in. In this way the juice is reabsorbed by
the tissues, whereupon the pressure is again applied. This operation
is repeated daily for ten consecutive days, and at longer intervals
thereafter. A very dark product is thus obtained, but it is not strong,
as the oxidation of the nicotine has been carried very far.
One of the most interesting features of the sweating of tobacco is
the destruction of a part of the nicotine, this part yielding up its
nitrogen probably as ammonia, which is indeed a product of the sweat-
ing. It was, of course, of importance to prove that the oxidizing
enzyms contained in the tobacco leaf can decompose nicotine, and for
this purpose 50 grams of cured tobacco from Connecticut which had
not yet been subjected to fermentation and showed a strong reaction
for peroxidase, but none for oxidase, was thoroughly moistened with
water. After two hours 250 cc. of alcohol of 50 per cent was added
and the mixture allowed to stand for two days. The liquid obtained
by pressing was now mixed with one and a half times its volume of
absolute alcohol and the brown-colored precipitate washed upon the
filter with some alcohol. After pressing between filter paper, the pre-
cipitate, containing a large proportion of the peroxidase, was dissolved
farmers' Bull. No. 60, U. S. Dept. of Agr.
32
in 20 cc. of water and 0.5 gram of nicotine tartrate added. This mix-
ture was digested for two days at from 50° to 60° 0. (122° to 140° F.)
in a large flask, holding about 500 cc. of air, to enable oxidation to
go on. An addition of a small amount of thymol prevented bacterial
growth. A small U tube, holding 10 cc. of dilute chemically pure sul-
phuric acid of 0.2 per cent, was attached to the flask. The examina-
tion of this acid after two days with JSessler's reagent indicated that
ammonia was present, but the colorimetric comparison showed that
the amount was hardly more than 0.1 milligram. However, the amount
of ammonia formed but not volatilized was much larger, as indicated
by the strong reaction obtained after the addition of a little potassium
carbonate to the mixture and warming for a short time in order to liber-
ate the ammonia in the form of carbonate from other less volatile salts. 1
Thus there can be no doubt that the tobacco peroxidase can attack
nicotine with formation of ammonia, but this process is exceedingly
slow. Indeed, the sweating, lasting fully eight weeks, can diminish
the nicotine content on an average only by about one-third. 2
What the products of destruction of nicotine are besides ammonia
can be determined only when the purified enzyms and a pure nicotine
salt serve in large quantities for the experiment. It may be men-
tioned, however, that the writer has examined in vain an aqueous
extract of fermented tobacco for nicotyrin and nicotinic acid — known
oxidation products of nicotine.
The writer has now fully established the presence of oxidizing
enzyms in the tobacco leaf. 3 That such enzyms can exert a powerful
action upon certain compounds, leading even to the formation of car-
bonic acid, is known. 4 Oxidations produce heat, hence it can safely be
inferred that the so-called tobacco fermentation consists in the activity
of oxidases, while the curing of tobacco consists in the combined work
of oxidases, diastase, and peptase. As the use of the term "fermen-
tation" might lead in this case to an entirely erroneous conception, the
writer proposes "oxidizing enzymosis" or "oxidizing enzymation" as
correct scientific designations.
There has already been mentioned an interesting case of oxidase
action in a technical branch, viz, the preparation of the Japanese lac.
Furthermore, in the manufacture of the natural indigo bacteria are
not concerned (Molisch), but simply an oxidizing enzym (Br^audat).
1 The oxidase might have exerted a more powerful action on the nicotine than the
peroxidase.
2 A control experiment was made with a colorless peroxidase solution (p. 29) npon
highly diluted free nicotine at the ordinary temperature, in order to observe whether
a brown solution is produced by a change of the nicotine, but the mixture remained
colorless after one day under these conditions. However, it may be mentioned that
nicotine, when exposed a long time to air and light, will turn brown.
3 He has already pointed out (p. 15) that there is sufficient access of air possible
to enable oxidation in the tobacco piles.
4 The further inference is certainly justified that certain basic compounds might
thus give up their nitrogen in the form of ammonia.
33
Another instance where an oxidizing enzym plays a part in a technical
branch is the "fermentation" of the olive, which is practiced in certain
parts of Italy. It is believed that by this operation an oil of superior
quality is obtained and that the yield by pressure is larger, but this
has not been confirmed. It has been shown by Tolomei 1 that this olive
"fermentation" is due to the action of an oxidizing enzym, to which
also is due the fact that olive oil is bleachable by sunlight.
When the freshly gathered olives are kept in sacks their temperature
gradually rises far above that of the rooms, oxygen is absorbed, and
carbonic, acetic, and sebacic acids, and small quantities of the higher
volatile fatty acids are formed. This process goes on to a larger
extent when a temperature of 35° C. (95° F.) is reached. These changes
do not occur if the olives are kept in nitrogen or carbonic acid gas;
neither do they occur when the olives have been heated to 75° C. (167° F.)
for forty-five minutes. For obvious reasons the spontaneous rising of
temperature is noticed only when a large number of olives are kept
together. Tolomei showed that the oxidase extracted with water and
purified by a repeated precipitation with alcohol produces guaiac blue
from guaiac tincture, forms purpurogallol from pyrogallol without the
aid of peroxide of hydrogen, quinhydrone from hydroquinone, and
a brown substance from gallic acid. He calls this oxidizing enzym
olease. As unripe olives do not contain this olease, the oil pressed from
them will not bleach upon exposure to the sunlight, but will do so after
being shaken with an aqueous extract of the ripe olives. On the other
hand, olive oil will sooner acquire rancidity in the presence of the olease
than when free from it.
Finally, still another case may be pointed out where oxidases might
possibly play a part — that is, in the so called fermentation of the cacao'
beans, by which a bitter principle is destroyed.
SUMMARY.
(1) The so-called tobacco fermentation is not caused by bacteria.
(2) The amount of water present in normally fermenting tobacco
leaves is insufficient to bring nourishment for the microbes from the
interior of the cells to the surface of the leaves. It barely suffices for
imbibition of the organic matter.
(3) There exists no bacterial coating on the fermenting tobacco leaves
under normal conditions, but some spores may occur.
(4) In the so-called petuning of tobacco an immense number of bac-
teria may be transferred to the leaves. These bacteria, however, are
not essential for the fermentation, but on the contrary, may prove
noxious as soon as a small surplus of water enables them to further
develop.
(5) Suchsland's theory that the aroma of tobacco is caused by spe-
cific bacteria is incorrect.
1 Atti della Reale Accademia dei Lincei, 1896, p. 122.
15846 3