ZYMASE AND ITS PROPERTIES.
DISCOVERY OF ZYMASE.
The history of Buchner's discovery is of great interest [Gruber, 1908;
Hahn, 1908]. As early as 1893 Hans and Eduard Buchner found that the
cells of even the smallest micro-organism could be broken by being
ground with sand [Buchner, E. and H., and Hahn, 1903, p. 20], and in
1896 the same process was applied by these two investigators to yeast,
with the object of obtaining a preparation for therapeutic purposes.
Difficulties arose in the separation of the cell contents from the
ground-up mixture of cell membranes, unbroken cells, and sand, but
these were overcome by carrying out the suggestion of Martin Hahn (at
that time assistant to Hans Buchner) that kieselguhr should be added
and the liquid squeezed out by means of a hydraulic press [Buchner,
E. and H., and Hahn, 1903, p. 58]. The yeast-juice thus obtained
was, in the first instance, employed for animal experiments, but
underwent change very rapidly. The ordinary antiseptics were found to
be unsuitable, and hence sugar was added as a preservative, and it
was the marked action of the juice upon this added cane sugar that
drew Eduard Buchner's attention to the fact that fermentation was
proceeding in the absence of yeast-cells.
As in the case of so many discoveries, the new phenomenon was brought
to light, apparently by chance, as the result of an investigation
directed to quite other ends, but fortunately fell under the eye of
an observer possessed of the genius which enabled him to realise its
importance and give to it the true interpretation.
In his first papers [1897, 1, 2; 1898], Buchner established the
following facts: (1) yeast-juice free from cells is capable of
producing the alcoholic fermentation of glucose, fructose, cane
sugar, and maltose; (2) the fermenting power of the juice is neither
destroyed by the addition of chloroform, benzene, or sodium arsenite
[Hans Buchner, 1897], by filtration through a Berkefeld filter, by
evaporation to dryness at 30° to 35°, nor by precipitation with
alcohol; (3) the fermenting power is completely destroyed when the
liquid is heated to 50°. [p019]
From these facts he drew the conclusion "that the production of
alcoholic fermentation does not require so complicated an apparatus as
the yeast cell, and that the fermentative power of yeast-juice is due
to the presence of a dissolved substance". To this active substance he
gave the name of zymase.
Buchner's discovery was not received without some hesitation. A
number of investigators prepared yeast-juice, but failed to obtain an
active product [Will, 1897; Delbrück, 1897; Martin and Chapman, 1898;
Reynolds Green, 1897; Lintner, 1899]. A more accurate knowledge of the
necessary conditions and of the properties of yeast-juice, however,
led to more successful results [Will, 1898; Reynolds Green, 1898;
Lange, 1898], and it was soon established that, given suitable yeast,
an active preparation could be readily procured by Buchner's method.
Criticism was then directed to the effect of the admitted presence
of a certain number of micro-organisms in yeast-juice [Stavenhagen,
1897], but Buchner [Buchner and Rapp, 1897] was able to show by
experiments in the presence of antiseptics and with juice filtered
through a Chamberland candle that the fermentation was not due to
living organisms of any kind.
The most weighty criticism of Buchner's conclusion consisted in an
attempt to show that the properties of yeast-juice might be due to the
presence, suspended in it, of fragments of living protoplasm, which,
although severed from their original surroundings in the cell, might
retain for some time the power of producing alcoholic fermentation.
This, it will be seen, was an endeavour to extend Nägeli's theory to
include in it the newly discovered fact.
In favour of this view were adduced the similarity between the effects
of many antiseptics on living yeast and on the juice, the ephemeral
nature of the fermenting agent present in the juice, the effect of
dilution with water, and the phenomenon of autofermentation which is
exhibited by the juice in the absence of added sugar [Abeles, 1898;
v. Kupffer, 1897; v. Voit, 1897; Wehmer, 1898; Neumeister, 1897;
Macfadyen, Morris, and Rowland, 1900; Bokorny, 1906; Fischer, 1903;
Beijerinck, 1897, 1900; Wroblewski, 1899, 1901].
A brief general description of the actual properties of yeast-juice
and of the phenomena of fermentation by its means is sufficient to
show the great improbability of this view.
The juice prepared by Buchner's method forms a somewhat viscous
opalescent brownish-yellow liquid, which is usually faintly acid in
reaction [compare Ahrens, 1900] and almost optically inactive. It has
a specific gravity of 1·03 to 1·06, contains 8·5 to 14 per cent.
[p020] of dissolved solids, and leaves an ash amounting to 1·4 to 2
per cent. About 0·7 to 1·7 per cent. of nitrogen is present, nearly
all in the form of protein, which coagulates to a thick white mass
when the juice is heated.
A powerful digestive enzyme of the type of trypsin is also present, so
that when the juice is preserved its albumin undergoes digestion at
a rate which depends on the temperature [Hahn, 1898; Geret and Hahn,
1898, 1, 2; 1900; Buchner, E. and H., and Hahn, 1903, pp. 287-340],
and is converted into a mixture of bases and amino-acids. After
about six days at 37°, or 10 to 14 days at the ordinary temperature,
the digestion is so complete that no coagulation occurs when the
juice is boiled. As this proteoclastic enzyme, like the alcoholic
enzyme, cannot be extracted from the living cells, it is termed
yeast endotrypsin or endotryptase. Fresh yeast-juice produces a slow
fermentation of sugar, which lasts for forty-eight to ninety-six
hours at 25° to 30°, about a week at the ordinary temperature, and
then ceases, owing, not to exhaustion of the sugar, but to the
disappearance of the fermenting agent. When the juice is preserved or
incubated in the absence of a fermentable sugar this disappearance
occurs considerably sooner, so that even after standing for a single
day at room temperature, or two days at 0°, no fermentation may
occur when sugar is added. The reason for this behaviour has not
been definitely ascertained. As will be seen later on (p. 64) the
phenomenon is a complex one, but the disappearance of the enzyme was
originally ascribed by Buchner to the digestive action upon it of
the endotrypsin of the juice [1897, 2], and no better explanation
has yet been found. Confirmation of this view is afforded by the
fact that the addition of a tryptic enzyme of animal origin greatly
hastens the disappearance of the alcoholic enzyme [Buchner, E. and
H., and Hahn, 1903, p. 126], and that some substances which hinder
the tryptic action favour fermentation [Harden, 1903]. The amount of
fermentation produced is almost unaffected by the presence of such
antiseptics as chloroform or toluene, although some others, such as
arsenites and fluorides, decrease it when added in comparatively high
concentrations, and it is only slightly diminished by dilution with
three or four volumes of sugar solution, somewhat more considerably
by dilution with water. When it is filtered through a Chamberland
filter the first portions of the filtrate are capable of bringing
about fermentation, but the fermenting power diminishes in the
succeeding portions and finally disappears. The juice can be spun
in a centrifugal machine without being in any way altered, and no
separation into more or less active layers takes place under these
conditions. [p021]
The amorphous powder obtained by drying the precipitate produced when
the juice is added to a mixture of alcohol and ether is also capable
of producing fermentation, and the process of precipitation may be
repeated without seriously diminishing the fermenting power of the
product.
These facts clearly show that the various phenomena adduced by
the supporters of the theory of protoplasmic fragments are quite
consistent with the presence of a dissolved enzyme as the active agent
of the juice, and at the same time that the properties demanded of
the living fragments of protoplasm to which fermentation is ascribed
are such as cannot be reconciled with our knowledge of living
matter. If living protoplasm is the cause of alcoholic fermentation
by yeast-juice, a new conception of life will be necessary; the
properties of the postulated fragments of protoplasm must be so
different from those which the protoplasm of the living cell possesses
as to deprive the theory of all real value [Buchner, 1900, 2; Buchner,
E. and H., and Hahn, 1903, p. 33].
Further and very convincing evidence against the protoplasm theory
is afforded by the behaviour of yeast towards various desiccating
agents. When yeast is dried at the ordinary temperature it retains
its vitality for a considerable period. If, however, the dried yeast
be heated for six hours at 100° it loses the power of growth and
reproduction but still retains that of fermenting sugar, and when
ground with sand, kieselguhr and 10 per cent. glycerol solution yields
an active juice [Buchner, 1897, 2; 1900, 1]. Preparations (known as
zymin) obtained by treating yeast with a mixture of alcohol and ether
[Albert, 1900, 1901, 1], or with acetone and ether [Albert, Buchner,
and Rapp, 1902], show precisely similar properties (p. 38). The
proof in this case has been carried a step further, for the active
juice obtained by grinding such acetone-yeast, when precipitated
with alcohol and ether, yields an amorphous powder, still capable of
fermenting sugar.
THE PREPARATION OF YEAST-JUICE.
Buchner's process for the preparation of active yeast-juice is
characterised by extreme simplicity. The yeast employed, which
should be fresh brewery yeast, is washed two or three times by being
suspended in a large amount of water and allowed to settle in deep
vessels. It is then collected on a filter cloth, wrapped in a press
cloth, and submitted to a pressure of about 50 kilos, per sq. cm. for
five minutes. The resulting friable mass contains about 70 per cent.
of water and is free from adhering wort. The washed yeast is then
[p022] mixed with an equal weight of silver sand and 0·2 to 0·3 parts
of kieselguhr, care being taken that this is free from acid. The
correct amount of kieselguhr to be added can only be ascertained by
experience, and varies with different samples of yeast. The dry powder
thus obtained is brought in portions of 300 to 400 grams into a large
porcelain mortar and ground by hand by means of a porcelain pestle
fastened to a long iron rod which passes through a ring fixed in the
wall (Fig. 1). The mortar used by Buchner has a diameter of 40 cm. and
the pestle and rod together weigh 8 kilos.
[Illustration: FIG. 1. (Mortar and Pestle--Ed.)]
As the grinding proceeds the light-coloured powder gradually darkens
and becomes brown, and the mass becomes moist and adheres to the
pestle, until finally, after two to three minutes' grinding, it takes
the consistency of dough, at which stage the process is stopped. The
mass is next enveloped in a press cloth and submitted to a pressure
of 90 kilos, per sq. cm. in a hydraulic hand press, the pressure
being very gradually raised in order to avoid rupture of the cloth.
The cloth required for 1000 grams of yeast measures 60 by 75 cm. and
is previously soaked in water and then submitted to a pressure of 50
kilos, per sq. cm., retaining about 35 to 40 c.c. of water.
The juice runs from the press on to a folded filter paper, to remove
kieselguhr and yeast cells, and passes into a vessel standing in ice
water.
The yield of juice obtained by Buchner in an operation of this kind
from 1 kilo. of yeast amounts to 320 to 460 c.c. It may be increased
by re-grinding the press cake and again submitting it to pressure, and
then amounts on the average to 450 to 500 c.c.
Since the cell membranes constitute about 20 per cent. of the weight
of the dry yeast, this yield corresponds to more than 60 per cent.
of the total cell contents of the yeast. It has been computed by
Will [quoted by Buchner, E. and H., and Hahn, 1903, p. 66] that
[p023] only about 20 per cent. of the cells are left unaltered by one
grinding and pressing, and only 4 per cent. after a repetition of the
process, at least 57 per cent. of the cells being actually ruptured by
the double process, and the remainder to some extent altered. It seems
probable from these figures that a certain amount of the juice may be
derived from the unbroken cells, and Will expressly states that many
unbroken cells have lost their vacuoles.
[Illustration: FIG. 2. (Hydraulic Hand Press--Ed.)]
If the yeast be submitted to a process of regeneration, which consists
in exposure to a well-aerated solution of sugar and mineral salts
until fermentation is complete, the juice subsequently obtained
[p024] is more active than that yielded by the original yeast
[Albert, 1899, 1].
A modified method of grinding yeast was introduced by Macfadyen,
Morris, and Rowland [1900], who placed a mixture of yeast and sand
in a jacketed and cooled vessel, in which a spindle carrying brass
flanges was rapidly rotated [Rowland, 1901]. One kilo. of yeast ground
in this way for 3·5 hours yielded 350 c.c. of juice.
This grinding process was at first adopted by Harden and Young in
their experiments but was afterwards abandoned in favour of Buchner's
hand-grinding process, as it was found liable to yield juices of low
fermenting power, probably on account of inefficient cooling during
the grinding process. A slight modification of Buchner's process has,
however, been introduced, the hand-ground mass being mixed with a
further quantity of kieselguhr until a nearly dry powder is formed,
and the mass packed between two layers of chain cloth in steel
filter plates and pressed out in a hydraulic press at about 2 tons
to the square inch (300 kilos. per sq. cm.). The press and plates
are shown in section in Fig. 2. It has also been found convenient to
remove yeast cells and kieselguhr from the freshly pressed juice by
centrifugalisation instead of by filtration through paper, and to wash
the yeast before grinding by means of a filter-press.
Working with English top yeasts Harden and Young have found the yield
of juice extremely variable, the general rule being that the amount
of juice obtainable from freshly skimmed yeast is smaller than that
yielded by the same yeast after standing for a day or two after being
skimmed. The yield for 1000 grams of pressed brewer's yeast varies
from 150 to 375 c.c., and is on the average about 250 c.c.
Very fresh yeast occasionally presents the peculiar phenomenon that
scarcely any juice can be expressed from the ground mass, although the
latter does not differ in appearance or consistency from a mass which
gives a good yield.
EXTRACTION OF ZYMASE FROM UNGROUND YEAST.
/1. Maceration of Dried Yeast./
A valuable addition to the methods of obtaining an active solution
of zymase was made in 1911 by Lebedeff [1911, 2; 1912, 2; see also
1911, 3, 7, and 1912, 1]. This investigator had been in the habit of
grinding dried yeast with water for preparing samples of yeast-juice
of uniform character and observed that when the dried yeast was
digested with sugar solution and the mixture heated, coagulation
[p025] took place throughout the whole liquid, the proteins of the
yeast having passed out of the cells. Further examination revealed the
interesting fact that dried yeast readily yielded an active extract
when macerated in water for some time. The quality of the resulting
"maceration extract" depends on a considerable number of factors, the
chief of which are: (1) the temperature of drying of the yeast; (2)
the temperature of maceration; (3) the duration of maceration; and (4)
the nature of the yeast, as well as, of course, the amount of water
added in maceration.
In general the yeast should be dried at 25°-30° and then macerated
with 3 parts of water for 2 hours at 35°.
The temperature of maceration may as a rule be varied, without
detriment to the product provided that the time of maceration is
also suitably altered; thus with dried Munich yeast, maceration for
4·5 hours at 25° is about as effective as 2 hours at 35°, whereas
treatment for a shorter time at 25° or a longer time at 35° produces
in general a less efficacious extract. Yeast dried at a lower
temperature than 25° tends to yield an extract poor in co-enzyme (p.
59) and hence of low fermenting power, this being especially marked at
air temperature.
The subsequent treatment of the yeast during maceration may, however,
be of great influence in such cases. Thus a yeast dried at 15° gave by
maceration at 25° for 4·5 hours a weak extract (yielding with excess
of sugar 0·33g. CO{2}), whereas when macerated at 35° for 2 hours it
yielded a normal extract (1·36g. CO{2}).
The nature of the yeast is of paramount importance. Thus while Munich
(bottom) yeast usually gives a good result, a top yeast from a Paris
brewery was found to yield extracts containing neither zymase nor its
co-enzyme in whatever way the preparation was conducted. The existence
of such yeasts is of great interest, and it was probably due to the
unfortunate selection of such a yeast for his experiments that Pasteur
was unable to prepare active fermenting extracts and therefore failed
to anticipate Buchner by more than 30 years (see p. 15). The English
top yeasts as a rule give poor results [see Dixon and Atkins, 1913]
and sometimes yield totally inactive maceration extract. It is not
understood why the enzyme passes out of the cell during the process of
maceration and the whole method gives rise to a number of extremely
interesting problems.
/Method./--A suitable yeast is washed by decantation, filtered through
a cloth, lightly pressed by means of a hand press, and then passed
through a sieve of 5mm. mesh, spread out in a layer 1-1·5cm. thick
and left at 25°-35° for two days. Fifty grams of the dried yeast is
[Pg 026] thoroughly and carefully mixed with 150 c.c. of water in
a basin by means of a spatula and the whole digested for two hours
at 35°. The mass often froths considerably. It is then filtered
through ordinary folded filter paper, preferably in two portions,
and collected in a vessel cooled by ice. The separation may also be
effected by centrifuging or pressing out the mass, and the maceration
may be conveniently conducted in a flask immersed in the water of a
thermostat. It is not advisable to macerate more than 50 grams in one
operation. Under these conditions 25-30 c.c. of extract are obtained
after 20 minutes' filtration, 70-80 c.c. in twelve hours. Dried Munich
yeast can be bought from Messrs. Schroder of Munich and serves as a
convenient source of the extract.[1]
[1] The material supplied is occasionally found to yield an inactive
extract and every sample should be tested.
This extract closely resembles in properties the juice obtained by
grinding the same yeast, but it is usually more active and contains
more inorganic phosphate (see p. 46).
/2. Other Methods./
Attempts to prepare active extracts from undried yeast in an analogous
manner have so far not been very successful. Thus Rinckleben [1911]
found that plasmolysis by glycerol (8 per cent.) or sodium phosphate
(5 per cent.) sometimes yielded an active juice and sometimes a juice
which contained enzyme but no co-enzyme, but more often an inactive
juice incapable of activation (p. 64) [see also Kayser, 1911].
Giglioli [1911] by the addition of chloroform also obtained an active
liquid. It appears in fact as though almost any method of plasmolysing
the yeast cell may yield a certain proportion of zymase in the exudate.
An ingenious process has been devised by Dixon and Atkins [1913] who
applied the method of freezing in liquid air which they had found
efficacious for obtaining the sap from various plant organs. They thus
succeeded in obtaining from yeast, derived from Guinness' brewery in
Dublin, liquids capable of fermenting sugar and of about the same
efficacy as the maceration extracts prepared by Lebedeff's method from
the same yeast. The results were, however, in both cases very low, the
maximum total production of CO{2} by 25 c.c. of liquid from excess
of sugar being 32·5 c.c. (air temperature) or about 0·06g. Munich
yeast on the other hand yields, either by maceration or grinding, a
liquid giving as much as 1·5-2g. of CO{2} per 25 c.c., whilst [p027]
English yeast-juice prepared by grinding often gives as much as
0·5-0·7g. of CO{2}.
No direct comparison with the juice prepared by grinding was made by
Dixon and Atkins, but it may be concluded from their results that the
best method of obtaining an active preparation from the top yeasts
used in this country is that of grinding. Maceration, freezing and
plasmolysis alike yield poor results. With Munich yeast on the other
hand the maceration process yields excellent results, whilst the
liquid air process has not so far been tried.
PRACTICAL METHODS FOR THE ESTIMATION OF THE FERMENTING POWER OF
YEAST-JUICE.
In order to estimate the amount of carbon dioxide evolved in a given
time and the total amount evolved by the action of yeast-juice on
sugar, Buchner adopted an extremely simple method, which consisted in
carrying out the fermentation in an Erlenmeyer flask provided with
a small wash-bottle, which contained sulphuric acid and was closed
by a Bunsen valve, and ascertaining the loss of weight during the
experiment. Corrections are necessary for the carbon dioxide present
in the original juice and retained in the liquid at the close of the
experiment and for that present in the air space of the apparatus,
but it was found that for most purposes these could be neglected.
In cases in which greater accuracy was desired, the carbon dioxide
was displaced by air before the weighings were made. A typical
experiment of this kind, without displacement of carbon dioxide, is
the following:--
March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane
sugar + 0·2 c.c. toluene as antiseptic at 16°. Grams of carbon dioxide
after
24 48 72 96 hours.
0·40 0·64 0·99 1·11
The total weight of carbon dioxide evolved under these conditions is
termed the fermenting power of the juice (Buchner).
A more accurate method [Macfadyen, Morris, and Rowland, 1900]
consists in passing the carbon dioxide into caustic soda solution and
estimating it by titration. The yeast-juice, sugar, and antiseptic are
placed in an Erlenmeyer flask provided with a straight glass tube,
through which air can be passed over the surface of the liquid, and a
conducting tube leading into a second flask which contains 50 c.c. of
10 per cent. caustic soda solution and is connected with the air by a
guard tube containing soda lime. The juice can be freed from carbon
dioxide by agitation in a current of air before the flask is connected
to [p028] that containing the caustic soda solution, and at the end
of the period of incubation air is passed through the apparatus, the
liquid being boiled out if great accuracy is required. The absorption
flask is then disconnected and the amount of absorbed carbon dioxide
estimated by titration. This is carried out by making up the contents
of the flask to 200 c.c., taking out an aliquot portion, rendering
this exactly neutral to phenophthalein by the addition first of normal
and finally of decinormal acid, adding methyl orange and titrating
with decinormal acid to exact neutrality. Each c.c. of decinormal acid
used in this last titration represents 0·0044 gram of carbon dioxide
in the quantity of solution titrated.
[Illustration: FIG. 3. (Schiff's Azotometer--Ed.)]
These methods are only suitable for observations at considerable
intervals of time. For the continuous observation of the course
of fermentation Harden, Thompson and Young [1910] connect the
fermentation flask with a Schiff's azotometer filled with mercury and
measure the volume of gas evolved, the liquid having been previously
saturated with carbon dioxide (Fig. 3). The level of the mercury in
the reservoir is kept constant by a syphon overflow, as shown in the
figure, or, according to a modification introduced by S. G. Paine,
by a specially constructed bottle provided with two tubulures near
the bottom. This ensures that no change in the pressure in the flask
occurs, and the volume of gas observed is reduced to normal pressure
by means of a table. Before making a reading it is necessary to shake
the fermenting mixture thoroughly, as the albuminous liquid very
readily becomes greatly supersaturated with carbon dioxide, so much
so in fact that very little gas is evolved in the intervals between
the shakings. The exact procedure in making an observation consists in
shaking the flask [p029] thoroughly, replacing in the thermostat,
allowing to remain for one minute, and then reading the level of the
mercury in the azotometer. After the required time, say five minutes,
has elapsed from the time at which the flask was first shaken, it is
again removed from the bath, shaken as before, replaced, allowed to
remain for one minute and the reading then taken. In this way readings
can be conveniently made at intervals of three or five minutes or even
less, and much more detailed information obtained about the course
of the reaction than is possible by means of observations made at
intervals of several hours.
Another form of volumetric apparatus, designed by Walton [1904], has
been used by Lebedeff [1909].
An apparatus on a different principle has been designed by Slator
[1906] for use with living yeast, but is equally applicable to
yeast-juice, and a very similar form has been more recently employed
by Iwanoff [1909, 2]. In this apparatus the change of pressure
produced by the evolution of carbon dioxide is measured at constant
volume, and comparative rates of evolution can be obtained with
considerable accuracy, although the method has the disadvantage that
the absolute volume of gas evolved is not measured. The apparatus
consists of a bottle or flask connected with a mercury manometer. The
fermenting mixture is placed in the bottle along with glass beads to
facilitate agitation, the pressure is reduced to a small amount by the
water-pump, and the rise of pressure is then observed at intervals,
this being proportional to the volume of gas produced. As in the
preceding case, the liquid must be well shaken before a reading is
made.
THE ALCOHOLIC FERMENTATION OF THE SUGARS BY YEAST-JUICE.
Yeast-juice brings about a slow fermentation of those sugars which
are fermented by the yeast from which it is prepared as well as of
dextrin, and of starch and glycogen, which are not fermented by living
yeast.
(/a/) /Relation to Fermentation by living Yeast./
Both in rate of fermentation and in the total fermentation produced,
yeast-juice stands far behind the equivalent amount of living yeast.
Taking 25 c.c. of yeast-juice to be equivalent to at least 36 grams of
pressed yeast containing 70 per cent. of moisture, it is found that
whereas the yeast-juice (from English top yeast) gives with glucose
a maximum rate of fermentation of about 3 c.c. in five minutes, the
living yeast ferments the sugar at the rate of about 126 c.c. in the
same time, or [p030] about forty times as quickly. The total carbon
dioxide obtainable from the yeast-juice, moreover, corresponds to the
fermentation of only 2 to 3 grams of sugar, whilst the living yeast
will readily ferment a much larger quantity, although the exact limit
in this respect has not been accurately determined. The reasons for
this great difference in behaviour will be discussed later on, after
the various factors concerned in fermentation have been considered (p.
123).
(/b/) /Relation of Alcohol to Carbon Dioxide./
In all cases of fermentation by yeast-juice and zymin, the relative
amounts of carbon dioxide and alcohol produced are substantially in
the ratio of the molecular weights of the compounds, that is as 44:
46, so that for 1 part of carbon dioxide 1·04 of alcohol are formed.
This has been shown for the juice and zymin from bottom yeasts by
Buchner [Buchner, E. and H., and Hahn, 1903, pp. 210, 211], who
obtained the ratios 1·01, 0·98, 1·01, and 0·99 from experiments in
which from 8 to 15 grams of alcohol were produced. Similar numbers,
0·90, 1·12, 0·95, 0·91 and 0·92, have been obtained for the juice from
top yeasts by Harden and Young [1904], who worked with much smaller
quantities. The variable results obtained with juice from top yeast by
Macfadyen, Morris and Rowland [1900], have not been confirmed.
(/c/) /Relation of Carbon Dioxide and Alcohol Produced to the Amount
of Sugar Fermented./
The construction of a balance-sheet between the sugar fermented and
the products formed is of special interest in the case of alcoholic
fermentation by yeast-juice, because, there being no cell growth as
in the case of living yeast, an opportunity appears to be afforded of
ascertaining whether the whole of the sugar is converted into alcohol
and carbon dioxide, or whether some fraction of the sugar passes into
any of the well-known subsidiary products of alcoholic fermentation by
yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately
the question cannot be settled in this way. When the loss of sugar
during the fermentation is estimated directly, it is usually found
to be considerably greater than the sum of the alcohol and carbon
dioxide produced from it. This fact was first observed by Macfadyen,
Morris and Rowland [1900], and was then confirmed by Buchner [Buchner,
E. and H., and Hahn, 1903, p. 212], in one instance, the excess of
sugar lost over products being in this case about 15 per cent. of the
total sugar which had disappeared. The matter was then more thoroughly
investigated by Harden and Young [1904]. [p031]
The conditions under which the experiment must be carried out are not
very favourable to the attainment of extreme accuracy. Yeast-juice
contains glycogen and a diastatic enzyme which converts this into
dextrins and finally into sugar. This process goes on throughout
fermentation, tending to increase the sugar present and to make the
apparent loss of sugar less than the sum of the products. In spite
of this it was found that a certain amount of sugar invariably
disappeared without being accounted for as alcohol or carbon dioxide,
and this whether the fermentation lasted sixty or a hundred and
eight hours, and independently of the dilution of the juice. This
disappearing sugar amounted in some cases to 44 per cent. of the
total loss of sugar, and on the average of twenty-five experiments
was 38 per cent. Further information was sought by converting all the
sugar-yielding constituents of the juice into sugar by hydrolysis
before and after the fermentation. This process revealed the fact that
when the glucose equivalent of the juice before and after fermentation
was determined after hydrolysis with three times normal acid for
three hours (and a correction made for the loss of reducing power
experienced by glucose itself when submitted to this treatment),
the difference was almost exactly equal to the alcohol and carbon
dioxide produced. In other words, accompanying fermentation, a change
proceeds by which sugar is converted into a less reducing substance,
reconvertible into sugar by hydrolysis with acids. Similar results
were subsequently obtained by Buchner and Meisenheimer [1906], who
employed 1·5 normal acid and observed a small nett loss of sugar.
Still more recently Lebedeff [1909, 1910, see also 1913, 2] has
carried out similar estimations with the same result. It is doubtful
whether the experiments which have so far been made on this point are
sufficiently accurate to decide with certainty whether or not the
loss of sugar is exactly equal to the sum of the carbon dioxide and
alcohol produced. It has been shown by Buchner and Meisenheimer [1906]
that glycerol is a constant product of alcoholic fermentation by
yeast-juice (p. 95), and no other source for this than the sugar has
yet been found, so that it is not improbable that a small amount of
sugar is converted into non-carbohydrate substances other than carbon
dioxide and alcohol.
It has also been shown [Harden and Young, 1913] that the deficit of
sugar is not due to the formation of hexosephosphate (p. 47), which
has a lower reduction than glucose, and that the solution from which
the sugar (either glucose or fructose) has disappeared actually
contains some substance of relatively high dextrorotation and of low
reducing power. [p032]
However this may be, it may be considered as established that
during alcoholic fermentation sugar is converted by an enzyme into
some compound of less reducing power, which again yields sugar on
hydrolysis with acids. The exact nature of this substance has not been
ascertained, but it appears likely that the process is a synthetical
one resulting in the formation of some polysaccharide, possibly
intermediate between the hexoses and glycogen.
A similar phenomenon has been observed with living yeast by Euler
and Johansson [1912, 1], and Euler and Berggren [1912], whose
interpretation of the observation is discussed later on (p. 57).
(/d/) /Fermentation of Different Carbohydrates. Autofermentation./
Yeast-juice and zymin ferment all the sugars which are fermented by
the yeast from which they are prepared, and, in addition, a number
of colloidal substances which cannot pass through the membrane of
the living yeast cell, but which are hydrolysed by enzymes in the
juice and thus converted into simpler sugars capable of fermentation
[Buchner and Rapp, 1898, 3; 1899, 2]. Of the simple sugars which have
been examined, glucose, fructose, and mannose are freely fermented,
l-arabinose not at all, whilst the case of galactose is doubtful.
Galactose is, however, fermented by juice prepared from a yeast which
has been "trained" to ferment galactose [Harden and Norris, 1910]. As
regards both the rate of fermentation and the total amount of carbon
dioxide evolved from glucose and fructose by the action of a definite
amount of yeast-juice, Buchner and Rapp obtained practically identical
numbers. Harden and Young [1909], using juice from top yeast, found
that fructose was slightly more rapidly fermented and gave a somewhat
larger total than glucose, whilst mannose was initially fermented at
almost the same rate as glucose, but gave a decidedly lower total, the
following being the average result:--
Sugar. Relative Rates. Relative Totals.
Glucose 1 1
Fructose 1·29 1·15
Mannose 1·04 0·67
Among the disaccharides, cane sugar and maltose are freely fermented,
and the juice can be shown like living yeast to contain invertase and
maltase. The extent of fermentation does not differ materially from
that attained with glucose. Lactose is not fermented.
Of the higher sugars raffinose is fermented by juice from bottom
yeast, but more slowly than cane sugar or maltose. No experiments seem
to have been made with juice from top yeast. [p033]
As regards the fermentation of the higher carbohydrates, very little
experimental work has been carried out. Buchner and Rapp found that
the fermentation of starch paste was doubtful, but that soluble starch
and commercial dextrin were fermented with some freedom. No special
study has been made of the diastatic enzymes which bring about the
hydrolysis of these substances.
The fermentation of glycogen by yeast-juice is of considerable
interest, since it is known that the characteristic reserve
carbohydrate of the yeast cell is glycogen [see Harden and Young,
1902, where the literature is cited], and moreover that in living
yeast the intracellular fermentation of glycogen proceeds readily,
whereas glycogen added to a solution in which yeast is suspended is
not affected. Yeast-juice contains a diastatic enzyme which hydrolyses
glycogen to a reducing and fermentable sugar, so that in a juice poor
in zymase to which glycogen has been added, the amount of sugar is
found to increase, the hydrolysis of the glycogen proceeding more
quickly than the fermentation of the resulting sugar [Harden and
Young, 1904], but the course of this enzymic hydrolysis of glycogen by
yeast-juice has not yet been studied. As a rule, it is found both with
juices from top and bottom yeast that the evolution of carbon dioxide
from glycogen proceeds less rapidly and reaches a lower total than
from an equivalent amount of glucose.
Since nearly all samples of yeast contain glycogen, yeast-juice and
also zymin usually contain this substance as well as the products of
its hydrolysis. These provide a source of sugar which enters into
alcoholic fermentation, so that a slow spontaneous production of
carbon dioxide and alcohol proceeds when yeast-juice is preserved
without any addition of sugar. The extent of this autofermentation
varies considerably, as might be expected, with the nature of the
yeast employed or the preparation of the material, but is generally
confined within the limits of 0·06 to 0·5 gram of carbon dioxide for
25 c.c. of juice.
In juice from bottom yeast it amounts to about 5 to 10 per cent.
of the total fermentation obtainable with glucose [Buchner, 1900,
2], whereas in juice from top yeasts, which gives a smaller total
fermentation with glucose, it may occasionally equal, or even exceed,
the glucose fermentation, and frequently amounts to 30 to 50 per cent.
of it. It is therefore generally advisable in studying the effect of
yeast-juice on any particular substance to ascertain the extent of
autofermentation by means of a parallel experiment.
The maceration extract of Lebedeff (p. 24) is usually, but not
invariably [Oppenheimer, 1914, 2], free from glycogen, which is
hydrolysed [p034] and fermented during the processes of drying
and macerating, and therefore as a rule shows no appreciable
autofermentation.
(/e/) /Effect of Concentration of Sugar on the Total Amount of
Fermentation./
The kinetics of fermentation by zymase will be considered later on
(p. 120), but the effect on the total fermentation of different
concentrations of sugar, this substance being present throughout in
considerable excess, may be advantageously discussed at this stage.
The subject has been investigated by Buchner [Buchner, E. and H., and
Hahn, 1903, pp. 150-8; Buchner and Rapp, 1897] using cane sugar, and
he has found both for yeast-juice and for dried yeast-juice dissolved
in water that (/a/) the total amount of fermentation increases with
the concentration of the sugar; (/b/) the initial rate of fermentation
decreases with the concentration of the sugar. The following are the
results of a typical experiment, 20 c.c. of yeast-juice being employed
in presence of toluene at 22°:--
Cane Sugar. │ CO{2} in grams after
─────────┬───────────┼──────────┬──────────┬──────────
Weight. │ Per cent. │ 6 hours. │24 hours. │ 96 hours.
─────────┼───────────┼──────────┼──────────┼──────────
2·2 │ 10 │ 0·17 │ 0·50 │ 0·55
3·52 │ 15 │ 0·14 │ 0·53 │ 0·64
5 │ 20 │ 0·13 │ 0·54 │ 0·73
6·66 │ 25 │ 0·13 │ 0·52 │ 0·80
8·56 │ 30 │ 0·12 │ 0·46 │ 0·81
10·76 │ 35 │ 0·12 │ 0·40 │ 0·82
13·33 │ 40 │ 0·11 │ 0·36 │ 0·82
10·76 │ 35 │ 0·12 │ 0·40 │ 0·82
13·33 │ 40 │ 0·11 │ 0·36 │ 0·82
The results as to the total fermentations in experiments of this kind
are liable to be vitiated by the circumstance that when a low initial
concentration of sugar is employed, the supply of sugar may be so
greatly exhausted before the close of the experiment as to cause a
marked diminution in the rate of fermentation and hence an unduly
low total. Even allowing, however, for any effect of this kind, the
foregoing table clearly shows the increase in total fermentation
and the decrease in initial rate accompanying the increase of sugar
concentration from 10 to 40 per cent. Working with a greater range
of concentrations (3·3-53·3 grm. per 100 c.c.) Lebedeff has obtained
similar results with maceration extract [1911, 4], but has found
that the total amount fermented diminishes after a certain optimum
concentration (about 33·3 grm. per 100 c.c.) is reached.
A practical conclusion from these experiments is that a high [p035]
concentration of sugar tends to preserve the enzyme in an active state
for a longer time. Simultaneously it prevents the development of
bacteria and yeast cells.
(/f/) /Effect of Varying Concentration of Yeast-Juice./
This subject, which is of considerable importance with reference to
the question of the protoplasmic or enzymic nature of the active agent
in yeast-juice, has been examined in some detail by Buchner [Buchner,
E. and H., and Hahn, 1903, pp. 158-65] and by Meisenheimer [1903]
for juices from bottom yeast, by Harden and Young [1904] for those
from top yeast, and by Lebedeff [1911, 4] for maceration extract, the
results obtained being in substantial agreement.
Dilution of yeast-juice with sugar solution, so that the concentration
of the sugar remains constant, produces a small progressive diminution
in the total fermentation, which only becomes marked when more than 2
volumes are added, and this independently of the actual concentration
of the sugar. Dilution with water produces a somewhat more decided
diminution, which, however, does not exceed 50 per cent. of the total
for the addition of 3 volumes of water. The effect on maceration
extract is somewhat greater but of the same kind. The autofermentation
of juice from top yeast is scarcely affected by dilution with 4
volumes of water.
┌───────────────────────────────────────────────────────────────────┐
│ Nature of Per cent. Volumes of Volumes of Total │
│ Juice of Sugar Sugar Water Added. Fermentation │
│ (1,2,3) Employed Solution in g. │
│ by Weight Added. of CO{2}. │
│ Bottom Yeast──────────────────────────────────────────────────────┤
│ 1 29 0 -- 0·99 │
│ 1 -- 1·13 │
│ 2 -- 0·92 │
│ 4 -- 0·79 │
│ 2 9 0 -- 0·43 │
│ 1 -- 0·60 │
│ 2 -- 0·53 │
│ 4 -- 0·41 │
│ 3 9 -- 0 0·46 │
│ -- 1 0·32 │
│ -- 2 0·33 │
│ -- 3 0·36 │
│ Top Yeast─────────────────────────────────────────────────────────┤
│ 1 0 -- 0 0·29 │
│ (Autofermentation) -- 2 0·29 │
│ -- 3 0·28 │
│ 2 29 0 -- 0·31 │
│ 1 -- 0·34 │
│ 2 -- 0·31 │
│ 4 -- 0·35 │
│ 6 -- 0·28 │
│ 3 7·4 -- 0 0·44 │
│ -- 1 0·35 │
│ -- 2 0·30 │
│ -- 3 0·28 │
└───────────────────────────────────────────────────────────────────┘
[p036]
On the whole, therefore, yeast-juice may be said to be only slightly
affected by dilution even with pure water, and the effect of the
latter can in no way be regarded as comparable with the poisonous
effect which it exerts on living protoplasm, as suggested by
Macfadyen, Morris, and Rowland [1900].
(/g/) /The Effect of Antiseptics on the Fermentation of Sugars by
Yeast-Juice./
Buchner has paid special attention to the effect of antiseptics
on the course of fermentation by yeast-juice [Buchner and Rapp,
1897; 1898, 2, 3; 1899, 1; Buchner and Antoni, 1905, 1; Buchner and
Hoffmann, 1907; Buchner, E. and H., and Hahn, 1903, pp. 169-205; see
also Albert, 1899, 2; Gromoff and Grigorieff, 1904; Duchaček, 1909]
in order (1) to obtain evidence as to the possibility of the active
agent in yeast-juice consisting of fragments of protoplasm and not of
a soluble enzyme, and (2) also to provide a safe method of avoiding
contamination, by the growth of bacteria or yeasts, of the liquids
used which were often kept at 25° for several days. The results of
these experiments are briefly summarised in the following table, in
which the effect of each substance on the total fermentation produced
is noted:--
Substance. Effect on Total Fermentation.
Concentrated solution of glycerol Slight diminution
" " " sugar " increase
Toluene (to saturation or excess) Less than 10 per cent.
diminution
Chloroform 0·5 per cent. Slight increase
" 0·8 per cent. (saturation) No change
" Large excess (17 per cent.) 64 per cent. diminution
Chloral hydrate 0·7 per cent. Increase up to 27 per cent.
" 3·5-5·4 per cent. Completely destroyed
Phenol 0·1 per cent. No change
" 0·5 " 40 per cent. diminution
" 1·2 " Completely destroyed
Thymol 1 " Slight diminution
" 5 " Marked "
Benzoic acid 0·1 " 7 per cent. diminution
" 0·25 " 26 " "
Salicylic acid 0·1 " 10 " "
" 0·27 " 35 " "
Formaldehyde 0·12 " 20 " "
" 0·24 " 30-60 " "
Acetone 6 " 20 " "
" 14 " 80 " "
Alcohol 6 " 0-20 " "
" 14 " 75 " "
Sodium fluoride 0·5 " 90 " "
" 2 " Almost completely destroyed
Ammonium fluoride 0·55 per cent. Completely destroyed
Sodium azoimide, NaN{3}, 0·36 per cent. Slight diminution
" " 0·71 " Marked "
Quinine hydrochloride 1 " Slight increase
Ozone 10·4-34·8 milligrams per 20 c.c. Marked diminution
Hydrocyanic acid 1·2 per cent. Completely destroyed
[p037]
The general result of these experiments is to show that quantities
of antiseptics which are sufficient to inhibit the characteristic
action of living cells have only a slight effect on the fermentative
activity of yeast-juice. A large excess of the antiseptic in many
cases produces a very decided diminution or total destruction of
the fermenting power, and accompanying this a precipitation of the
constituents of the juice. The decided increase of activity produced
by small quantities of chloral hydrate, and to a less marked extent by
chloroform and a few other substances, is of considerable interest.
It is ascribed by Duchaček to a selective action on the proteoclastic
enzyme, but without satisfactory evidence.
Hydrocyanic acid, even in dilute solution, completely suspends the
fermenting power of the juice, without, however, producing any
permanent change in the fermenting complex, as is shown by the fact
that when the hydrocyanic acid is removed by a current of air, the
juice regains its fermenting power. In this respect hydrocyanic acid
behaves precisely as with many other enzymes and with colloidal
platinum [Bredig, 1901]. Sodium arsenite is a pronounced protoplasmic
poison, which rapidly destroys the power of growth and reproduction
in living cells, and was therefore applied to yeast-juice to
differentiate between protoplasmic and enzymic action. It was,
however, found that the action of this substance was complicated by
some unknown factor and very irregular results were obtained [Buchner,
E. and H., and Hahn, 1903, pp. 193 ff.]. These phenomena appear to be
of the same order as those produced by the addition of arsenates to
yeast-juice [Harden and Young, 1906, 3], and will be discussed along
with the latter (p. 77).
PERMANENT PREPARATIONS CONTAINING ACTIVE ZYMASE.
A considerable number of preparations have been obtained in the dry
state which retain some proportion of the fermenting power of yeast or
yeast-juice.
Starting with yeast-juice, it is possible to arrive at this result
either by evaporation or precipitation. When the juice is very rapidly
evaporated to a syrup at 20° to 25° and then further dried at 35°,
either in the air or in a vacuum and finally exposed over sulphuric
acid in a vacuum desiccator, a dry brittle mass is obtained which is
soluble in water and retains practically the whole of the fermenting
power of the juice. The success of the preparation depends on the
nature of the yeast from which the juice is derived, Berlin yeasts V
and S yielding much less satisfactory results than Munich yeast. The
powder when [p038] thoroughly dry is found to retain its properties
almost unimpaired for at least a year, and can be heated to 85° for
eight hours without undergoing any serious loss of fermenting power
[Buchner and Rapp, 1898, 4; 1901; Buchner, E. and H., and Hahn, 1903,
pp. 132-9].
Active powders can also be obtained by precipitating yeast-juice
with alcohol, alcohol and ether, or acetone. The preparation is
best effected by bringing the juice into 10 volumes of acetone,
centrifuging at once and as rapidly as possible, washing, first with
acetone and then with ether, and finally drying over sulphuric acid.
The white powder thus obtained is not completely soluble in water
but is almost entirely dissolved by aqueous glycerol (2·5 to 20 per
cent.), forming a solution which has practically the same fermenting
power as the original juice. The precipitation can be repeated without
any serious loss of fermenting power. Prolonged contact of the
precipitate with the supernatant liquid, especially when alcohol or
alcohol and ether are used, causes a rapid loss of the characteristic
property [Albert and Buchner, 1900, 1, 2; Buchner, E. and H., and
Hahn, 1903, pp. 228-246; Buchner and Duchaček, 1909].
Dry preparations capable of fermenting sugar can also be readily
obtained from yeast without any preliminary rupture of the cells.
Heat alone (yielding a product known as hefanol) or treatment with
dehydrating agents may be used for this purpose, and a brief allusion
has already been made (p. 21) to the different varieties of permanent
yeast (Dauerhefe) obtainable in these ways. The most important of
these products are the dried Munich yeast (Lebedeff, see p. 25), and
the material known as zymin, which is now made under patent rights for
medicinal purposes by Schroder of Munich. The latter has proved of
value in the investigation of the production of zymase in the yeast
cell [Buchner and Spitta, 1902], and of many other problems concerned
with alcoholic fermentation. In order to prepare it 500 grams of
finely divided pressed brewer's yeast, containing about 70 per cent.
of water, are brought into 3 litres of acetone, stirred for ten
minutes, and filtered and drained at the pump. The mass is then well
mixed with 1 litre of acetone for two minutes and again filtered and
drained. The residue is roughly powdered, well kneaded with 250 c.c.
of ether for three minutes, filtered, drained, and spread on filter
paper or porous plates. After standing for an hour in the air it is
dried at 45° for twenty-four hours. About 150 grams of an almost white
powder containing only 5·5 to 6·5 per cent. of water are obtained.
This is quite incapable of growth or reproduction but produces a very
considerable amount of alcoholic fermentation, far greater indeed than
a corresponding [p039] quantity of yeast-juice. Two grams of the
powder corresponding to 6 grams of yeast and about 3·5 to 4 c.c. of
yeast-juice, are capable of fermenting about 2 grams of sugar, whereas
the 4 c.c. of yeast-juice would on the average only ferment from
one-quarter to one-sixth of this amount of sugar. The rate produced
by this amount of zymin is about one-eighth of that given by the
corresponding amount of living yeast [Albert, 1900; Albert, Buchner,
and Rapp, 1902]. The proteoclastic ferment is still present in zymin,
which undergoes autolysis in presence of water in a similar manner to
yeast-juice [Albert, 1901, 2].
As already mentioned an active juice can be prepared by grinding
acetone-yeast with water, sand, and kieselguhr, and this process
presents the advantage that samples of yeast-juice of approximately
constant composition can be prepared at intervals from successive
portions of a uniform supply of acetone-yeast.
Preparations of acetone-yeast, made from yeast freed from glycogen
by exposure in a thin layer to the air for three or four hours at
35° to 45°, or eight hours at the ordinary temperature [Buchner and
Mitscherlich, 1904], show practically no autofermentation and may be
used analytically for the estimation of fermentable sugars.
All the foregoing preparations exhibit the same general properties as
yeast-juice, as regards their behaviour towards the various sugars,
antiseptics, etc.
When zymin is mixed with sugar solution without being previously
ground, it exhibits a peculiarity which is of some practical interest.
The time which elapses before the normal rate of fermentation is
attained and the total fermentation obtainable vary with the amount of
sugar solution added, the time increasing and the total diminishing
as the quantity of this increases. This phenomenon appears to have
been noticed by Trommsdorff [1902], and a single experiment of Buchner
shows the influence of the same conditions [Buchner, E. and H., and
Hahn, 1903, p. 265, Nos. 700-1]. Harden and Young have found that when
2 grams of zymin are mixed with varying quantities of 10 per cent.
sugar solution the following results are obtained:--
───────────┬──────────────────────────────────────────
Volumes of │ Total Gas Evolved in
Sugar │ 1 2 3 4 22·5 hours.
Solution │
───────────┼──────────────────────────────────────────
5 c.c. │ 15·7 31·6 44·8 56·5 233·3
10 " │ 2·2 10·5 23 31·8 202·3
20 " │ 0·9 2·4 13·6 23·7 125·5
40 " │ 1·4 1·7 2·3 2·9 56·3
───────────┴──────────────────────────────────────────
[p040]
This behaviour appears to be due to the removal of soluble matter
essential for fermentation from the cell, which is discussed later
on. It follows that when zymin is being tested for fermenting power,
a uniform method should be adopted, and all comparative tests should
be made with the same volumes of added sugar solution. Ground zymin
appears to begin to ferment somewhat more slowly than unground (2 grm.
to 12·4 c.c. of sugar solution in each case), but eventually produces
the same total volume of gas [Buchner and Antoni, 1905, 1]. [p041]