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ALCOHOLIC FERMENTATION
2nd Edition, 1914
by
Arthur Harden
MONOGRAPHS ON BIOCHEMISTRY
EDITED BY
R. H. A. PLIMMER, D.Sc.
AND
F. G. HOPKINS, M.A., M.B., D.Sc., F.R.S.
GENERAL PREFACE.
The subject of Physiological Chemistry, or Biochemistry, is
enlarging its borders to such an extent at the present time
that no single text-book upon the subject, without being
cumbrous, can adequately deal with it as a whole, so as to
give both a general and a detailed account of its present
position. It is, moreover, difficult, in the case of the larger
text-books, to keep abreast of so rapidly growing a science
by means of new editions, and such volumes are therefore
issued when much of their contents has become obsolete.
For this reason, an attempt is being made to place this
branch of science in a more accessible position by issuing
a series of monographs upon the various chapters of the
subject, each independent of and yet dependent upon the
others, so that from time to time, as new material and
the demand therefor necessitate, a new edition of each monograph
can be issued without reissuing the whole series. In
this way, both the expenses of publication and the expense
to the purchaser will be diminished, and by a moderate
outlay it will be possible to obtain a full account of any
particular subject as nearly current as possible.
The editors of these monographs have kept two objects
in view: firstly, that each author should be himself working
at the subject with which he deals; and, secondly, that a
Bibliography, as complete as possible, should be included,
in order to avoid cross references, which are apt to be
wrongly cited, and in order that each monograph may yield
full and independent information of the work which has been
done upon the subject.
It has been decided as a general scheme that the volumes
first issued shall deal with the pure chemistry of physiological
products and with certain general aspects of the subject.
Subsequent monographs will be devoted to such questions
as the chemistry of special tissues and particular aspects of
metabolism. So the series, if continued, will proceed from
physiological chemistry to what may be now more properly
termed chemical physiology. This will depend upon the
success which the first series achieves, and upon the divisions
of the subject which may be of interest at the time.
R. H. A. P.F. G. H.
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By Gilbert T. Morgan, D.Sc., F.I.C.
LONGMANS, GREEN AND
CO., LONDON, NEW YORK, BOMBAY,
CALCUTTA, AND MADRAS.
ALCOHOLIC FERMENTATION
BY
ARTHUR HARDEN, Ph.D., D.Sc., F.R.S.
PROFESSOR OF BIOCHEMISTRY, LONDON UNIVERSITY
HEAD OF THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA
SECOND EDITION
LONGMANS, GREEN AND CO.
39 PATERNOSTER ROW, LONDON
FOURTH AVENUE & 30TH
STREET, NEW YORK,
BOMBAY, CALCUTTA, AND MADRAS
1914
PREFACE.
The following chapters are based on courses of lectures
delivered at the London University and the Royal Institution
during 1909–1910. In them an account is given of the work
done on alcoholic fermentation since Buchner's epoch-making
discovery of zymase, only in so far as it appears to throw light
on the nature of that phenomenon. Many interesting subjects,
therefore, have perforce been left untouched, among them the
problem of the formation of zymase in the cell, and the vexed
question of the relation of alcoholic fermentation to the metabolic
processes of the higher plants and animals.
My thanks are due to the Council of the Royal Society,
and to the Publishers of the "Journal of Physiology" for
permission to make use of blocks (Figs. 2, 4 and 7) which
have appeared in their publications.
A. H.
PREFACE TO THE SECOND EDITION.
In the New Edition no change has been made in the scope
of the work. The rapid progress of the subject has,
however, rendered necessary many additions to the text and
a considerable increase in the bibliography.
A. H.
May,1914.
CONTENTS.
CHAPTER
PAGE
I.
Historical Introduction
II.
Zymase and its Properties
III.
The Function of Phosphates in
Alcoholic Fermentation
IV.
The Co-Enzyme of Yeast-Juice
V.
Action of Some Inhibiting and
Accelerating Agents on the Enzymes of Yeast-Juice
VI.
Carboxylase
VII.
The By-Products of Alcoholic Fermentation
VIII.
The Chemical Changes involved in
Fermentation
IX.
The Mechanism of Fermentation
Bibliography
Index
CHAPTER I.
HISTORICAL INTRODUCTION.
[p001]
The problem of alcoholic fermentation, of the origin and nature of that
mysterious and apparently spontaneous change which converted the
insipid juice of the grape into stimulating wine, seems to have exerted
a fascination over the minds of natural philosophers from the very
earliest times. No date can be assigned to the first observation of the
phenomena of the process. History finds man in the possession of
alcoholic liquors, and in the earliest chemical writings we find fermentation,
as a familiar natural process, invoked to explain and illustrate the
changes with which the science of those early days was concerned.
Throughout the period of alchemy fermentation plays an important
part; it is, in fact, scarcely too much to say that the language of the
alchemists and many of their ideas were founded on the phenomena of
fermentation. The subtle change in properties permeating the whole
mass of material, the frothing of the fermenting liquid, rendering evident
the vigour of the action, seemed to them the very emblems of the
mysterious process by which the long sought for philosopher's stone
was to convert the baser metals into gold. As chemical science
emerged from the mists of alchemy, definite ideas about the nature
of alcoholic fermentation and of putrefaction began to be formed.
Fermentation was distinguished from other chemical changes in which
gases were evolved, such as the action of acids on alkali carbonates
(Sylvius de le Boë, 1659); the gas evolved was examined and termed
gas vinorum, and was distinguished from the alcohol with which it
had at first been confused (van Helmont, 1648); afterwards it was
found that like the gas from potashes it was soluble in water (Wren,
1664). The gaseous product of fermentation and putrefaction was
identified by MacBride, in 1764, with the fixed air of Black, whilst
Cavendish in 1766 showed that fixed air alone was evolved in alcoholic
fermentation and that a mixture of this with inflammable air was produced
by putrefaction. In the meantime it had been recognised that
only sweet liquors could be fermented ("Ubi notandum, nihil fermentare
quod non sit dulce," Becher, 1682), and finally Cavendish
[p002]
[] determined the proportion of fixed air obtainable from
sugar by fermentation and found it to be 57 per cent. It gradually
became recognised that fermentation might yield either spirituous or acid
liquors, whilst putrefaction was thought to be an action of the same
kind as fermentation, differing mainly in the character of the products
(Becher).
As regards the nature of the process very confused ideas at first
prevailed, but in the time of the phlogistic chemists a definite theory of
fermentation was proposed, first by Willis (1659) and afterwards by
Stahl [],
the fundamental idea of which survived the overthrow
of the phlogistic system by Lavoisier and formed the foundation
of the views of Liebig. To explain the spontaneous origin of fermentation
and its propagation from one liquid to another, they supposed that
the process consisted in a violent internal motion of the particles of the
fermenting substance, set up by an aqueous liquid, whereby the combination
of the essential constituents of this material was loosened and
new particles formed, some of which were thrust out of the liquid (the
carbon dioxide) and others retained in it (the alcohol).
Stahl specifically states that a body in such a state of internal disquietude
can very readily communicate the disturbance to another,
which is itself at rest but is capable of undergoing a similar change, so
that a putrefying or fermenting liquid can set another liquid in putrefaction
or fermentation.
Taking account of the gradual accumulation of fact and theory we
find at the time of Lavoisier, from which the modern aspect of the
problem dates, that Stahl's theoretical views were generally accepted.
Alcoholic fermentation was known to require the presence of sugar and
was thought to lead to the production of carbon dioxide, acetic acid,
and alcohol.
The composition of organic compounds was at that time not understood,
and it was Lavoisier who established the fact that they consisted
of carbon, hydrogen, and oxygen, and who made systematic analyses
of the substances concerned in fermentation (1784–1789). Lavoisier
[] applied the results of these analyses to the study of alcoholic
fermentation, and by employing the principle which he regarded as the
foundation of experimental chemistry, "that there is the same quantity
of matter before and after the operation," he drew up an equation between
the quantities of carbon, hydrogen, and oxygen in the original
sugar and in the resulting substances, alcohol, carbon dioxide, and
acetic acid, showing that the products contained the whole matter of
the sugar, and thus for the first time giving a clear view of the chemical
[p003]
change which occurs in fermentation. The conclusion to which he
came was, we now know, very nearly accurate, but the research must
be regarded as one of those remarkable instances in which the genius
of the investigator triumphs over experimental deficiencies, for the
analytical numbers employed contained grave errors, and it was only by
a fortunate compensation of these that a result so near the truth was
attained.
Lavoisier's equation or balance sheet was as follows:—
Carbon.
Hydrogen.
Oxygen.
95·9 pounds of sugar (cane sugar) consist of
26·8
7·7
61·4
These yield:—
57·7 pounds of alcohol containing
16·7
9·6
31·4
35·3 pounds of carbon dioxide containing
9·9
—
25·4
2·5 pounds of acetic acid containing
0·6
0·2
1·7
―――
―――
―――
Total contained in products
27·2
9·8
58·5
The true composition of the sugar used was carbon 40·4, hydrogen
6·1, oxygen 49·4.
Lavoisier expressed no view as to the agency by which fermentation
was brought about, but came to a very definite and characteristic
conclusion as to the chemical nature of the change. The sugar, which
he regarded in harmony with his general views as an oxide, was split
into two parts, one of which was oxidised at the expense of the other
to form carbonic acid, whilst the other was deoxygenised in favour of
the former to produce the combustible substance alcohol, "so that if it
were possible to recombine these two substances, alcohol and carbonic
acid, sugar would result".
From this point commences the modern study of the problem.
Provided by the genius of Lavoisier with the assurance that the hitherto
mysterious process of fermentation was to be ranked along with
familiar chemical changes, and that it proceeded in harmony with the
same quantitative laws as these simpler reactions, chemists were stimulated
in their desire to penetrate further into the mysteries of the phenomenon,
and the importance and interest of the problem attracted
many workers.
So important indeed did the matter appear to Lavoisier's countrymen
that in the year 8 of the French Republic (1800) a prize—consisting
of a gold medal, the value of which, expressed in terms of the
newly introduced metric system, was that of one kilogram of gold—was
offered by the Institute for the best answer to the question:
"What are the characteristics by which animal and vegetable substances
which act as ferments can be distinguished from those which they are
capable of fermenting?"
[p004]
This valuable prize was again offered in 1802 but was never
awarded, as the fund from which it was to be drawn was sequestrated
from the Institute in 1804. The first response to this stimulating
offer was an important memoir by citizen Thenard [], which
provided many of the facts upon which Liebig subsequently based his
views. Thenard combats the prevailing idea, first expressed by
Fabroni (1787–1799), that fermentation is caused by the action of
gluten derived from grain on starch and sugar, but is himself uncertain
as to the actual nature of the ferment. He points out that all fermenting
liquids deposit a material resembling brewer's yeast, and he
shows that this contains nitrogen, much of which is evolved as
ammonia on distillation. His most important result is, however,
that when yeast is used to ferment pure sugar, it undergoes a gradual
change and is finally left as a white mass, much reduced in weight,
which contains no nitrogen and is without action on sugar. Thenard,
moreover, it is interesting to note, differs from Lavoisier, inasmuch
as he ascribes the origin of some of the carbonic acid to the carbon
of the ferment, an opinion which was still held in various degrees by
many investigators (see Seguin, quoted by Thenard).
Thenard's memoir was followed by a communication of fundamental
importance from Gay-Lussac [].
A process for preserving
food had been introduced by Appert, which consisted in placing
the material in bottles, closing these very carefully and exposing
them to the temperature of boiling water for some time. Gay-Lussac
was struck by the fact that when such a bottle was opened
fermentation or putrefaction set in rapidly. Analysis of the air left
in such a sealed bottle showed that all the oxygen had been absorbed,
and these facts led to the view that fermentation was set up by the
action of oxygen on the fermentable material. Experiment appeared
to confirm this in the most striking way. A bottle of preserved grape-juice
was opened over mercury and part of its contents passed
through the mercury into a bell-jar containing air, the remainder into
a similar vessel free from air. In the presence of air fermentation set
in at once, in the absence of air no fermentation whatever occurred.
This connection between fermentation and the presence of air was
established by numerous experiments and appeared incontestable.
Fermentation, it was found, could be checked by boiling even after
the addition of oxygen, and hence food could be preserved in free
contact with the air, provided only that it was raised to the temperature
of boiling water at short intervals of time. Gay-Lussac's opinion
was that the ferment was formed by the action of the oxygen on the
[p005]
liquid, and that the product of this action was altered by heat and
rendered incapable of producing fermentation, as was also brewer's
yeast, which, however, he regarded, on account of its insolubility, as
different from the soluble ferment which initiated the change in the
limpid grape-juice. Colin, on the other hand [], recognised that
alcoholic fermentation by whatever substance it was started, resulted
in the formation of an insoluble deposit more active than the
original substance, and he suggested that this deposit might possibly
in every case be of the same nature.
So far no suspicion appears to have arisen in the minds of those
who had occupied themselves with the study of fermentation that this
change differed in any essential manner from many other reactions
familiar to chemists. The origin and properties of the ferment were
indeed remarkable and involved in obscurity, but the uncertainty regarding
this substance was no greater than that surrounding many, if
not all, compounds of animal and vegetable origin. Although, however,
the purely chemical view as to the nature of yeast was generally
recognised and adopted, isolated observations were not wanting which
tended to show that yeast might be something more than a mere chemical
reagent. As early as 1680 in letters to the Royal Society Leeuwenhoek
described the microscopic appearance of yeast of various origins
as that of small, round, or oval particles, but no further progress seems
to have been made in this direction for nearly a century and a half,
when we find that Desmazières [] examined the film formed
on beer, figured the elongated cells of which it was composed, and
described it under the name of Mycoderma Cerevisiæ. He, however,
regarded it rather as of animal than of vegetable origin, and does not
appear to have connected the presence of these cells with the process
of fermentation.
Upon this long period during which yeast was regarded merely as
a chemical compound there followed, as has so frequently occurred in
similar cases, a sudden outburst of discovery. No less than three
observers hit almost simultaneously upon the secret of fermentation and
declared that yeast was a living organism.
First among these in strict order of time was Cagniard-Latour
[], who made a number of communications to the Academy
and to the Société Philomatique in 1835–6, the contents of which were
collected in a paper presented to the Academy of Sciences on 12 June,
1837, and published in 1838. The observations upon which this
memoir was based were almost exclusively microscopical. Yeast was
recognised as consisting of spherical particles, which were capable of
[p006]
reproduction by budding but incapable of motion, and it was therefore
regarded as a living organism probably belonging to the vegetable
kingdom. Alcoholic fermentation was observed to depend on the
presence of living yeast cells, and was attributed to some effect of their
vegetative life (quelque effet de leur végétation). It was also noticed
that yeast was not deprived of its fermenting power by exposure to
the temperature of solid carbonic acid, a sample of which was supplied
to Cagniard-Latour by Thilorier, who had only recently prepared it for
the first time.
Theodor Schwann [], whose researches were quite independent
of those of Cagniard-Latour, approached the problem from an
entirely different point of view. During the year 1836 Franz Schulze
[] published a research on the subject of spontaneous generation,
in which he proved that when a solution containing animal or
vegetable matter was boiled, no putrefaction set in provided that all
air which was allowed to have access to the liquid was previously
passed through strong sulphuric acid. Schwann performed a very
similar experiment by which he showed that this same result, the
absence of putrefaction, was attained by heating all air which came into
contact with the boiled liquid. Wishing to show that other processes
in which air took part were not affected by the air being heated, he
made experiments with fermenting liquids and found, contrary to his
expectation, that a liquid capable of undergoing vinous fermentation
and containing yeast did not undergo this change after it had been
boiled, provided that, as in the case of his previous experiments,
only air which had been heated was allowed to come into contact
with it.
Schwann's experiments on the prevention of putrefaction were unexceptionable
and quite decisive. The analogous experiments dealing
with alcoholic fermentation were not quite so satisfactory. Yeast was
added to a solution of cane sugar, the flask containing the mixture
placed in boiling water for ten minutes, and then inverted over mercury.
About one-third of the liquid was then displaced by air and the
flasks corked and kept inverted at air temperature. In two flasks the
air introduced was ordinary atmospheric air, and in these flasks fermentation
set in after about four to six weeks. Into the other two flasks
air which had been heated was led, and in these no fermentation
occurred. As described, the experiment is quite satisfactory, but
Schwann found on repetition that the results were irregular. Sometimes
all the flasks showed fermentation, sometimes none of them.
This was correctly ascribed to the experimental difficulties, but none
[p007]
the less served as a point of attack for hostile and damaging criticism
at the hands of Berzelius (p. ).
The origin of putrefaction was definitely attributed by Schwann to
the presence of living germs in the air, and the similarity of the result
obtained with yeast suggested the idea that alcoholic fermentation was
also brought about by a living organism, a conception which was at
once confirmed by a microscopical examination of a fermenting
liquid. The phenomena observed under the microscope were similar
to those noted by Cagniard-Latour, and in accordance with these
observations alcoholic fermentation was attributed to the development
of a living organism, the fermentative function of which was found to
be destroyed by potassium arsenite and not by extract of Nux vomica,
so that the organism was regarded rather as of vegetable than of
animal nature. This plant received the name of "Zuckerpilz" or
sugar fungus (which has been perpetuated in the generic term Saccharomyces).
Alcoholic fermentation was explained as "the decomposition
brought about by this sugar fungus removing from the sugar
and a nitrogenous substance the materials necessary for its growth and
nourishment, whilst the remaining elements of these compounds, which
were not taken up by the plant, combined chiefly to form alcohol".
Kützing's memoir, the third of the trio [], also dates from
1837, and his opinions, like those of Cagniard-Latour, are founded on
microscopical observations. He recognises yeast as a vegetable
organism and accurately describes its appearance. Alcoholic fermentation
depends on the formation of yeast, which is produced when the
necessary elements and the proper conditions are present and then
propagates itself. The action on the liquid thus increases and the
constituents not required to form the organism combine to form unorganised
substances, the carbonic acid and alcohol. "It is obvious,"
says Kützing, in a passage which roused the sarcasm of Berzelius,
"that chemists must now strike yeast off the roll of chemical compounds,
since it is not a compound but an organised body, an
organism."
These three papers, which were published almost simultaneously,
were received at first with incredulity. Berzelius, at that time the
arbiter and dictator of the chemical world, reviewed them all in his
"Jahresbericht" for 1839 [] with impartial scorn. The microscopical
evidence was denied all value, and yeast was no more to be
regarded as an organism than was a precipitate of alumina. Schwann's
experiment (p. ) was criticised on the ground that the fermenting
power of the added yeast had been only partially destroyed in the
[p008]
flasks in which fermentation ensued, completely in those which remained
unchanged, the admission of heated or unheated air being
indifferent, a criticism to some extent justified by Schwann's statement,
already quoted, of the uncertain result of the experiment.
Berzelius himself regarded fermentation as being brought about
by the yeast by virtue of that catalytic force, which he had supposed
to intervene in so many reactions, both between substances of mineral
and of animal and vegetable origin [], and which enabled "bodies,
by their mere presence, and not by their affinity, to arouse affinities
ordinarily quiescent at the temperature of the experiment, so that the
elements of a compound body arrange themselves in some different
way, by which a greater degree of electro-chemical neutralisation is
attained".
To the scorn of Berzelius was soon added the sarcasm of Wöhler
and Liebig [, ].
Stimulated in part by the publications of the
three authors already mentioned, and in part by the report of Turpin
[], who at the request of the Academy of Sciences had satisfied
himself by observation of the accuracy of Cagniard-Latour's conclusions,
Wöhler prepared an elaborate skit on the subject, which he
sent to Liebig, to whom it appealed so strongly that he added some
touches of his own and published it in the "Annalen," following immediately
upon a translation of Turpin's paper. Yeast was here described
with a considerable degree of anatomical realism as consisting
of eggs which developed into minute animals, shaped like a distilling
apparatus, by which the sugar was taken in as food and digested into
carbonic acid and alcohol, which were separately excreted, the whole
process being easily followed under the miscroscope.
Close upon this pleasantry followed a serious and important communication
from Liebig [], in which the nature of fermentation,
putrefaction, and decay was exhaustively discussed. Liebig did not admit
that these phenomena were caused by living organisms, nor did he
attribute them like Berzelius to the catalytic action of a substance which
itself survived the reaction unchanged. As regards alcoholic fermentation,
Liebig's chief arguments may be briefly summarised. As the
result of alcoholic fermentation, the whole of the carbon of the sugar
reappears in the alcohol and carbon dioxide formed. This change is
brought about by a body termed the ferment, which is formed as the result
of a change set up by the access of air to plant juices containing sugar,
and which contains all the nitrogen of the nitrogenous constituents of the
juice. This ferment is a substance remarkably susceptible of change,
which undergoes an uninterrupted and progressive metamorphosis, of
[p009]
the nature of putrefaction or decay, and produces the fermentation of
the sugar as a consequence of the transformation which it is itself
undergoing.
The decomposition of the sugar is therefore due to a condition of
instability transferred to it from the unstable and changing ferment, and
only continues so long as the decomposition of the ferment proceeds.
This communication of instability from one substance undergoing
chemical change to another is the basis of Liebig's conception, and is
illustrated by a number of chemical analogies, one of which will suffice
to explain his meaning. Platinum is itself incapable of decomposing
nitric acid and dissolving in it; silver, on the other hand, possesses this
power. When platinum is alloyed with silver, the whole mass dissolves
in nitric acid, the power possessed by the silver being transferred to
the platinum. In like manner the condition of active decomposition
of the ferment is transferred to the sugar, which by itself is quite stable.
The central idea is that of Stahl (p. ) which was thus reintroduced
into scientific thought.
In a pure sugar solution the decomposition of the ferment soon
comes to an end and fermentation then ceases. In beer wort or vegetable
juices, on the other hand, more ferment is continually formed in
the manner already described from the nitrogenous constituents of the
juice, and hence the sugar is completely fermented away and unexhausted
ferment left behind. Liebig's views were reiterated in his
celebrated "Chemische Briefe," and became the generally accepted
doctrine of chemists. There seems little doubt that both Berzelius
and Liebig in their scornful rejection of the results of Cagniard-Latour,
Schwann and Kützing, were influenced, perhaps almost unconsciously,
by a desire to avoid seeing an important chemical change relegated to
the domain of that vital force from beneath the sway of which a large
part of organic chemistry had just been rescued by Wöhler's brilliant
synthetical production of urea and by the less recognised synthesis of
alcohol by Hennell (see on this point Ahrens []). A strong
body of evidence, however, gradually accumulated in favour of the
vegetable nature of yeast, so that it may be said that by 1848 a
powerful minority adhered to the views of Cagniard-Latour, Schwann,
and Kützing [see Schrohe, , p. 218, and compare Buchner,
]. Among these must be included Berzelius
[], who had
so forcibly repudiated the idea only ten years before, whereas Liebig
in the 1851 edition of his letters does not mention the fact that yeast
is a living organism (Letter XV).
The recognition of the vegetable nature of yeast, however, by no
[p010]
means disproved Liebig's view of the nature of the change by which
sugar was converted into carbon dioxide and alcohol, as was carefully
pointed out by Schlossberger [] in a research on the nature of
yeast, carried out in Liebig's laboratory but without decisive results.
Mitscherlich was also convinced of the vegetable character of yeast,
and showed [] that when yeast was placed in a glass tube
closed by parchment and plunged into sugar solution, the sugar
entered the glass tube and was there fermented, but was not fermented
outside the tube. He regarded this as a proof that fermentation only
occurred at the surface of the yeast cells, and explained the process by
contact action in the sense of the catalytic action of Berzelius, rather
than by Liebig's transference of molecular instability. Similar results
were obtained with an animal membrane by Helmholtz [],
who also expressed his conviction that yeast was a vegetable organism.
In 1854 Schröder and von Dusch [,
] strongly reinforced
the evidence in favour of this view by succeeding in preventing
the putrefaction and fermentation of many boiled organic liquids by
the simple process of filtering all air which had access to them through
cotton-wool. These experiments, which were continued until 1861,
led to the conclusion that the spontaneous alcoholic fermentation of
liquids was due to living germs carried by the air, and that when the
air was passed through the cotton-wool these germs were held back.
At the middle of the nineteenth century opinions with regard to
alcoholic fermentation, notwithstanding all that had been done, were
still divided. On the one hand Liebig's theory of fermentation was
widely held and taught. Gerhardt, for example, as late as 1856 in the
article on fermentation in his treatise on organic chemistry [],
gives entire support to Liebig's views, and his treatment of the matter affords
an interesting glimpse of the arguments which were then held to be
decisive. The grounds on which he rejects the conclusions of Schwann
and the other investigators who shared the belief in the vegetable nature
of yeast are that, although in some cases animal and vegetable matter
and infusions can be preserved from change by the methods described
by these authors, in others they cannot, a striking case being that of
milk, which even after being boiled becomes sour even in filtered air,
and this without showing any trace of living organisms. The action
of heat, sulphuric acid, and filtration on the air is to remove, or destroy,
not living organisms but particles of decomposing matter, that is to
say, ferments which would add their activity to that of the oxygen of
the air. Moreover, many ferments, as for example diastase, act without
[p011]
producing any insoluble deposit whatever which can be regarded
as an organism.
"Evidemment," he concludes, "la théorie de M. Liebig explique
seule tous les phénomènes de la manière la plus complète et la plus
logique; c'est à elle que tous les bons esprits ne peuvent manquer de
se rallier."
On the other hand it was held by many to have been shown that
Liebig's view of the origin of yeast by the action of the air on a vegetable
infusion was erroneous, and that fermentation only arose when
the air transferred to the liquid an active agent which could be removed
from it by sulphuric acid (Schulze), by heat (Schwann), and by cotton-wool
(Schröder and von Dusch). Accompanying alcoholic fermentation
there was a development of a living organism, the yeast, and
fermentation was believed, without any very strict proof, to be a phenomenon
due to the life and vegetation of this organism. This doctrine
seems indeed [Schrohe, ] to have been widely taught in Germany
from 1840–56, and to have established itself in the practice of
the fermentation industries.
In 1857 commenced the classical researches of Pasteur which finally
decided the question as to the origin and functions of yeast and led
him to the conclusion that "alcoholic fermentation is an act correlated
with the life and organisation of the yeast cells, not with the death or
putrefaction of the cells, any more than it is a phenomenon of contact,
in which case the transformation of sugar would be accomplished in
presence of the ferment without yielding up to it or taking from it
anything" []. It is impossible here to enter in detail into
Pasteur's experiments on this subject, or indeed to do more than indicate
the general lines of his investigation. His starting-point was the
lactic acid fermentation.
The organism to which this change was due had hitherto escaped
detection, and as we have seen the spontaneous lactic fermentation of
milk was one of the phenomena adduced by Gerhardt (p. ) in favour of
Liebig's views. Pasteur [] discovered the lactic acid producing
organism and convinced himself that it was in fact a living organism
and the active cause of the production of lactic acid. One of the
chief buttresses of Liebig's theory was thus removed, and Pasteur next
proceeded to apply the same method and reasoning to alcoholic fermentation.
Liebig's theory of the origin of yeast by the action of the oxygen
of the air on the nitrogenous matter of the fermentable liquid was conclusively
and strikingly disproved by the brilliant device of producing
a crop of yeast in a liquid medium containing only comparatively
[p012]
simple substances of known composition—sugar, ammonium tartrate and
mineral phosphate. Here there was obviously present in the original
medium no matter which could be put into a state of putrefaction by
contact with oxygen and extend its instability to the sugar. Any such
material must first be formed by the vital processes of the yeast. In
the next place Pasteur showed by careful analyses and estimations that,
whenever fermentation occurred, growth and multiplication of yeast accompanied
the phenomenon. The sugar, he proved, was not completely
decomposed into carbon dioxide and alcohol, as had been assumed by
Liebig (p. ). A balance-sheet of materials and products was constructed
which showed that the alcohol and carbon dioxide formed
amounted only to about 95 per cent. of the invert sugar fermented, the
difference being made up by glycerol, succinic acid, cellulose, and other
substances [, p. 347]. In every case of fermentation, even
when a paste of yeast was added to a solution of pure cane sugar in
water, the yeast was found by quantitative measurements to have taken
something from the sugar. This "something" was indeterminate in
character, but, including the whole of the extractives which had passed
from the yeast cells into the surrounding liquid, it amounted to as much
as 1·63 per cent. of the weight of the sugar fermented [,
p. 344].
Pasteur was therefore led to consider fermentation as a physiological
process accompanying the life of the yeast. His conclusions were
couched in unmistakable words: "The chemical act of fermentation is
essentially a phenomenon correlative with a vital act, commencing and
ceasing with the latter. I am of opinion that alcoholic fermentation
never occurs without simultaneous organisation, development, multiplication
of cells, or the continued life of cells already formed. The
results expressed in this memoir seem to me to be completely opposed
to the opinions of Liebig and Berzelius. If I am asked in what consists
the chemical act whereby the sugar is decomposed and what is
its real cause, I reply that I am completely ignorant of it.
"Ought we to say that the yeast feeds on sugar and excretes alcohol
and carbonic acid? Or should we rather maintain that yeast in its
development produces some substance of the nature of a pepsin, which
acts upon the sugar and then disappears, for no such substance is found
in fermented liquids? I have nothing to reply to these hypotheses.
I neither admit them nor reject them, and wish only to restrain myself
from going beyond the facts. And the facts tell me simply that all
true fermentations are correlative with physiological phenomena."
Liebig felt to the full the weight of Pasteur's criticisms; his reply
[p013]
was long delayed [], and, according to his biographer,
Volhard [], caused him much anxiety. In it he admits the
vegetable nature of yeast, but does not regard Pasteur's conclusion
as in any way a solution of the problem of the nature of alcoholic
fermentation. Pasteur's "physiological act" is for Liebig the very
phenomenon which requires explanation, and which he still maintains
can be explained by his original theory of communicated instability.
On some of Pasteur's results, notably the very important one of the
cultivation of yeast in a synthetic medium, he casts grave doubt,
whilst he explains the production of glycerol and succinic acid as due
to independent reactions. The phenomenon of fermentation is still for
him one which accompanies the decomposition of the constituents
of the cell, rather than their building up by vegetative growth.
"When the fungus ceases to grow, the bond which holds together
the constituents of the cell contents is relaxed, and it is the motion
which is thus set up in them which is the means by which the yeast
cells are enabled to bring about a displacement or decomposition
of the elements of sugar or other organic molecules." Pasteur replied
in a brief and unanswerable note []. All his attention was
concentrated on the one question of the production of yeast in a
synthetic medium, which he recognised as fundamental. The validity
of this experiment he emphatically reaffirmed, and finally undertook,
from materials supplied by Liebig himself, to produce as much yeast as
could be reasonably desired. This challenge was never taken up, and
this communication formed the last word of the controversy. Pasteur
had at this time firmly established his thesis, no fermentation without
life, both for alcoholic fermentation and for those other fermentations
which are produced by bacteria, and had put upon a sound and permanent
basis the conclusions drawn by Schulze, Cagniard-Latour,
Schwann, and Kützing from their early experiments. It became
generally recognised that putrefaction and other fermentative changes
were due to specific organisms, which produced them in the exercise
of their vital functions.
Pasteur subsequently [] came to the conclusion that fermentation
was the result of life without oxygen, the cells being able,
in the absence of free oxygen, to avail themselves of the energy liberated
by the decomposition of substances containing combined oxygen.
This view, which did not involve any alteration of Pasteur's original
thesis but was an attempt to explain the physiological origin and
function of fermentation, gave rise to a prolonged controversy, which
cannot be further discussed in these pages.
[p014]
Nevertheless, Liebig's desire to penetrate more deeply into the
nature of the process of fermentation remained in many minds, and
numerous endeavours were made to obtain further insight into the
problem. In spite of an entire lack of direct experimental proof, the
conception that alcoholic fermentation was due to the chemical action
of some substance elaborated by the cell and not directly to the
vital processes of the cell as a whole found strenuous supporters even
among those who were convinced of the vegetable character of yeast.
As early as 1833 diastase, discovered still earlier by Kirchhoff and
Dubrunfaut, had been extracted by means of water from germinating
barley and precipitated by alcohol as a white powder, the solution of
which was capable of converting starch into sugar, but lost this power
when heated [Payen and Persoz, ]. Basing his ideas in part
upon the behaviour of this substance, Moritz Traube [] enunciated
in the clearest possible manner the theory that all fermentations
produced by living organisms are caused by ferments, which are
definite chemical substances produced in the cells of the organism. He
regarded these substances as being closely related to the proteins and
considered that their function was to transfer the oxygen and hydrogen
of water to different parts of the molecule of the fermentable
substance and thus bring about that apparent intramolecular oxidation
and reduction which is so characteristic of fermentative
change and had arrested the attention of Lavoisier and, long after
him, of Liebig.
Traube's main thesis, that fermentation is caused by definite ferments
or enzymes, attracted much attention, and received fresh support
from the separation of invertase in 1860 from an extract of yeast
by Berthelot, and from the advocacy and authority of this great
countryman of Pasteur, who definitely expressed his opinion that
insoluble ferments existed which could not be separated from the
tissues of the organism, and further, that the organism could not itself
be regarded as the ferment, but only as the producer of the ferment
[, ].
Hoppe-Seyler [] also supported the enzyme
theory of fermentation, but differed in some respects from Traube
as to the exact function of the ferment [see Traube,
; Hoppe-Seyler,
].
Direct experimental evidence was, however, still wanting, and
Pasteur's reiterated assertion [] that all fermentation phenomena
were manifestations of the life of the organism remained uncontroverted
by experience.
Numerous and repeated direct experimental attacks had been made
[p015]
from time to time upon the problem of the existence of a fermentation
enzyme, but all had yielded negative or unreliable results.
As early as 1846 a bold attempt had been made by Lüdersdorff
[] to ascertain whether fermentation was or was not bound up
with the life of the yeast by grinding yeast and examining the ground
mass. A single gram of yeast was thoroughly ground, the process
lasting for an hour, and the product was tested with sugar solution.
Not a single bubble of gas was evolved. A similar result was
obtained in a repetition of the experiment by Schmidt in Liebig's
laboratory [], the grinding being continued in this case for six
hours, but the natural conclusion that living yeast was essential for
fermentation was not accepted, on the ground that during the lengthy
process of trituration in contact with air the yeast had become altered
and now no longer possessed the power of producing alcoholic fermentation,
but instead had acquired that of changing sugar into lactic
acid [see Gerhardt, , p. 545].
Similar experiments made in 1871 by Marie von Manasseïn
[,
], in which yeast was ground for six to fifteen hours with
powdered rock crystal, yielded products which fermented sugar, but
they contained unbroken yeast cells, so that the results obtained could
not be considered decisive [Buchner and Rapp, ], although
Frau von Manasseïn herself drew from them and from others in which
sugar solution was treated with heated yeast, but not under aseptic conditions,
the conclusion that living yeast cells were not necessary for
fermentation.
Quite unsuccessful were also the attempts made to accomplish the
separation of fermentation from the living cell by Adolf Mayer [,
p. 66], and, as we learn from Roux, by Pasteur himself, grinding,
freezing, and plasmolysing the cells, having in his hands proved alike
in vain. Extraction by glycerol or water, a method by which many
enzymes can be obtained in solution, gave no better results [Nägeli
and Loew, ], and the enzyme theory of alcoholic fermentation
appeared quite unjustified by experiment.
Having convinced himself of this, Nägeli [] suggested a
new explanation of the facts based on molecular-physical grounds.
According to this view, which unites in itself some of the conceptions
of Liebig, Pasteur, and Traube, fermentation is the transference of a
state of motion from the molecules, atomic groups, and atoms of the
compounds constituting the living plasma of the cell to the fermentable
material, whereby the equilibrium existing in the molecules of the
latter is disturbed and decomposition ensues [, p. 29].
[p016]
This somewhat complex idea, whilst including, as did Liebig's
theory, Stahl's fundamental conception of a transmission of a state of
motion, satisfies Pasteur's contention that fermentation cannot occur
without life, and at the same time explains the specific action of different
organisms by differences in the constitution of their cell contents.
The really essential part of Nägeli's theory consisted in the limitation
of the power of transference of molecular motion to the living plasma,
by which the failure of all attempts to separate the power of fermentation
from the living cell was explained. This was the special phenomenon
which required explanation; to account for this the theory
was devised, and when this was experimentally disproved, the theory
lost all significance.
For nearly twenty years no further progress was made, and then
in 1897 the question which had aroused so much discussion and conjecture,
and had given rise to so much experimental work, was finally
answered by Eduard Buchner, who succeeded in preparing from yeast
a liquid which, in the complete absence of cells, was capable of effecting
the resolution of sugar into carbon dioxide and alcohol [].
In the light of this discovery the contribution to the truth made
by each of the great protagonists in the prolonged discussion on the
problem of alcoholic fermentation can be discerned with some degree
of clearness. Liebig's main contention that fermentation was essentially
a chemical act was correct, although his explanation of the nature
of this act was inaccurate. Pasteur, in so far as he considered the act
of fermentation as indissolubly connected with the life of the organism,
was shown to be in error, but the function of the organism has only
been restricted by a single stage, the active enzyme of alcoholic fermentation
has so far only been observed as the product of the living
cell. Nearest of all to the truth was Traube, who in 1858 enunciated
the theorem, which was only proved for alcoholic fermentation in
1897, that all fermentations produced by living organisms are due to
ferments secreted by the cells.
Buchner's discovery of zymase has introduced a new experimental
method by means of which the problem of alcoholic fermentation can
be attacked, and the result has been that since 1897 a considerable
amount of information has been gained with regard to the nature and
conditions of action of the enzymes of the yeast cell. It has been
found that the machinery of fermentation is much more complex than
had been surmised. The enzyme zymase, which is essential for fermentation,
cannot of itself bring about the alcoholic fermentation of
sugar, but is dependent on the presence of a second substance, termed, for
[p017]
want of a more reasonable name, the co-enzyme. The chemical nature
and function of this mysterious coadjutor are still unknown, but as it
withstands the temperature of boiling water and is dialysable, it is
probably more simple in constitution than the enzyme. This, however,
is not all; for the decomposition of sugar a phosphate is also indispensable.
It appears that in yeast-juice, and therefore also most probably
in the yeast cell, the phosphorus present takes an active part in
fermentation and goes through a remarkable cycle of changes. The
breakdown of sugar into alcohol and carbon dioxide is accompanied
by the formation of a complex hexosephosphate, and the phosphate
is split off from this compound and thus again rendered available
for action by means of a special enzyme, termed hexosephosphatase.
In addition to this complex of ferments, the cell also possesses special
enzymes by which the zymase and the co-enzyme can be destroyed, and,
further, at least one substance, known as an anti-enzyme, which
directly checks this destructive action. It seems probable, moreover,
that the decomposition of the sugar molecule takes place in stages,
although much doubt yet exists as to the nature of these.
At the present moment the subject remains one of the most
interesting in the whole field of biological chemistry, the limited
degree of insight which has already been gained into the marvellous
complexity of the cell lending additional zest to the attempt to penetrate
the darkness which shrouds the still hidden mysteries.
CHAPTER II.
ZYMASE AND ITS PROPERTIES.
Discovery of Zymase.
[p018]
The history of Buchner's discovery is of great interest [Gruber,
;
Hahn, ]. 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, ,
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, , 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 [,
; ], 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, ], 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, ; Delbrück,
; Martin and Chapman,
; Reynolds Green,
; Lintner,
]. A more accurate knowledge
of the necessary conditions and of the properties of yeast-juice,
however, led to more successful results [Will, ; Reynolds Green,
; Lange, ],
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,
], but Buchner [Buchner and Rapp,
] 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, ;
v. Kupffer, ; v.
Voit, ; Wehmer,
; Neumeister, ; Macfadyen, Morris, and Rowland, ;
Bokorny, ;
Fischer, ;
Beijerinck, ,
; Wroblewski,
, ].
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, ] 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, ;
Geret and Hahn, ,
; ; Buchner, E. and H., and Hahn, ,
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. ) 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 [], 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, , p. 126],
and that some substances which hinder the tryptic action favour
fermentation [Harden, ]. 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, ; Buchner, E. and H., and Hahn, ,
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, ; ].
Preparations (known as zymin) obtained by treating
yeast with a mixture of alcohol and ether [Albert, , ], or with acetone and ether [Albert, Buchner, and Rapp, ], show precisely
similar properties (p. ). 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.
Fig. 1.
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, , 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.
Fig. 2.
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,
].
A modified method of grinding yeast was introduced by Macfadyen,
Morris, and Rowland [], 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, ]. 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 [; ;
see also , , and ]. 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. ) 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. CO2), whereas when macerated at 35° for
2 hours it yielded a normal extract (1·36g. CO2).
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, ] 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
[p026]
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.
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. ).
2. Other Methods.
Attempts to prepare active extracts from undried yeast in an
analogous manner have so far not been very successful. Thus Rinckleben
[] 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. ) [see also Kayser,
].
Giglioli [] 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
[] 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 CO2 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 CO2 per 25 c.c., whilst
[p027]
English yeast-juice prepared by grinding often gives as much as 0·5–0·7g.
of CO2.
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, ]
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.
Fig. 3.
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 [] 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 [],
has been used by Lebedeff [].
An apparatus on a different principle has been designed by Slator
[] for use with living yeast, but is equally applicable to yeast-juice,
and a very similar form has been more recently employed by
Iwanoff []. 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. ).
(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, , 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 [], who worked with much smaller quantities.
The variable results obtained with juice from top yeast by Macfadyen,
Morris and Rowland [], 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 [], and was then confirmed by Buchner [Buchner, E.
and H., and Hahn, , 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 [].
[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
[],
who employed 1·5 normal acid and observed a small
nett loss of sugar. Still more recently Lebedeff
[, , see also ] 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
[]
that glycerol is a constant product of alcoholic fermentation by
yeast-juice (p. ), 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, ] that the deficit
of sugar is not due to the formation of hexosephosphate (p. ), 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 [],
and Euler and Berggren [],
whose interpretation of the observation is discussed later on (p. ).
(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, ; ]. 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,
].
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 [], 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.
RelativeRates.
RelativeTotals.
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, ,
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, ], 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, ], 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. )
is usually, but not invariably [Oppenheimer, ], 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. ), 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, ,
pp. 150–8; Buchner and Rapp, ] 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.
CO2 in grams after
Weight.
Per cent.
6 hours.
24 hours.
96 hours.
2·210
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
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 [], 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, , pp.
158–65] and by Meisenheimer []
for juices from bottom yeast, by Harden and Young [] for those from top
yeast, and by Lebedeff [] 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.
Natureof Juice.
Per cent. of SugarEmployed by Weight.
Volumes ofSugarSolutionAdded.
Volumesof WaterAdded.
TotalFermentationin g. of CO2.
BottomYeast
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
TopYeast
1
0(Auto-ferment-ation)
—
0
0·29
—
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 [].
(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, ;
, ; ; Buchner and Antoni, ; Buchner and
Hoffmann, ; Buchner, E. and H., and Hahn, , pp. 169–205; see
also Albert, ; Gromoff and Grigorieff, ; Duchaček, ]
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 onTotal Fermentation.
Concentrated solution of glycerol
Slight diminution
Concentrated solution of sugar
Slight 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, NaN3,
0·36 per cent.
Slight diminution
0·71 "
Marked "
Quinine hydrochloride
1 "
Slight increase
Ozone
10·4–34·8 mgs. 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, ]. 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, , 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, ], and will be discussed
along with the latter (p. ).
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, ; ;
Buchner, E. and H., and Hahn, , 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, , ; Buchner, E. and H., and Hahn, ,
pp. 228–246; Buchner and Duchaček, ].
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. ) 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, ], 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, ; Albert, Buchner, and Rapp, ]. The
proteoclastic ferment is still present in zymin, which undergoes autolysis
in presence of water in a similar manner to yeast-juice [Albert,
].
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,
], 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 [], and a single experiment of Buchner
shows the influence of the same conditions [Buchner, E. and H., and
Hahn, , 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 Sugar Solution
Total Gas Evolved in
1
2
3
4
22·5hours.
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,
].
CHAPTER III.
THE FUNCTION OF PHOSPHATES IN ALCOHOLIC
FERMENTATION.
[p041]
In the course of some preliminary experiments (commenced by the
late Allan Macfadyen, but subsequently abandoned) on the production
of anti-ferments by the injection of yeast-juice into animals, the
serum of the treated animals was tested for the presence of such antibodies
both for the alcoholic and proteoclastic enzymes of yeast-juice,
and it was then observed that the serum of normal and of treated
animals alike greatly diminished the autolysis of yeast-juice.
As the explanation of the comparatively rapid disappearance of
the fermenting power from yeast-juice had been sought, as already
mentioned (p. ), in the hydrolytic action of the tryptic enzyme
which always accompanies zymase, the experiment was made of
carrying out the fermentation in the presence of serum, with the result
that about 60 to 80 per cent. more sugar was fermented than in the
absence of the serum [Harden, ].
This fact was the starting-point of a series of attempts to obtain a
similar effect by different means, in the course of which a boiled and
filtered solution of autolysed yeast-juice was used, in the hope that the
products formed by the action of the tryptic enzyme on the proteins
of the juice would, in accordance with the general rule, prove to be an
effective inhibitant of that enzyme. This solution was, in fact, found
to produce a very marked increase in the total fermentation effected
by yeast-juice, the addition of a volume of boiled juice equal to that
of the yeast-juice doubling the amount of carbon dioxide evolved
[Harden and Young, ]. This effect was found to be common
to the filtrates from boiled fresh yeast-juice and from boiled autolysed
yeast-juice, and was ultimately traced in the main, not to the antitryptic
effect which had been surmised, but to two independent factors,
either of which was capable in some degree of bringing about the
observed result.
Boiled yeast-juice was indeed found to possess a decided anti-autolytic
effect, as determined by a comparison of the amounts of nitrogen
rendered non-precipitable by tannic acid in yeast-juice alone [p042]
and in a mixture of yeast-juice and boiled juice on preservation
[Harden, ]. The anti-autolytic
effect, however, appeared to vary independently of the effect on the
fermentation, and the conclusion was drawn, as stated above, that the
increase in the alcoholic fermentation was not directly dependent on
the decrease in the action of the proteoclastic enzyme but was due to
some independent cause. The property possessed by boiled yeast-juice of
diminishing the autolysis of yeast-juice has now been carefully examined by
Buchner and Haehn [] and ascribed by them to a soluble antiprotease (p. ).
The two factors to which the increase in fermentation produced
by the addition of boiled juice were ultimately traced were (1)
the presence of phosphates in the liquid, and (2) the existence
in boiled fresh yeast-juice of a co-ferment or co-enzyme, the
presence of which is indispensable for fermentation [Harden and
Young, , ].
The former of these factors will be here discussed and the co-enzyme
will form the subject of the following chapter.
The general fact that sodium phosphate increases the total
fermentation produced by a given volume of yeast juice was observed on
several occasions by Wroblewski []
and also by Buchner [Buchner, E. and H., and Hahn, ,
pp. 141–2], who ascribed the action of this salt to its alkalinity,
comparing it in this respect with potassium carbonate and remarking that
the increase in both cases took place chiefly in the first twenty hours
of fermentation. The increased amount of fermentation following the
addition of boiled yeast-juice was also noted by Buchner and Rapp [, No. 265, p. 2093] in
a single experiment.
Observations made at intervals of a few minutes instead of twenty
hours have, however, revealed the fact that phosphates play a part of
fundamental importance in alcoholic fermentation and that their presence
is absolutely essential for the production of the phenomenon.
Effect of the Addition of Phosphate to a Fermenting Mixture
of Yeast-Juice and Sugar.
When a suitable quantity of a soluble phosphate is added to a
fermenting mixture of glucose, fructose, or mannose with yeast-juice,
the rate of fermentation rapidly rises, sometimes increasing as much
as twenty-fold, continues at this high value for a certain period and
then falls again to a value approximately equal to, but generally
[p043]
somewhat higher than, that which it originally had. Careful experiments
have shown that during this period of enhanced fermentation
the amounts of carbon dioxide and alcohol produced exceed those
which would have been formed in the absence of added phosphate by
a quantity exactly equivalent to the phosphate added in the ratio CO2
or C2H6O:R′2HPO4
[Harden and Young, ].
The effect of an excess of phosphate is discussed later on, p.
.
This result is of fundamental importance, and the evidence on
which it rests deserves some consideration. Quantitative experiments
on this subject require certain preliminary precautions. The acid
phosphates are too acid to permit of any extended fermentation and
the phosphates of the formula R′2HPO4
absorb a considerable volume
of carbon dioxide with production of a bicarbonate, according to the
reaction:—
R2HPO4 + H2CO3 ⇌ RHCO3 + RH2PO4.
The method which has been adopted, therefore, is to employ either
a secondary phosphate saturated with carbon dioxide at the temperature
of the experiment, or a mixture of five molecular proportions of
the secondary phosphate with one molecular proportion of a primary
phosphate, in which the amount of bicarbonate formed is negligible.
In the former case it is necessary to ascertain whether any of the
carbon dioxide evolved is derived from the bicarbonate by the action
of acid originally present or produced in the yeast-juice or by a disturbance
of the original equilibrium owing to the chemical change
which occurs. This is done by acidifying duplicate samples with
hydrochloric acid before and after the fermentation and measuring the
gas evolved in each case. Any necessary correction can then be made.
The calculation of the extra amount of carbon dioxide evolved from
yeast-juice containing sugar when a phosphate is added involves an
estimation of the amount which would have been evolved in the
absence of added phosphate, and this is a matter of some difficulty.
Since the final steady rate of fermentation attained is often slightly
different from the initial rate, the practice has been adopted of ascertaining
this final rate and then calculating the total evolution corresponding
to it for the whole period from the time of the addition of
the phosphate to the end of the observations. This amount deducted
from the observed total leaves the extra amount of carbon dioxide
formed, and it is this quantity which is equivalent to the phosphate
added. Alcohol is simultaneously produced in the normal ratio.
The justification for this method of calculation will be found later
(p. ).
The following table, containing the results of experiments with
[p044]
glucose, fructose, and mannose, indicates very clearly the nature of the
method of calculation and also of the agreement between observation
and theory.
Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution containing
1 gram of the sugar to be examined (a large excess) were incubated
with toluene at 25° for one hour, in order to remove all free
phosphate, and to each were then added 5 c.c. of a solution of sodium
phosphate corresponding to 0·1632 gram of Mg2P2O7 and equivalent
to 32·6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were
then observed until they had passed through the period of acceleration
and had fallen and attained a steady value, the gases being measured
moist at 19·3° and 760·15 mm.
Glucose.
Mannose.
Fructose.
Maximum rate attained, c.cs. per five minutes
9·6
7 11·3
Final rate of fermentation
1·1
0·96
1·08
Total carbon dioxide produced by fermentation in fifty-five minutes after addition of phosphate
49·7
47·847·6
Correction for evolution in absence of phosphate in fifty-five minutes
12·1
10·611·9
Extra carbon dioxide equivalent to phosphate
37·6
37·235·7
Extra carbon dioxide equivalent to phosphate at N.T.P.
34·4
34 32·6
These numbers agree well with the value calculated from the phosphate
added, viz. 32·6 [Harden and Young, ].
Another experiment is illustrated graphically in Fig. 4, in which
the volume of carbon dioxide evolved is plotted against time. The
determination was in this case made by adding 25 c.c. of an aqueous
solution containing 5 grams of glucose to one quantity of 25 c.c. of
yeast-juice (curve A) and 5 c.c. of 0·3 molar solution of the mixed primary
and secondary sodium phosphates, and 20 c.c. of a solution containing
5 grams of glucose to a second equal quantity of yeast-juice (curve B).
Curve A shows the normal course of fermentation of yeast-juice with
glucose. There is a slight preliminary acceleration during the first
twenty minutes, due to free phosphate in the juice, and the rate then
becomes steady at about 1·4 c.c. in five minutes. During this preliminary
acceleration 10 c.c. of extra carbon dioxide are evolved, this number
being obtained graphically by continuing the line of steady rate back
to the axis of zero time. Curve B shows the effect of the added phosphate.
The rate rises to about 9·5 c.c. in five minutes, i.e. to more
than six times the normal rate, and then gradually falls until after an
hour it is again steady and almost exactly equal to 1·4 c.c. per five
minutes. Continuing the line of steady rate back to the axis of zero
[p045]
time it is found that the extra amount of carbon dioxide is 48 c.c.
Subtracting from this the 10 c.c. shown in curve A as due to the juice
alone, a difference of 38 c.c. is obtained due to the added phosphate.
The amount calculated from the phosphate added in this case is, at
atmospheric temperature and pressure, 38·9 c.c.
Fig. 4.
After the expiration of seventy minutes from the commencement
of the experiment, a second addition is made of an equal amount of
phosphate. The whole phenomenon then recurs, as shown in curve C,
the maximum rate being slightly lower than before, about 6 c.c. per five
minutes, and the rate again becoming finally steady at 1·4 c.c. as before.
The extra amount of carbon dioxide evolved in this second period
obtained graphically as in the former case, is 107–68 = 39 c.c.
It may be noted that in this case the observations after each addition
last fifty to seventy minutes, so that an error of 0·1 c.c. per five
minutes in the estimated final rate would make an error of 1 to 1·4 c.c.
in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent. of
the total, and this is approximately the limit of accuracy of the method.
[p046]
The results are more precise when the yeast-juice employed is an active
one, since, when the fermenting power of the juice is low, the initial
period of accelerated fermentation is unduly prolonged and the calculation
of the extra amount of carbon dioxide is rendered uncertain.
Zymin (p. ) yields precisely similar results to yeast-juice, but in
this case the rate of fermentation is not so largely increased. This has
the effect that the extra amount of carbon dioxide cannot be quite so
accurately estimated for zymin, because a slight error in the determination
of the final rate of fermentation has a greater influence on the result.
The equivalence between the extra amount of carbon dioxide evolved
and the phosphate added is, however, unmistakable, as is shown by the
following results of an experiment with zymin, in which 6 grams of
zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water
were incubated at 25° in presence of toluene until a steady rate had been
attained. Five c.c. of a solution of sodium phosphate equivalent to
32·2 c.c. carbon dioxide at N.T.P. were then added.
Maximum rate attained, c.c. per five minutes
14·1
Final rate of fermentation
6·2
Total evolved by fermentation in eighty minutes after addition of phosphate
131
Correction for evolution in absence of phosphate in eighty minutes
99·2
Extra carbon dioxide at 16° and 767·1 mm
31·8
Extra carbon dioxide at N.T.P
29·8
Considering the small proportional rise in rate and the long period of
accelerated fermentation, the agreement between the volume observed,
29·8 c.c., and that calculated from the phosphate, 32·2, is quite satisfactory
[Harden and Young, .] Precisely the same relations
hold for maceration extract, but in this case it must be remembered that
a large amount of free phosphate is present in the extract, as
much as 0·3129 grm. Mg2P2O7 being obtained from 20 c.c. in one preparation,
so that the original extract had the concentration of a 0·14
molar solution of sodium phosphate. It is in fact not improbable
that the delay in the onset of fermentation sometimes observed with
maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal,
1913) may be due to the presence of phosphate in so great an excess of the
amount which can be rapidly esterified by the enzymes that the rate of
fermentation is at first greatly lowered (see p. ). When this phosphate
is removed by incubation with glucose or fructose, the subsequent addition
of phosphate produces the characteristic action and the extra
carbon dioxide evolved is, as with other yeast preparations, equivalent
to the phosphate added. An actual estimation carried out in this way
gave 35 c.c. of CO2 for an addition of phosphate equivalent to 32·9
c.c. [Harden and Young, ].
[p047]
Within the limits imposed by the experimental conditions, then, the
fact is well established that the addition of a soluble phosphate to a
fermenting mixture of a hexose with yeast-juice, maceration extract,
dried yeast, or zymin causes the production of an equivalent amount
of carbon dioxide and alcohol.
This fact indicates that a definite chemical reaction occurs in which
sugar and phosphate are concerned, and this conclusion is confirmed
when the fate of the added phosphate is investigated. If an experiment,
such as one of those described above, be interrupted as soon as the
rate of fermentation has again become normal, and the liquid be boiled
and filtered, it is found that nearly the whole of the phosphorus present
passes into the filtrate, but that only a small proportion of this exists
as mineral phosphate, whilst the remainder, including that added in
the form of a soluble phosphate, is no longer precipitable by magnesium
citrate mixture [Harden and Young, ].
A similar observation was made at a later date by Iwanoff [],
who had previously observed [] that living yeast, like many
other vegetable organisms, converted mineral phosphates into organic
derivatives. Iwanoff employed zymin and hefanol (p. ) instead of
yeast-juice, and found that phosphates were thereby rendered non-precipitable
by uranium acetate solution, but did not observe the
accelerated fermentation caused by their addition.
The foregoing conclusions have been strikingly confirmed by experiments
with maceration extract carried out by Euler and Johansson
[], in which both the carbon dioxide evolved and the phosphate
rendered non-precipitable by magnesia were determined at intervals.
When dried yeast is employed as the fermenting agent, the amount
of phosphate esterified in the earlier stages is greater than would be
expected, but ultimately becomes exactly equivalent to the carbon
dioxide evolved.
Nature of the Phospho-organic Compound formed by Yeast-Juice
and Zymin from the Hexoses and Phosphate.
The formation and properties of the compound produced from phosphates
in the manner just described have been investigated by
Harden and Young
[;
;
;
],
Young
[;
],
Iwanoff
[;
],
Lebedeff
[;
;
, ;
;
];
and Euler
[;
Euler and Fodor, ;
Euler and Kullberg, ;
Euler and Ohlsén, ;
;
Euler and Johansson, ;
Euler and Bäckström, ], but its exact constitution cannot
as yet be regarded as definitely known.
[p048]
Phosphates undergo this characteristic change when the sugar
undergoing fermentation is glucose, mannose, or fructose, and it may
be said at once that no distinction can be established between the
products formed from these various hexoses; they all appear to be
identical. The compound produced is, as already mentioned, not precipitated
by ammoniacal magnesium citrate mixture, nor by uranium
acetate solution. It can, however, be precipitated by copper acetate
(Iwanoff) and by lead acetate (Young). The preparation of the pure
lead salt from the liquid obtained by fermenting a sugar with yeast-juice
or zymin in presence of phosphate is commenced by boiling and
filtering the liquid. Magnesium nitrate solution and a small quantity
of caustic soda solution are then added to precipitate any free phosphate,
and the liquid well stirred and allowed to stand over night. To
the neutralised filtrate lead acetate is then added together with sufficient
caustic soda solution to maintain the reaction neutral to litmus, until
no further precipitate is formed. The liquid is then filtered or, better,
centrifugalised, and the precipitate repeatedly washed with water until
a portion of the clear filtrate gives no reduction when boiled with Fehling's
solution. It is essential that this washing should be thorough as
evidence has recently been obtained of the formation under certain conditions
of a hexosephosphate, the lead salt of which is not so sparingly
soluble as that of the hexosediphosphate [Harden and Robison, ].
The lead precipitate is then suspended in water, decomposed by a current
of sulphuretted hydrogen, the clear filtrate freed from sulphuretted
hydrogen by a current of air, and finally neutralised with caustic soda.
The removal of phosphate and conversion into lead salt are repeated
twice, and the resulting lead salt is then found to be free from nitrogen
and to have a composition represented by the formula
C6H10O4(PO4Pb)2.
Lebedeff carries out the preparation in a somewhat different manner.
The fermentation is effected by means of air-dried yeast (150 grams
to 1 litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2
parts Na2HPO4 and 1 part NaH2PO4) and the liquid (about 700 c.c.)
after boiling and filtering, is treated with an equal volume of acetone.
About 300 c.c. of a thick liquid is precipitated and this is redissolved in
water and precipitated by an equal volume of acetone two or three times.
The final liquid is then precipitated with warm lead acetate solution
and filtered and washed with dilute lead acetate solution until the filtrate
is clear and no longer reduces Fehling's solution after removal
of the lead []. Euler and Fodor [] on the other hand
precipitate the free phosphate with magnesia mixture and then add
acetone, dissolve the syrup thus precipitated in water and add copper
[p049]
acetate solution. A blue copper salt is precipitated which is thoroughly
washed with water and used for the preparation of solutions of the
acid. A solution of the free acid can readily be prepared by the action
of sulphuretted hydrogen on the lead salt suspended in water. It
forms a strongly acid liquid, which requires exactly two equivalents
of base for each atom of phosphorus present to render it neutral to
phenolphthalein. It decomposes when evaporated, leaving a charred
mass containing free phosphoric acid. The acid is slightly optically
active, and has [aD] = + 3·4°. A number of amorphous salts have been
prepared by precipitation from a solution of the sodium salt, and of these
the silver, barium, and calcium salts have been analysed with results
agreeing with the general formula
C6H10O4(PO4R′2)2.
The magnesium,
calcium, barium, and manganese salts, which are only sparingly soluble,
are all precipitated when their solutions are boiled but re-dissolve on
cooling, and this property can be utilised for their purification. The
alkali salts have only been obtained as viscid residues.
A difference of opinion exists as to the molecular weight and
constitution of this substance. Iwanoff [] regards it as a
triosephosphoric acid,
C3H5O2(PO4H2),
basing this view on the preparation
of an osazone which melted at 142°, but when recrystallised
from benzene gave a product melting at 127°–8°, which had the same
appearance, melting-point, and nitrogen content as the triosazone
formed by the action of phenylhydrazine on the oxidation products of
glycerol. Neither Lebedeff [] nor Young could obtain Iwanoff's
osazone, and all attempts to reduce the acid with formation of
glycerol either by sodium amalgam or hydriodic acid were unsuccessful
(Young). There is therefore practically no serious experimental
evidence in favour of Iwanoff's view.
On the other hand, Harden and Young regard the acid as a diphosphoric
ester of a hexose. This view is based on the fact that when the
acid is boiled with water, or an acid, free phosphoric acid is produced
along with a levo-rotatory solution containing fructose and possibly a
small proportion of some other sugar or sugars. (Euler and Fodor
however did not obtain a hexose in this way [].) The acid itself
only reduces Fehling's solution after some hours in the cold, rapidly
when boiled, whereas when its solution is first boiled, and then treated
with Fehling's solution in the cold, the products of decomposition bring
about reduction in a few minutes. The reduction brought about when
the acid is boiled with Fehling's solution is considerably less (33 per cent.)
than that produced by an equivalent amount of glucose. The behaviour
of the compound towards phenylhydrazine is also in complete agreement
[p050]
with this view. Lebedeff found [, ] that the acid or its
salts heated with phenylhydrazine in presence of acetic acid gave an
insoluble compound which was ultimately found to be the phenylhydrazine
salt of hexosemonophosphoric acid osazone
C6H5NH·NH2·H2PO4·C4H5(OH)3·C(N2HC6H5)CH(N2HC6H5)
[Lebedeff, ;
; Young, ]. After recrystallisation from alcohol this
compound forms yellow needles, melting at 151°–152°. It is decomposed by
caustic soda yielding a sodium salt
Na2PO4·C4H5(OH)3·(CN2HC6H5)·CH(N2HC6H5)
and on boiling with caustic soda decomposes giving a
hexosazone (free from phosphorus) which is probably glucosazone, and
in addition glyoxalosazone, probably as the result of a secondary
decomposition. Towards acids it is remarkably stable yielding with
hydrochloric acid a hexosonephosphoric ester from which the
original osazone can be regenerated (Lebedeff). Lebedeff at first [] argued from the formation of this
osazone that the original hexosephosphate contained only one phosphoric
acid group per molecule of hexose. It was however shown by Young [] and subsequently confirmed by Lebedeff
[] that one molecule of
phosphoric acid is split off during the formation of the osazone,
even in neutral solution. Moreover it has been found that in the cold
hexosediphosphoric acid reacts with 3 molecules of phenylhydrazine forming
the diphenylhydrazine salt of hexosediphosphoric acid phenylhydrazone
(C6H5NH·NH2·H2PO4)2·C6H7(OH)3·N2HC6H5.
This compound crystallises out when 1 volume of alcohol is added
to a solution of 3 molecules of phenylhydrazine in one of the acid and
forms colourless needles melting at 115°–117°. p-Bromophenylhydrazine
yields an analogous compound melting at 127°–128°.
Precisely the same products are given with phenylhydrazine by the
hexosephosphoric acid prepared from glucose, mannose, and fructose,
proving that all these sugars yield the same hexosediphosphoric acid,
a point of fundamental importance.
Direct measurements of the molecular weight of the acid by the
freezing-point method, combined with the determination of the degree
of dissociation by the rate of cane-sugar inversion, are indecisive, but
indicate that the acid has a molecular weight considerably higher than
that required for a triosephosphoric acid.
A similar uncertainty attaches to the determination of the molecular
weight from the freezing-point depression and conductivity of the acid
potassium salt [Euler and Fodor, ]. Euler however concludes
[p051]
that both a hexosediphosphoric acid and a triosemonophosphoric acid
are formed, but has not prepared any derivatives of the latter.
As regards the constitution of the hexosephosphoric ester several
suggestions have been made by Young, but no decisive evidence at
present exists. The identity of the products from glucose, mannose,
and fructose may be explained by regarding the acid as a derivative
of the enolic form common to these three sugars (p. ), or by supposing
that portions of two sugar molecules may be concerned in its
production. The formation and composition of the hydrazone and
osazone are of great importance as they indicate that in all probability
one of the phosphoric acid residues is united with the carbon atom
adjacent to the carbonyl group of the hexose. They moreover render
it certain that the original phosphoric ester is a hexosediphosphoric
ester and not a triosemonophosphoric ester.
Hexosediphosphoric acid has not as yet been discovered in the
animal body. The action of a number of enzymes upon it has been
examined [Euler, ;
Euler and Funke, ;
Harding, ;
Plimmer, ] with the following results.
The lipase of castor oil seeds, a glycerol extract of the intestinal
mucous membrane of the rabbit and pig, and an aqueous extract
of bran have a slow hydrolytic action, whereas pepsin and trypsin
are without effect. Feeding experiments with rabbits and dogs
indicate that the ester is capable of hydrolysis in the animal body,
a large proportion of the phosphorus being excreted as inorganic
phosphate. The ester is also decomposed by Bacillus coli communis.
It is remarkable that the hexosephosphate is not fermented nor
hydrolysed by living yeast, a fact observed by Iwanoff, Harden and
Young, and Euler, although, according to the experiments of Paine
[], the yeast cell is at all events partially permeable to the sodium
salt.
The Equation of Alcoholic Fermentation.
An equation can readily be constructed for the reaction in which
hexosephosphate is formed, the data available being the formula of the
product and the relation between the phosphate added and the
carbon dioxide and alcohol produced:—
(1) 2 C6H2O6 + 2 PO4HR3 =
2 CO2 + 2 C2H6O + 2 H2O + C6H10O4(PO4R2)2.
According to this, two molecules of sugar are concerned in the
change, the carbon dioxide and alcohol being equal in weight to one
[p052]
half of the sugar used, and the hexosephosphate and water representing
the other half.
Additional confirmation of this equation is afforded by the determination
of the ratio between sugar used and carbon dioxide evolved
when a known weight of sugar together with an excess of phosphate
is added to yeast-juice at 25°. The phenomena then observed are
precisely similar to those which occur when a phosphate is added to
a fermenting mixture of yeast-juice and excess of sugar as described
above. The rate of fermentation rapidly rises and then gradually
falls until a rate is attained approximately equal to that of the autofermentation
of the juice in presence of phosphate. At this point it is
found that the extra amount of carbon dioxide evolved, beyond that
which would have been given off in the absence of added sugar, bears
the ratio expressed in equation (1) to the sugar added [Harden and Young,
].
The results of four estimations made in this way
were (a) 0·2 grams of glucose gave 26·5 and 27·9 c.c. of carbon dioxide
at N.T.P.; (b) 0·2 grams of fructose gave 27·9 and 28·9 c.c. The
carbon dioxide calculated from the sugar added in each of the four
cases is 26·96 c.c.
It has also been shown by Euler and Johansson [] that in
the fermentation of a mixture of equivalent amounts of phosphate and
glucose, the whole of the glucose had disappeared when the whole of
the phosphate had become esterified.
Cycle of Changes Undergone by Phosphate in Alcoholic
Fermentation.
According to equation (1) the free phosphate present is used up
in the reaction, and the question at once arises whether there is any
source from which a constant supply of free phosphate can be elaborated
in the juice, or whether some other change occurs which results in the
formation of carbon dioxide and alcohol in the absence of free phosphate.
The experimental evidence points in the direction of the
former of these alternatives, but the question is a very difficult one to
decide with absolute certainty.
When a mixture of a phosphate with yeast-juice and sugar is
examined at intervals and the amount of free phosphate estimated,
the following stages are observed:—
1. During the initial period of accelerated fermentation following
the addition of the phosphate, the concentration of free phosphate
rapidly diminishes.
2. At the close of this period, the amount of free phosphate
[p053]
present is very low, and, as long as active fermentation continues, no
marked increase in it occurs.
3. As alcoholic fermentation slackens and finally ceases, a marked
and rapid rise in the amount of free phosphate occurs at the expense
of the hexosephosphate, which steadily diminishes in amount, and
this change is brought about by an enzyme in the juice and ceases if
the liquid be boiled.
This last reaction may be represented by the equation
(2) C6H10O4(PO4R2)2 + 2 H2O = C6H12O6 + 2 PO4HR2.
In the light of this equation, together with equation No. 1, given above,
all these facts can be simply and easily understood.
The rapid diminution in the amount of free phosphate during
stage 1 corresponds with the occurrence of reaction (1). During the
whole period of fermentation the enzymic hydrolysis of the hexosephosphate
is proceeding according to equation (2). Up to the end of
stage 2 the phosphate thus produced enters into reaction, according
to equation (1), with the sugar which is present in excess and is thus
reconverted into hexosephosphate, so that as long as alcoholic fermentation
is proceeding freely, no accumulation of free phosphate can
occur.
As soon as alcoholic fermentation ceases, however, it is no longer
possible for the phosphate to pass back into hexosephosphate, and
hence it accumulates in the free state.
A similar hydrolysis of hexosephosphate and accumulation of
phosphate occur when a solution of hexosephosphate is treated with
yeast-juice which has been deprived of the power of fermentation by
dialysis, or with zymin freed from co-enzyme by washing (p. ).
The actual rate of fermentation observed in any particular case in
presence of excess of sugar, enzyme, and co-enzyme must on this view
depend on the supply of phosphate which is available.
In presence of an adequate amount of phosphate, as well as of
sugar, the highest rate attained represents the maximum velocity at
which reaction (1) can proceed in that sample of yeast-juice or zymin,
and this high rate is characteristic of the initial period of accelerated
fermentation which follows the addition of a suitable quantity of phosphate.
By the simple expedient of renewing the supply of phosphate
as rapidly as it is converted into hexosephosphate, this high rate can
be maintained for a considerable time [Harden and Young, ].
In this way, for example, an average rate of evolution of carbon
dioxide of 15 c.c. in five minutes was maintained for an hour and a
[p054]
quarter, whereas the normal rate in the absence of added phosphate
was 3 c.c.
As soon as all the free phosphate has entered into the reaction,
however, the supply of phosphate depends in the main on the rate at
which the resulting hexosephosphate is decomposed, and the rate of
fermentation now attained is conditioned by the rate at which reaction
(2) proceeds, and this evidently depends on the existing concentration
of the hydrolytic enzyme, which may be provisionally termed
hexosephosphatase.
The rates attained during the initial period of rapid fermentation
and the subsequent period of slow fermentation are thus seen to
represent the velocities of two entirely different chemical reactions.
These considerations also explain why it is the extra carbon
dioxide evolved during the initial period, and not the total carbon
dioxide, which is equivalent to the added phosphate. As the production
of phosphate is proceeding throughout the whole period at
a rate which is equivalent to the normal rate of fermentation, it is
obviously necessary to deduct the corresponding amount of carbon
dioxide from the total evolved in order to ascertain the amount equivalent
to the added phosphate.
An explanation is also afforded of the fact that a considerable
increase in the concentration of hexosephosphate does not materially
increase the normal rate of fermentation. This is probably due to the
circumstance that, in accordance with the general behaviour of enzymes
in presence of excess of the fermentable substance, the hexosephosphatase
hydrolyses approximately equal amounts of hexosephosphate
in equal times whatever the concentration of the latter may be, above a
certain limit.
According to the experiments of Euler and Johansson [] the
hydrolytic activity of the hexosephosphatase is greatly diminished by
the presence of toluene.
Effect of Phosphate on the Total Fermentation Produced by
Yeast-Juice.
The addition of a phosphate to yeast-juice not only produces the
effect already described, but also enables a given volume of yeast-juice
to effect a larger total fermentation, even after allowance is made for
the carbon dioxide equivalent to the quantity of phosphate added.
The increase in the case of ordinary yeast-juice has been found to
amount to from 10 to 150 per cent. of the original total fermentation
[p055]
produced by the juice in the absence of added phosphate. The numbers
contained in columns 1 and 2 of the table on p. illustrate this
effect, the ratio of the total in the presence of phosphate to that
obtained in its absence being given, as well as that of the total in presence
of phosphate less the equivalent of the phosphate added, to the
original fermentation. The cause of this increase in the total fermentation
is probably to be sought mainly in a protective action of the excess
of hexosephosphate on the various enzymes, for, as has been
stated above, the rate of fermentation after the termination of the
initial period, is practically the same as in the absence of added
phosphate (see p. ).
Now it follows from equation (1) (p. ) that in the total absence
of phosphate no fermentation should occur, and the experimental
realisation of this result would afford very strong evidence in favour
of this interpretation of the phenomenon.
Hitherto, however, it has not been found possible to free the
materials employed completely from phosphorus compounds which
yield phosphates by enzymic hydrolysis during the experiment, but it
has been found that when the phosphate contents are reduced to as low
a limit as possible, the amount of sugar fermented becomes correspondingly
small, and, further, that in these circumstances the addition of a
small amount of phosphate or hexosephosphate produces a relatively
large increase in the fermenting power of the enzyme.
When the total phosphorus present is thus largely reduced, the increase
produced by the addition of a small amount of phosphate may
amount to as much as eighty-eight times the original, in addition to the
quantity equivalent to the phosphate, whilst the actual total evolved,
including this equivalent, may be as much as twenty times the original
fermentation. This result must be regarded as strong evidence in
favour of the view that phosphates are indispensable for alcoholic fermentation.
The results indicated above were experimentally obtained in three
different ways and are exhibited in the following table. In the first
place (cols. 3 and 4), advantage was taken of the fact that the residues
obtained by filtering yeast-juice through a Martin gelatin filter (p. )
are sometimes found to be almost free from mineral phosphates, whilst
they still contain a small amount of co-enzyme. The experiment then
consists in comparing the fermentation produced by such a residue poor
in phosphate with that observed when a small amount of phosphate is
added. The second method (col. 5) consisted in carrying out two
parallel fermentations by means of a residue rendered inactive by filtration
[p056]
and a solution of co-enzyme free from phosphate and hexosephosphate
(p. ) [Harden and Young, ].
The third method (col. 6) consisted in washing zymin with water,
to remove soluble phosphates, and then adding to it a solution of co-enzyme
containing only a small amount of phosphate, and ascertaining
the effect of the addition of a small known amount of hexosephosphate
upon the fermentation produced by this mixture [Harden and Young,
].
1c.c.
2c.c.
3c.c.
4c.c.
5c.c
6c.c.
Gas evolved in absence of added phosphate
369 220 1·4
1·2
20·3
1·5
In the presence of added phosphate
629 629 25·8
26·8
92·3
132·7
Increase due to phosphate
260 409 24·4
25·6
72·0
131·2
Carbonic acid equivalent to phosphate
63 61 16·9
16·8
16·8
—
Increase after initial period
197 348 7·5
8·8
55·2
—
Ratio of totals
1·7
2·9
18·4
21·3
4·5
88
Ratio of increase after initial period to original fermentation
0·5
1·6
5·3
7·3
2·7
—
Production of a Fermentable Sugar from Hexosephosphate by
the Action of an Enzyme Contained in Yeast-Juice.
The sugar which, according to equation (2) accompanies the phosphate
formed by the enzymic hydrolysis of hexosephosphate is under ordinary
circumstances fermented by the alcoholic enzyme of the juice
and thus escapes detection.
When, however, a solution of a hexosephosphate is exposed to the
action of either yeast-juice or zymin, entirely or partially freed from co-enzyme,
this sugar, being no longer fermented, accumulates and can
be examined. It has thus been found [Harden and Young, ]
that a sugar is in fact produced in this way which can be fermented by
living yeast and exhibits the reactions of fructose, although the presence
of other hexoses is not excluded. The products of the enzymic hydrolysis
of the hexosephosphates therefore appear to be the same as, or
similar to, those formed by the action of acids [Young, ].
A further consequence of these facts is that a hexosephosphate will
yield carbon dioxide and alcohol when it is added to yeast-juice or
zymin, and this has also been found to be the case [Harden and Young,
; Iwanoff, ].
[p057]
Mechanism of the Formation of Hexosediphosphoric Acid.
On this subject little is yet known, but a number of extremely
interesting results, the interpretation of which is still doubtful, have
been obtained by Euler and his colleagues. Euler has obtained a
yeast [Yeast H of the St. Erik's brewery in Stockholm] which differs
from Munich yeast in several respects. A maceration extract prepared
from the yeast dried at 40° in a vacuum produces no effect on a glucose
solution containing phosphate. If, however, the glucose solution be
previously partially fermented with living yeast and then boiled and
filtered, the addition of the extract prepared from Yeast H brings
about the esterification of phosphoric acid without any accompanying
evolution of carbon dioxide [Euler and Ohlsén, , ].
Euler interprets this as follows: (a) Glucose itself is not directly
esterified, but must first undergo some preliminary change, which is
brought about by the action of living yeast. No proof of the existence
of a new modification of glucose in this solution has however been advanced,
other than its behaviour to extract of Yeast H, so that Euler's
conclusion cannot be unreservedly accepted. It is moreover possible
and even more probable that some thermostable catalytic substance
(perhaps a co-enzyme) passes from the yeast into the glucose solution
and enables the yeast extract to attack the glucose and phosphoric
acid. A very small degree of esterification was also produced when
an extract having no action on glucose and phosphate was added to
glucose which had been treated with 2 per cent. caustic soda for forty
hours, but the nature of the compound formed was not ascertained
[Euler and Johansson, ]. (b) The esterification of phosphoric
acid without the evolution of carbon dioxide implies that the enzyme
by which this process is effected is distinct from that which causes the
actual decomposition of the sugar. Euler goes further than this and
regards the enzyme as a purely synthetic one, giving it the name of
hexosephosphatese to distinguish it from the hexosephosphatase which
hydrolyses the hexosephosphate.
The evidence on which this conclusion is based cannot be regarded
as satisfactory, inasmuch as it consists in the observation that in presence
of sugar yeast extract does not hydrolyse the phosphoric ester. This,
however, could not be expected since hydrolysis and synthesis under
these conditions would ultimately proceed at equal rates.
In any case the adoption of this nomenclature is inconsistent with
the conception of an enzyme as a catalyst and is therefore inadvisable
until the reaction has been much more thoroughly studied.
[p058]
It may further be pointed out that no proof has yet been advanced
that the phosphoric ester produced without evolution of carbon
dioxide is identical with hexosediphosphoric acid produced with
evolution of carbon dioxide. It is by no means improbable that it
represents some intermediate stage in the production of the latter
(see p. ).
Euler's other results on this subject may be briefly summarised as
follows:—
(1) In presence of excess of sugar the esterification of the phosphoric
acid proceeds by a monomolecular reaction and is most rapid
in faintly alkaline reaction [Euler and Kullberg, ].
(2) When yeast extract has been heated for 30 minutes to 40° it
effects the esterification of phosphoric acid at a much greater rate than
the unheated extract (2–10 times). Heating at 50° for 30 minutes
however completely inactivates the extract. The cause of the activation
is as yet unknown. The temperature coefficient for the unheated
extract (17·5°–30°) is 1·4–1·5 for 10° rise of temperature [Euler and
Ohlsén, ].
(3) Yeasts which in the dried state all produce rapid esterification
of phosphoric acid, yield extracts of very unequal powers in this
respect [Euler, ].
CHAPTER IV.
THE CO-ENZYME OF YEAST-JUICE.
[p059]
In the previous chapter reference was made to the fact that the addition
of boiled yeast-juice greatly increases the amounts of carbon
dioxide and alcohol formed from sugar by the action of a given volume
of yeast-juice.
Fig. 5.
When the boiled juice is dialysed the substance or substances to
which this effect is due pass into the dialysate, the residue being quite
inactive. In order to ascertain the effect on the process of alcoholic
fermentation of the complete removal of these unknown substances
from yeast-juice itself, dialysis experiments were instituted with fresh
yeast-juice, capable of bringing about an active production of carbon
dioxide and alcohol from sugar. It was already known from the experiments
of Buchner and Rapp [] that dialysis in parchment
paper for seventeen hours at 0° against water or physiological salt
solution only produced a diminution of about 20 per cent. in the
total amount of fermentation obtainable, and in view of the less permanent
character of the juice from top yeasts a more rapid method
of dialysis was sought. This was found in the process of filtration
under pressure through a film of gelatin, supported in the pores of a
Chamberland filter candle, which had been introduced by Martin
[].
In this way it was found possible to divide the juice into a residue
and a filtrate, each of which was itself incapable of setting up the
alcoholic fermentation of glucose, whereas, when they were reunited,
the mixture produced almost as active a fermentation as the original
juice [Harden and Young, ;
].
The apparatus employed for this purpose consists of a brass tube
provided with a *** in which the gelatinised candle is held by a
compressed india-rubber ring, and is shown in section in Fig. 5. Two
such apparatus are used, each capable of holding about 70 c.c. of the
liquid to be filtered. The tubes, after being filled with the yeast-juice,
are connected by means of a screw joint with a cylinder of compressed
air and the filtration carried out under a pressure of 50 atmospheres,
[p060]
the arrangement employed being shown in Fig. 6. In the earlier experiments
25 to 50 c.c. of yeast-juice were placed in
each tube and the filtration continued until no more
liquid passed through. The residue was then washed
several times in situ by adding water and forcing it
through the candle. The time occupied in this process
varied from six to twelve hours with different preparations
of yeast-juice. The candle was then removed
from the brass casing and the thick, brown-coloured
residue scraped off, dissolved in water, and at once
examined. It was subsequently found to be possible
to dry this residue in vacuo over sulphuric acid without
seriously altering the fermenting power, and this
led to a slight modification of the method, which is
now conducted as follows. Two quantities of 50 c.c.
each of yeast-juice are filtered, without washing, and
the residues spread on watch-glasses and dried in
vacuo. Two fresh quantities of 50 c.c. are then
filtered through the same candles and the residues
also dried. The 200 c.c. of juice treated in this way
give a dry residue of 17 to 24 grams. The residue
is then dissolved in 100 c.c. of water and filtered in
quantities of 50 c.c. through two fresh gelatinised
candles and the residue again dried. A considerable diminution in
weight occurs, partly owing to incomplete removal from the candle
and brass casing, and the final residue only amounts to about 9 to 12
grams. Occasionally it is necessary to repeat the processes of dissolving
in water, filtering, and drying, but a considerable loss both of
material and fermenting power attends each such operation.
The sticky residue dries up very rapidly in vacuo to a brittle, scaly
mass, which is converted by grinding into a light yellow powder.
The filtrate was invariably found to be quite devoid of fermenting
power, none of the enzyme passing through the gelatin.
Fig. 6.
Properties of the Filtered and Washed Residue.—The residue prepared
as described above consists mainly of the protein, glycogen, and dextrins
of the yeast-juice, and is almost free from mineral phosphates, but
contains a certain amount of combined phosphorus. It also contains
the enzymes of the juice, including the proteoclastic enzyme, and the
hexosephosphatase (p. ). Its solution in water is usually quite
inactive to glucose or fructose, but in some cases produces a small
and evanescent fermentation. When the original filtrate or a corresponding
[p061]
quantity of the filtrate from boiled fresh yeast-juice is added,
the mixture ferments glucose or fructose quite readily. The following
table shows the quantitative relations observed, the sugar being in all
cases present in excess:—
No.
Material.
Volume.
Fil-trate added.c.c.Boiled Juice added.c.c.Water added.c.c.CO2evolved.
1Undried and unwashed residue
15 c.c.
0
0
15
0 g.
15 "
15
0
0
0·035 "
2
15 "
0
0
15
0·024 "
15 "
0
15
0
0·282 "
3
Undried and washed residue
25 "
0
0
0
0·4 c.c.
25 "
0
25
0
268 "
420 "
0008·3 "
20 "
20
0090·3 "
5Washed and dried residue
1 gram in 15 c.c.0
000 "
0120108 "
6
1 gram in 25 c.c.
0000 "
0250364 "
[p062]
These experiments lead to the conclusion that the fermentation of
glucose and fructose by yeast-juice is dependent upon the presence, not
only of the enzyme, but also of another substance which is dialysable
and thermostable.
Precisely similar results were subsequently obtained by Buchner
and Antoni [] by the dialysis of yeast-juice. One hundred
c.c. of juice were dialysed for twenty-four hours at 0° against 1300 c.c.
of distilled water, and the dialysate was then evaporated at 40° to 50°
to 20 c.c. The fermenting power of 20 c.c. of the dialysed juice was
then determined with the following additions:—
(1) 20 c.c. of dialysed juice + 10 c.c. of water gave
0·02 gram CO2.
(2) 20 c.c. of dialysed juice + 10 c.c. of evaporated
dialysate gave 0·52 gram CO2.
(3) 20 c.c. of dialysed juice + 10 c.c. of boiled juice gave
0·89 gram CO2.
It was shown in the previous chapter that phosphates are essential
to fermentation, and hence it becomes necessary to inquire whether
the effect of dialysis is simply to remove these. Experiment shows
that this is not the case. Soluble phosphates do not confer the power
of producing fermentation on the inactive residue obtained by filtration.
Moreover, when yeast-juice is digested for some time before
being boiled, it is found, as will be subsequently described, that the
boiled autolysed juice is quite incapable of setting up fermentation in
the inactive residue, although free phosphates are abundantly present
[Harden and Young, ].
The filtration residue is never obtained quite free from combined
phosphorus, but the production from this of the phosphate necessary
for fermentation to proceed, may be so slow as to render the test for
co-enzyme uncertain, owing to the absence of sufficient phosphate.
When a filtration residue is being tested it is therefore necessary to
secure the presence of sufficient phosphate to enable the characteristic
reaction to proceed, and at the same time to avoid adding phosphate
in too great concentration, as this may, in the presence of only small
amounts of enzyme or co-enzyme, inhibit the fermentation (p. ).
The proof that a filtration residue or dialysed juice is quite free from
co-enzyme is therefore a somewhat complicated matter, and not only
involves the experimental demonstration that the material will not
ferment sugar, but also that this power is not imparted to it by the
addition of a small concentration of phosphate. As it has been
found (p. ) that the fermentation of fructose is less affected than
that of glucose by the presence of excess of phosphate, the practical
method of examining a filtration residue for co-enzyme is to test its
action on a solution of fructose (1) alone and (2) in presence of a
small concentration of phosphate. If the residue produces no action
[p063]
in either case, but produces fermentation when a solution of co-enzyme
is added in the presence of the same concentration of phosphate as
was previously employed, it may be concluded that this sample was
free from co-enzyme but contained enzyme; such an experiment also
affords a definite proof that the co-enzyme does not consist of phosphate.
This dialysable, thermostable substance, without which alcoholic
fermentation cannot proceed, has been provisionally termed the co-ferment
or co-enzyme of alcoholic fermentation. This expression was
first introduced by Bertrand [], to denote substances of this kind,
and he applied it in two instances—to the calcium salt which he considered
was necessary for the action of pectase on pecten substances,
and to the manganese which he supposed to be essential for the activity
of laccase. Without inquiring whether these substances are precisely
comparable in function with that contained in yeast-juice, the
term may be very well applied to signify the substance of unknown
constitution without the co-operation of which the thermolabile enzyme
of yeast-juice is unable to set up the process of alcoholic fermentation.
The active agent of yeast-juice consisting of both enzyme and co-enzyme
may be conveniently spoken of as the fermenting complex, and this
term will occasionally be employed in the sequel.
The co-enzyme is present alike in the filtrates from fresh yeast-juice
and from boiled yeast-juice, and is also contained in the liquids
obtained by boiling yeast with water and by washing zymin or dried
yeast with water.
Practically the only chemical property of the co-enzyme, other than
that of rendering possible the process of alcoholic fermentation, which
has so far been observed, is that it is capable of being decomposed,
probably by hydrolysis, by a variety of reagents, prominent among
which is yeast-juice. This was observed by Harden and Young in
the course of their attempts to prepare a completely inactive residue
by filtration. In many cases a residue was obtained which still possessed
a very limited power of fermentation, only a small amount of
carbon dioxide being formed and the action ceasing entirely after
the expiration of a short period; on the subsequent addition of boiled
juice, however, a very considerable evolution of carbon dioxide was
produced. This was interpreted to mean that the residue in question
contained an ample supply of enzyme but only a small proportion of
co-enzyme, and that the latter was rapidly destroyed, so that the fermentation
soon ceased. The boiled juice then added provided a further
proportion of co-enzyme by the aid of which the surplus enzyme was
[p064]
enabled to carry on the fermentation. This view was confirmed by
adding to a solution of a completely inactive filtration residue and
glucose successive small quantities of boiled juice and observing the
volumes of carbon dioxide evolved after each such addition. Thus in
one case successive additions of volumes of 3 c.c. of boiled juice produced
evolutions of 8·2, 6, and 6 c.c. of carbon dioxide. In another
case two successive additions of 15 c.c. of boiled juice produced evolutions
of 54 and 41·2 c.c. On the other hand, the enzyme itself also
gradually disappears from yeast-juice when the latter is incubated
either alone or with sugar (p. ).
The cessation of fermentation in any particular mixture of enzyme
and co-enzyme may, therefore, be due to the disappearance of either of
these factors from the liquid. If the amount of co-enzyme present be
relatively small it is the first to disappear, and fermentation can then
only be renewed by the addition of a further quantity, whilst the
addition of more enzyme produces no effect. If, on the other hand,
the amount of co-enzyme be relatively large, the inverse is true; the
enzyme is the first to disappear, and fermentation can only be renewed
by the addition of more enzyme, a further quantity of co-enzyme producing
no effect. It has, moreover, been found that the co-enzyme,
like the enzyme, disappears more rapidly in the absence of glucose
than in its presence, incubation at 25° for two days being as a rule
sufficient to remove all the co-enzyme from yeast-juice from top yeasts
in the absence of sugar, whilst in the presence of fermentable sugar
co-enzyme may still be detected at the end of four days.
In all the experiments carried out by Harden and Young with
juice from English top yeast it was found that when a mixture of the
juice with glucose was incubated until fermentation had ceased, the
further addition of co-enzyme in the form of boiled juice did not
cause any renewal of the action; in other words, the whole of the
enzyme had disappeared.
On the other hand, Buchner and Klatte [], working with juice
and zymin prepared from bottom yeast, observed the extremely interesting
fact that after the cessation of fermentation the addition of an
equal volume of boiled juice caused a renewed decomposition of sugar,
and that the processes of incubation until no further evolution of gas
occurred and re-excitation of fermentation by the boiled juice could be
repeated as many as six times. Thus in one experiment the duration
of the fermentation was extended from three to a total of twenty-four
days, and the total gas evolved from 0·73 gram to 2·19 grams. The
phenomenon has been found to be common to yeast from Munich and
[p065]
from Berlin as well as to zymin and maceration extract, and it was
further observed that the boiled juice from one yeast could regenerate
the juice from another, although the quantitative relations were
different.
In these samples of yeast-juice, therefore, there is present a natural
condition of affairs precisely similar to that obtaining in the artificial
mixtures of inactive filtration residue and co-enzyme solution made by
Harden and Young. The balance of quantities is such that the co-enzyme
disappears before the enzyme, leaving a certain amount of
enzyme capable of exercising its usual function as soon as sufficient
co-enzyme is added. This establishes an interesting point of contrast
with the juice prepared from top yeast in England, in which the
enzyme does not outlast the co-enzyme [Harden and Young, ].
The difference may be due to some variation in the relative proportions
of enzyme and co-enzyme or of the enzymes to which the disappearance
of each of these is presumptively due, or to a combination
of these two causes. It was, however, found, even in the juice from
bottom yeast, that incubation for three days at 22° without the addition
of sugar caused the disappearance of the enzyme as well as of the
co-enzyme, and left a residue alike incapable of being regenerated by
the addition of co-enzyme or of restoring the power of producing fermentation
to an inactive mixture containing enzyme and sugar.
If the fermenting power of the juice is to be preserved by repeated
regeneration for a long period, it is absolutely necessary to add the
co-enzyme solution each time as soon as fermentation has ceased, since
the enzyme in the absence of this addition rapidly disappears, even
in the presence of sugar.
This result is probably to be explained, at all events in the main, by
the presence in the co-enzyme solution of the antiprotease to which reference
has already been made [Buchner and Haehn, ]. This
agent, the constitution of which is still unknown, protects proteins in
general from the action of digestive enzymes, and on the assumption
that the alcoholic enzyme of yeast-juice belongs to the class of proteins,
may be supposed to lessen the rate at which this enzyme is destroyed
by the endotryptase of the juice. This antiprotease is, like the co-enzyme
(p. ), destroyed by lipase but is more stable than the co-enzyme
towards hydrolytic agents, and can be obtained free from
co-enzyme by boiling the solution for some hours alone or by heating
with dilute sulphuric acid. Such a solution possesses no regenerative
power, but still retains its power of protecting proteins against digestion
and of preserving the fermenting power of yeast-juice.
[p066]
It must, however, be remembered that the addition of a phosphate
alone may greatly prolong the period of fermentation of yeast-juice
(p. ), and sugar is well known to exert a similar action. It appears,
therefore, that the existence of the enzyme is prolonged not only by
the presence of the antiprotease but also by that of sugar and hexosephosphate,
into which phosphate passes in presence of sugar. Similar
effects are exerted on the co-enzyme by sugar and probably also by
hexosephosphate.
The fermenting complex, therefore, in the presence of these substances,
either separately or together, falls off more slowly in activity
and is present for a longer time, and for both of these reasons produces
an increased amount of fermentation. It seems probable also that the
hexosephosphatase is similarly affected, so that the supply of free
phosphate is at the same time better maintained, and the rate of fermentation
for this reason decreases more slowly than would otherwise
be the case.
It is in this way that an explanation may be found of the remarkable
increase in total fermentation, which is produced by the addition
to yeast-juice and sugar of boiled yeast-juice, containing free phosphate
(which passes into hexosephosphate) as well as co-enzyme, of
boiled autolysed yeast-juice, containing free phosphate but no co-enzyme,
or of phosphate solution alone.
In no case is the original rate of fermentation greatly increased
after the initial acceleration has disappeared, but in every case the
total fermentation is considerably augmented, and this is no doubt
mainly to be attributed, as just explained, to the diminished rate of
decomposition of the fermenting complex and probably of the hexosephosphatase.
Although both enzyme and co-enzyme are completely precipitated
from yeast-juice, as already described (p. ), by 10 volumes of acetone,
the co-enzyme is less easily precipitated than the enzyme, and a certain
degree of separation can therefore be attained by fractional precipitation
[Buchner and Duchaček, ]. The enzyme cannot, however,
be completely freed from co-enzyme in this manner, and the process
is attended by a very considerable loss of enzyme. This is probably
due to the fact that only small quantities of acetone can be added (1·5
to 3 volumes), in order to avoid precipitation of co-enzyme, and that
the precipitates thus formed contain a large proportion of water, a condition
which appears to be fatal to the preservation of the enzyme.
It is, however, not quite certain whether it is the zymase or the
hexosephosphatase which is destroyed in these cases, as no attempt
[p067]
was made to distinguish between them. In any case the precipitates
obtained by fractional treatment with acetone, even when reunited,
produce a much smaller fermentation than the original juice or the
powder prepared by bringing it into 10 volumes of acetone.
Attempts to isolate the co-enzyme from boiled yeast-juice have also
been hitherto unsuccessful. It has, however, been found possible to
remove a considerable amount of material from the solution without
affecting the co-enzyme. When 1 volume of alcohol is added to
boiled yeast-juice, a bulky precipitate, consisting largely of carbohydrates,
is produced, and the filtrate from this is found to contain the
co-enzyme and can be freed from alcohol by evaporation. Further
precipitation with alcohol has not led to useful results.
When a solution which has been treated in this way is precipitated
with lead acetate and kept neutral to litmus, the free phosphate and
hexosephosphate are thrown down and the co-enzyme remains in solution.
The filtrate can be freed from lead by means of sulphuretted
hydrogen and neutralised, and then forms a solution of co-enzyme free
from phosphate and hexosephosphate but still containing combined
phosphorus. More complete purification than this has not yet been
accomplished. Occasionally the precipitate of lead salts retains some
of the co-enzyme, apparently by adsorption, but usually the greater
part remains in the solution (Harden and Young).
The co-enzyme is partially removed from yeast-juice by means of a
colloidal solution of ferric hydroxide (Resenscheck). A precipitate
is thus obtained which contains phosphorus and resembles boiled
yeast-juice in its regenerative action on yeast-juice rendered
inactive by fermentation. It has not, however, so far been found
possible to isolate any definite compound from this precipitate. There
are also indications that when yeast-juice, either fresh or boiled,
is electrolysed, the co-enzyme tends to accumulate at the cathode
[Resenscheck, , ].
Buchner and Klatte [] made use of yeast-juice rendered free
from co-enzyme by incubation with sugar solution to examine the
nature of the agent by which the co-enzyme is destroyed. This agent
is certainly an enzyme, since boiled yeast-juice can be preserved with
unimpaired powers for a considerable length of time, and suspicion fell
naturally, in the first instance, on the endotryptase of the yeast cell.
Direct experiment showed, however, that yeast-juice, which, when fresh,
rapidly destroyed the co-enzyme of boiled juice, lost this power on
preservation, but retained its proteoclastic properties without diminution,
so that the tryptic enzyme could not be the one concerned. The
direct action of commercial trypsin on boiled yeast-juice also yielded
[p068]
a negative result, although this cannot strictly be regarded as an indication
of the effect of the specific proteoclastic enzymes of yeast-juice.
On the other hand, it was found that when boiled juice was treated
for some time with an emulsion containing the lipase of castor oil seeds,
the co-enzyme was completely destroyed. This is a result of great importance,
inasmuch as it probably indicates that the co-enzyme is
chemically allied to the class of substances hydrolysable by lipase, i.e.
to the fats and other esters.
Further, observations by Buchner and Haehn [] have shown
that digestion with potassium carbonate solution containing 2·5 grams
per 100 c.c. also brings about the destruction of the co-enzyme, and that
this is also slowly accomplished by the repeated boiling of the juice. The
co-enzyme is also destroyed both by acid and alkaline hydrolysis, and
when the solution is evaporated to dryness and the residue ignited.
Beyond this general indication nothing is known of the chemical
nature of the co-enzyme. The intimate relation of phosphoric acid to the
process of fermentation renders it not impossible that the co-enzyme
may contain this group, but there is no definite evidence for such
a belief. Purely negative results have been obtained with all the
substances of known composition which have yet been tested, among these
being soluble phosphates, hexosephosphates and a number of oxidisable
and reducible substances, such as quinol, p-phenylenediamine, methylene
blue, peptone beef broth, etc. (Harden and Young; Harden and Norris
[]; see also
Euler and Bäckström []),
glycero-phosphates (Buchner and Klatte).
The precise function of the co-enzyme is even more obscure than
its chemical nature. The system of reacting substances consisting of
fermentable material, enzyme and co-enzyme, bears, however, an obvious
superficial resemblance to many of the systems required for the accomplishment
of chemical changes in the animal or vegetable organism.
Such a triad of substances is, for example, requisite for the process by
which the red blood corpuscles of an animal are broken up by the
serum of a different animal into the blood of which the red corpuscles
of the first animal have been injected. This effect is only produced
when two substances are present, the amboceptor or immune body and
the complement. The analogy may be carried to a further stage since
the amboceptor is, like the co-enzyme, more thermostable than the
complement, which therefore corresponds with the enzyme. Immune
serum can, in fact, be freed from complement by being heated at 57–60°
for half an hour, whereas the amboceptor is unaffected by this treatment.
On the other hand, the complement and amboceptor do not
[p069]
appear to act like enzymes but rather like ordinary chemical
reagents, remaining in combination even after the blood corpuscle has
been broken up, whereas the enzyme and co-enzyme of yeast-juice
are again liberated when the reaction between sugar and phosphate
has been completed.
CHAPTER V.
ACTION OF SOME INHIBITING AND ACCELERATING AGENTS
ON THE ENZYMES OF YEAST-JUICE.
[p070]
One of the most interesting and at the same time most difficult
problems concerning enzyme action in general is the nature of the
inhibiting or accelerating effect produced by many substances upon
the rate or total result of the chemical process set up in presence of the
enzyme. Inhibition, it is usually supposed, involves either the decomposition
of the enzyme, in which case it is irreversible, its removal
from the sphere of action by some change in its mode of solution, or
the formation of an inactive or less active compound between the
enzyme and the inhibiting agent. This compound it may sometimes
be possible to decompose, with the result that the activity of the enzyme
is restored. A striking example of this, to which allusion has already
been made, is the effect of hydrocyanic acid on alcoholic fermentation
(p. ).
Acceleration of enzyme action can in some cases be ascribed to the
fact that the accelerating substance possesses an assignable chemical
function in the reaction, so that an increase in the concentration of this
substance causes an increase in the rate of the reaction. As we have
seen in Chapter III, this is the explanation of the accelerating effect
of phosphates on fermentation by yeast-juice. In many other cases,
however, no such chemical function can be traced, as, for example, in
the effect of neutral salts on the hydrolytic action of invertase, or the
effect of the addition of the co-enzyme to zymase, and it is necessary to
fall back on some assumption, such as that the accelerating agent acts
by increasing the effective concentration of the enzyme or by combining
either with the enzyme or the substrate, forming a compound
which undergoes the reaction more readily.
The interest in the following examples of inhibition and acceleration
of fermentation by yeast-juice lies not only in their relation to these
general problems but also, and perhaps chiefly, in their bearing on the
specific problem of the nature and mode of action of the various agents
concerned in the production of alcohol and carbon dioxide from sugar
in the yeast-cell.
[p071]
I. Influence of Concentration of Phosphate on the Course of
Fermentation.
Prominent among these instances of inhibition and acceleration are
the phenomena attendant on the addition of excess of phosphate to
yeast-juice.
When a phosphate is added to a fermenting mixture of a sugar
and yeast-juice, the effect varies with the concentration of the phosphate
and the sugar and with the particular specimen of yeast-juice
employed. With low concentrations of phosphate in presence of excess
of glucose the acceleration produced is so transient that no accurate
measurements of rate can be made. As soon as the amount of phosphate
added is sufficiently large, it is found that the rate of evolution
of carbon dioxide very rapidly increases from five to ten times, and
then quickly falls approximately to its original value.
As the concentration of phosphate is still further increased, it is
first observed that the maximum velocity, which is still attained almost
immediately after the addition of the phosphate, is maintained for a
certain period before the fall commences, and then, as the increase in
concentration of phosphate proceeds, that the maximum is only
gradually attained after the addition, the period required for this increasing
with the concentration of the phosphate. Moreover, with
still higher concentrations, the maximum rate attained is less than that
reached with lower concentrations, and further, the rate falls off more
slowly. The concentration of phosphate which produces the highest
rate, which may be termed the optimum concentration, varies very
considerably with different specimens of yeast-juice [Harden and
Young, ].
All these points are illustrated by the accompanying curves (Fig. 7)
which show the rate of evolution per five minutes plotted against the
time for four solutions in which the initial concentrations of phosphate
were (A) 0·033, (B) 0·067, (C) 0·1, and (D) 0·133 molar, the volumes of
0·3 molar phosphate being 5, 10, 15, and 20 c.c. in each case added to
25 c.c. of yeast-juice, and made up to 45 c.c, each solution containing
4·5 grams of glucose. The time of addition is taken as zero, the rate
before addition being constant, as shown in the curves.
Fig. 7.
It will be observed that 5 and 10 c.c. (A and B) give the same
maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20
c.c. (D) a still lower one, the rate at which the velocity diminishes
after the attainment of the maximum being correspondingly slow in
these last two cases. By calculating the amount of phosphate which
has disappeared as such from the amount of carbon dioxide evolved,
[p072]
it is found that the maximum does not occur at the same concentration
of free phosphate in each case.
These results suggest that the phosphate is capable of forming
two or more different unstable associations with the fermenting complex.
One of these, formed with low concentrations of the phosphate,
has the composition most favourable for the decomposition of sugar,
whilst the others, formed with higher concentrations of phosphate,
contain more of the latter, probably associated in such a way with the
fermenting complex as to render the latter partially or wholly incapable
of effecting the decomposition of the sugar molecule. As the fermentation
proceeds slowly in the presence of excess of phosphate, the concentration
of the latter is reduced by conversion into hexosephosphate,
and a re-distribution of phosphate occurs, resulting in the gradual
change of the less active into the more active association of phosphate
with fermenting complex, and a consequent rise in the rate of fermentation.
In those cases in which the maximum rate corresponding to the
optimum concentration of phosphate is never attained, some secondary
cause may be supposed to intervene, such as a permanent change in
a portion of the fermenting complex, accumulation of the products of
the reaction, etc.
It is also possible as suggested by Buchner for the analogous case
of arsenite (p. ) that the addition of increasing amounts of phosphate
causes a progressive but reversible change in the mode of dispersion
[p073]
of the colloidal enzyme, and that this has the secondary effect
of altering the rate of fermentation. No decisive evidence is as yet
available upon the subject.
The results obtained by Euler and Johansson [] to which
reference has already been made indicate that in presence of a
moderate excess of phosphate esterification is more rapid than production
of carbon dioxide. No explanation of this phenomenon has
yet been given, but it might obviously be due either to the production
of some phosphorus compound which subsequently takes part in the
production both of hexosediphosphate and of carbon dioxide, or, less
probably, to the entire independence of the two changes—esterification
of phosphate and production of carbon dioxide—which might then be
differently affected by the presence of excess of phosphate and therefore
take place at different rates.
II. Reaction of Fructose with Phosphates in Presence of
Yeast-Juice.
Although, as has been pointed out (p. ), glucose, mannose, and
fructose all react with phosphate in a similar manner in presence of
yeast-juice, there are nevertheless certain quantitative differences between
the behaviour of glucose and mannose on the one hand, and
fructose on the other, which appear to be of considerable importance.
Fructose differs from the other two fermentable hexoses in two particulars:
(1) the optimum concentration of phosphate is much
greater; (2) the maximum rate of fermentation attainable is much
higher [Harden and Young, ; ].
These points are clearly illustrated by the following results, which
all refer to 10 c.c. of yeast-juice, and show that the optimum concentration
of phosphate for the fermentation of fructose is from 1·5 to
10 times that of glucose, and that the maximum rate of fermentation
for fructose in presence of phosphate is 2 to 6 times that of glucose.
Sugar in Grams.
Total Volume.
Optimum Volume of 0·6 Molar Phosphate in c.c.
Maximum Rate in Cubic Centimetres of CO2 per Five minutes.
Glucose.
Fructose.Glucose.
Fructose.
2 35 2 5 7·5
32·2
4 50 1 10 5·4
28·4
1·6
23 2 5 8 17
1 25 1·75
5 5·2
25·9
2 25 5 7·5
16·2
31·2
2 20 2 3·5
7·9
22·6
2 22·5
0·75
2 3·4
22·2
[p074]
It is interesting to note that the two high rates, 32·2 and 31·2 c.c.
per five minutes, are equal to about half the rate obtainable with an
amount of living yeast corresponding to 10 c.c. of yeast-juice, assuming
that about 16 to 20 grams of yeast are required to yield this
volume of juice, and that this amount of yeast would give about 56
to 70 c.c. of carbon dioxide per five minutes at 25°, which has been
found experimentally to be about the rate obtainable with the top
yeast employed for these experiments.
III. Effect of the Addition of Fructose on the Fermentation of
Glucose or Mannose in Presence of a Large Excess of
Phosphate.
When the maximum rate of fermentation of glucose or mannose
by yeast-juice in presence of phosphate is greatly lowered by the
addition of a large excess of phosphate, the addition of a relatively
small amount of fructose (as little as 2·5 per cent. of the weight of the
glucose) causes rapid fermentation to occur. This induced activity is
not due solely to the selective fermentation of the added fructose, since
the amount of gas evolved may be greatly in excess of that obtainable
from the quantity added.
Another way of expressing the same thing is to say that the
optimum concentration of phosphate (p. ) is greatly raised when
2·5 per cent. of fructose is added to glucose, and that consequently the
rate of fermentation rises. The effect is extremely striking, since a
mixture of glucose and yeast-juice fermenting in the presence of a
large excess of phosphate at the rate of less than 1 c.c. of carbon
dioxide in five minutes may be made to ferment at six to eight times
this rate by the addition of only 0·05 gram of fructose (2·5 per cent.
of the glucose present), and to continue until the total gas evolved is at
least five to six times as great as that obtainable from the added fructose,
the concentration of the phosphate being the whole time at such a
height as would limit the fermentation of glucose alone to its original
value.
The effect is not produced when the concentration of the phosphate
is so high that the rate of fermentation of fructose is itself greatly
lowered.
This remarkable inductive effect is specific to fructose and is not
produced when glucose is added to mannose or fructose, or by mannose
when added to glucose or fructose, under the proper conditions of
concentration of phosphate in each case.
This interesting property of fructose, taken in connection with the
[p075]
facts that this sugar in presence of phosphate is much more rapidly
fermented than glucose or mannose, and that the optimum concentration
of phosphate for fructose is much higher than for glucose or mannose,
appears to indicate that fructose when added to yeast-juice does not
merely act as a substance to be fermented, but in addition, bears some
specific relation to the fermenting complex.
All the phenomena observed are, indeed, consistent with the supposition
that fructose actually forms a permanent part of the fermenting
complex, and that, when the concentration of this sugar in the yeast-juice
is increased, a greater quantity of the complex is formed. As the
result of this increase in the concentration of the active catalytic agent,
the yeast-juice would be capable of bringing about the reaction with
sugar in presence of phosphate at a higher rate, and at the same time
the optimum concentration of phosphate would become greater, exactly
as is observed. The question whether, as suggested above, fructose
actually forms part of the fermenting complex, and the further questions,
whether, if so, it is an essential constituent, or whether it can be replaced
by glucose or mannose with formation of a less active complex, remain
at present undecided, and cannot profitably be more fully discussed until
further information is available.
It must, moreover, be remembered that different samples of yeast-juice
vary to a considerable extent in their relative behaviour to glucose
and fructose, so that the phenomena under discussion may be expected
to vary with the nature and past history of the yeast employed.
IV. Effect of Arsenates on the Fermentation of Sugars by
Yeast-Juice and Zymin.
The close analogy which exists between the chemical functions of
phosphorus and arsenic lends some interest to the examination of the
action of sodium arsenate upon a mixture of yeast-juice and sugar, and
experiments reveal the fact that arsenates produce a very considerable
acceleration in the rate of fermentation of such a mixture [Harden and
Young, ; ]. The phenomena
observed, however, differ markedly from those which accompany the action of
phosphate.
The acceleration produced is of the same order of magnitude as
that obtained with phosphate, but it is maintained without alteration
for a considerable period, so that there is no equivalence between the
amount of arsenate added and the extra amount of fermentation effected.
Further, no organic arsenic compound corresponding in composition
with the hexosephosphates appears to be formed.
Increase of concentration of arsenate produces a rapid inhibition of
[p076]
fermentation, probably due to some secondary effect on the fermenting
complex, possibly to be interpreted as the formation of compounds incapable
of combining with sugar and hence unable to carry on the process
of fermentation. An optimum concentration of arsenate therefore
exists just as of phosphate, at which the maximum rate is observed,
and this optimum concentration and the corresponding rate vary with
different samples of juice and are less for glucose than for fructose.
The rate of fermentation by zymin is relatively less increased than that
by yeast-juice.
Owing to the fact that the rate is permanently maintained the addition
of a suitable amount of arsenate increases the total fermentation
produced to a much greater extent than phosphate.
The nature of these effects may be gathered from the result of a
few typical experiments. In one case the rate of fermentation of
glucose by yeast-juice was raised by the presence of 0·03 molar arsenate
from 2 to 23 c.c. per five minutes, and the total evolved in ninety-five
minutes from 51 to 459 c.c. The accelerating effect on 20 c.c. of
juice, of as little as 0·005 c.c. of 0·3 molar arsenate, containing 0·11
mgrm. of arsenic, can be distinctly observed, but the maximum effect
is usually produced by about 1 to 3 c.c., the concentration being
therefore 0·015 to 0·045 molar. Greater concentrations than this produce
a less degree of acceleration accompanied by a shorter duration
of fermentation, as shown by the following numbers which refer to 20
c.c. of yeast-juice in a total volume of 40 c.c. containing 10 per cent.
of glucose:—
C.cs. of 0·3 Molar Arsenate in 40 c.c.
Molar Concentration of Arsenate.
Maximum Rate of Fermentation.
0
0
3·5
0·005
0·0000375
6·3
0·01
0·000075
8
0·02
0·00015
14·2
0·04
0·0003
19·9
0·1
0·00075
29·7
0·2
0·0015
35
0·5
0·00375
34·9
1·0
0·0075
29·5
2·0
0·015
23·2
5·0
0·0375
14·5
10·0
0·075
8·7
15·0
0·1125
5·3
20
0·15
3·2
The contrast between glucose and fructose in their relations to
[p077]
arsenate are well exhibited in the following table, in which the rates of
fermentation produced by arsenate in presence of excess of glucose
and fructose respectively are given:—
Concentration of Arsenate.
Rate.
Glucose.
Fructose.
0·0075 molar12·126·6
0·0225 (opt. for glucose)13·4—
0·0525 (opt. for fructose)— 45·8
0·11255·139
Here the optimum concentration for fructose is more than twice
that for glucose, whilst the maximum rate of fermentation obtainable
with fructose is between three and four times the maximum given by
glucose.
V. Effect of Arsenites on the Fermentation Produced by
Yeast-Juice.
Effects somewhat similar to those produced by arsenates were
observed by Buchner
[Buchner and Rapp, ;
,
;
Buchner, E. and H., and Hahn, ,
pp. 184–205] when potassium
arsenite was added to yeast-juice. This substance, the action of which
on yeast had been adduced by Schwann as a proof of the vegetable nature
of this organism, was employed by Buchner on account of its poisonous
effect on vegetable cells as an antiseptic and as a means of testing for
the protoplasmic nature of the agent present in yeast-juice. Its effect
on the fermentation was, however, found to be irregular, and at the
same time it did not act as an efficient antiseptic in the concentrations
which could be employed. Even 2 per cent. of arsenious oxide, added as
the potassium salt, had in many cases a decided effect in diminishing the
total fermentation obtained with cane sugar, and this effect increased
with the concentration. A number of irregularities were also observed
which cannot here be discussed. It was further found that in some
cases 2 per cent. of arsenious oxide inhibited the fermentation of
glucose but not of saccharose, or of a mixture of glucose and fructose,
whilst its effect on fructose alone was of an intermediate character.
The important observation was also made by Buchner that the
addition of a suitable quantity of arsenite as a rule caused a greatly
increased fermentation during the first sixteen hours even in experiments
in which the total fermentation was diminished. By examining the
effect of arsenite on fermentation in a similar manner to that of arsenate,
Harden and Young []
have found that a close analogy exists
[p078]
between the effects and modes of action of these substances, but that
arsenite produces a much smaller acceleration than arsenate. An
optimum concentration of arsenite exists, just as in the case of arsenate,
which produces a maximum rate of fermentation. Further increase in
concentration leads to inhibition, and in no case is there any indication
of the production of an exactly equivalent amount of fermentation as
in the case of phosphate. In various experiments with dialysed,
evaporated, and diluted yeast-juice in which 2 per cent. of arsenious
oxide was found by Buchner to inhibit fermentation, it is probable
that, owing to the small amount of fermenting complex left, this
amount of arsenious oxide was considerably in excess of the optimum
concentration, although Buchner ascribes the effect to the removal of
some of the protective colloids of the juice, owing to the prolonged
treatment to which it had been subjected.
The extent of the action of arsenite appears from the following results.
In one case a rate of 1·7 c.c. was increased to 7 c.c. by 0·06 molar
arsenite. In another experiment it was found that the optimum concentration
was 0·04 molar arsenite, the addition of which increased the
rate three-fold. As in the case of arsenate the optimum concentration
and the corresponding maximum rate of fermentation are considerably
greater for fructose than for glucose. The relative rates produced by the
addition of equivalent amounts of arsenate and arsenite (1 c.c. of 0·3
molar solution in each case to 20 c.c. of yeast-juice) were 27·5 and 3·1,
the original rate of the juice being 1·7. In general the optimum concentration
of arsenite is considerably greater than that of arsenate.
The inhibiting effects of higher concentrations of arsenite and
arsenate also present close analogies, but this most interesting aspect
of the question has not yet been sufficiently examined to repay detailed
discussion. Buchner [Buchner, E. and H., and Hahn, , pp. 199–205]
has suggested that the inhibition is due primarily to some change in
the colloidal condition of the enzyme and has shown that certain
colloidal substances appear to protect it, as does also sugar. The
possibility is also present that inactive combinations of some sort are
formed between the fermenting complex and the inhibiting agent, in
the manner suggested to account for the inhibiting effect of excess of
phosphate (p. ). It seems most probable that the effect is a complex
one, in which many factors participate.
Nature of the Acceleration Produced by Arsenate and Arsenite.
In explanation of the remarkable accelerating action of arsenates
and arsenites two obvious possibilities present themselves. In the
[p079]
first place the arsenic compound may actually replace phosphate in the
reaction characteristic of alcoholic fermentation, the resulting arsenic
analogue of the hexosephosphate being so unstable that it undergoes
immediate hydrolysis, and is therefore only present in extremely small
concentration at any period of the fermentation and cannot be isolated.
In the second place it is possible that the arsenic compound may accelerate
the action of the hexosephosphatase of the juice, and thus
by increasing the rate of circulation of the phosphate produce the
permanent rise of rate. With this effect may possibly be associated
a direct acceleration of the action of the fermenting complex.
The experimental decision between these alternative explanations
is rendered possible by the use of a mixture of enzyme and co-enzyme
free from phosphate and hexosephosphate. As has already been
described (p. ) a mixture of boiled yeast-juice, which has been
treated with lead acetate, glucose or fructose, and washed zymin can
be prepared which scarcely undergoes any fermentation unless phosphate
be added. If now arsenates or arsenites can replace phosphate, they
should be capable of setting up fermentation in such a mixture.
Experiment shows that they do not possess this power. For fermentation
to proceed phosphate must be present and it cannot be replaced
either by arsenate or arsenite [Harden and Young, ].
The effect of these salts on the action of the hexosephosphatase
can also be ascertained by a modification of the foregoing experiment.
If a hexosephosphate be made the sole source of phosphate in such a
mixture as that described above, in which it must be remembered
abundance of sugar is present, the rate at which fermentation can
proceed will be controlled by the rate at which the hexosephosphate is
decomposed with formation of phosphate. Experiment shows that in
the presence of added arsenate or arsenite the rate of fermentation is
largely increased, so that the effect of these salts must be to increase
the rate of liberation of phosphate, or in other words, to accelerate the
hydrolytic action of the hexosephosphatase.
This conclusion is even more strikingly confirmed by a comparison
of the direct action of yeast-juice on hexosephosphate in presence and
in absence of arsenate, as measured by the actual production of free
phosphate. In a particular experiment this gave rise to 0·0707
gram of Mg2P2O7 in the absence of arsenate and 0·6136 gram of
Mg2P2O7 in the presence of arsenate.
The results obtained with arsenite are throughout very similar to
those given by arsenate, but are not quite so striking. It may therefore
be affirmed with some confidence that the chief action of arsenates
[p080]
and arsenites in accelerating the rate of fermentation of sugars by
yeast-juice or zymin, consists in an acceleration of the rate at which
phosphate is produced from the hexosephosphate by the action of
the hexosephosphatase.
It has further been found that arsenates, and to a less degree arsenites,
also produce an acceleration of the rate of autofermentation of yeast-juice
and of the rate at which glycogen is fermented. This turns out to
be due in all probability to an increase in the activity of the glycogenase
by the action of which the sugar is supplied which is the direct subject of
fermentation. Thus in one case an initial rate of fermentation of glycogen
of 1·9 c.c. per five minutes was increased by 0·05 molar arsenate
to 9·7 and the amount of carbon dioxide evolved in two hours from
38 to 158 c.c. Even this enhanced production of glucose from glycogen,
however, is not nearly sufficient for the complete utilisation of the
phosphate also being liberated by the action on the hexosephosphatase,
for the addition of an excess of sugar produces a much higher rate,
in this case 36 c.c. per five minutes. The effect of arsenate on the rate
of action of the glycogenase seems therefore to be much smaller than
on that of the hexosephosphatase.
No other substances have yet been found which share these interesting
properties with arsenates and arsenites, and no advance has
been made towards an understanding of the mechanism of the accelerating
action of these salts on the specific enzymes which are affected
by them.
CHAPTER VI.
CARBOXYLASE.
[p081]
An observation of remarkable interest, which promises to throw
light on several important features of the biochemistry of yeast, was
made in 1911, and has since then formed the subject of detailed
investigation by Neuberg and a number of co-workers.
It was found that yeast had the power of rapidly decomposing
a large number of hydroxy-and keto-acids [Neuberg and Hildesheimer,
;
Neuberg and Tir, ; see also
Karczag, , ]. The
most important among these are pyruvic acid,
CH3·CO·COOH, and a
considerable number of other aliphatic a-keto-acids which are decomposed
with evolution of carbon dioxide and formation of the corresponding
aldehyde:—
R·CO·COOH = R·CHO + CO2.
The reaction is produced by all races of brewer's yeast which
have been tried, as well as by active yeast preparations and extracts
and by wine yeasts [Neuberg and Karczag, ;
Neuberg and Kerb, ].
The phenomenon can readily be exhibited as a
lecture experiment by shaking up 2 g. of pressed yeast with 12 c.c. of
1 per cent. pyruvic acid, placing the mixture in a Schrötter's fermentation
tube, closing the open limb by means of a rubber stopper
carrying a long glass tube and plunging the whole in water of 38–40°.
Comparison tubes of yeast and water and yeast and 1 per cent.
glucose may be started at the same time, and it is then seen that glucose
and pyruvic acid are fermented at approximately the same rate
[Neuberg and Karczag, ].
If English top yeast be used it is
well to take 0·5 per cent. pyruvic acid solution and to saturate the liquids
with carbon dioxide before commencing the experiment. The production
of acetaldehyde can be readily demonstrated by distilling the
mixture at the close of fermentation and testing for the aldehyde
either by Rimini's reaction (a blue coloration with diethylamine and
sodium nitroprusside) or by means of p-nitrophenylhydrazine which
precipitates the hydrazone, melting at 128·5° [Neuberg and Karczag,
,
].
[p082]
As the result of quantitative experiments it has been shown that
80 per cent. of the theoretical amount of acetaldehyde can be recovered.
The salts of the acids are also attacked, the carbonate of the metal,
which may be strongly alkaline, being formed. Thus taking the case
of pyruvic acid, the salts are decomposed according to the following
equation:—
2 CH3·CO·COOK + H2O = 2 CH3·CHO + K2CO3 + CO2.
Under these conditions a considerable portion of the aldehyde
undergoes condensation to aldol [Neuberg, ]:—
2 CH3·CHO = CH3·CH(OH)·CH2·CHO.
This change appears to be due entirely to the alkali and not to
an enzyme since the aldol obtained yields inactive β-hydroxybutyric
acid on oxidation [Neuberg and Karczag, ;
Neuberg, ].
The various preparations derived from yeast which are capable of
producing alcoholic fermentation also effect the decomposition of
pyruvic acid in the same manner as living yeast. They are, however,
more sensitive to the acidity of the pyruvic acid, and it is therefore
advisable to employ a salt of the acid in presence of excess of a weak
acid, such as boric or arsenious acid, which decomposes the carbonate
formed but has no inhibiting action on the enzyme [Harden, ;
Neuberg and Rosenthal, ].
As already mentioned the action is exerted on α-ketonic acids
as a class and proceeds with great readiness with oxalacetic
acid, COOH·CH2·CO·COOH,
all the three forms of which are decomposed, with α-ketoglutaric
acid, and with α-ketobutyric acid. Hydroxypyruvic acid
CH2(OH)·CO·COOH
is slowly decomposed yielding glycolaldehyde, CH2(OH)·CHO,
and this condenses to a sugar [Neuberg and Kerb, ; ]. Positive
results have also been obtained with diketobutyric, phenylpyruvic,
p-hydroxyphenylpyruvic, phenylglyoxylic and acetonedicarboxylic acids
[Neuberg and Karczag, ].
Relation of Carboxylase to Alcoholic Fermentation.
With regard to the relation of carboxylase to the process of alcoholic
fermentation, nothing definite is yet known. As Neuberg points out [see
Neuberg and Kerb, ] the universal presence of the enzyme in yeasts capable of
producing alcoholic fermentation, and the extreme readiness with
which the fermentation of pyruvic acid takes place create a [p083]
strong presumption that the decomposition of pyruvic acid actually
forms a stage in the process of the alcoholic fermentation of the sugars.
On the other hand Ehrlich's alcoholic fermentation of the amino-acids (p.
) provides another function for carboxylase—that
of decomposing the α-ketonic acids produced by the deaminisation of the
amino-acids. It must be remembered in this connection that carboxylase is
not specific in its action, but catalyses the decomposition not only of
pyruvic acid but also of a large number of other α-ketonic acids, including
many of those which correspond to the amino-acids of proteins and are
doubtless formed in the characteristic decomposition of these amino-acids
by yeast. Carboxylase undoubtedly effects one stage in the production of
alcohols from amino-acids, whether it is also the agent by which one stage
in the alcoholic fermentation of sugar is brought about still remains to be
proved.
A comparison of the conditions of action of carboxylase and
zymase has revealed several interesting points of difference. Neuberg
and Rosenthal [] have observed that the fermentation of pyruvic
acid by maceration extract commences much more rapidly than that
of glucose and interpret this to mean that in the fermentation of glucose
a long preliminary process occurs before sufficient pyruvic acid has been
produced to yield a perceptible amount of carbon dioxide. The long delay
(3 hours) which they sometimes observed in the action of maceration
juice on glucose is however by no means invariable (see p. ),
but in any case indicates that the sugar fermentation can be affected
by conditions which are without influence on the pyruvic fermentation.
A similar conclusion is to be drawn from the fact that the pyruvic acid
fermentation is less affected by antiseptics than the glucose fermentation
[Neuberg and Karczag, ;
Neuberg and Rosenthal, ],
chloroform sufficient to stop the glucose fermentation brought about
by yeast or dried yeast being usually without effect on the fermentation
of the pyruvates either alone or in presence of boric or arsenious
acid. A more important difference is that carboxylase decomposes
pyruvic acid in the absence of the co-enzyme which is necessary for
the fermentation of glucose [Harden, ; Neuberg and Rosenthal,
]. This can be demonstrated experimentally by washing dried
yeast or zymin with water (see p. ) until it is no longer capable of
decomposing glucose (Harden), or by allowing maceration extract to
autolyse or dialyse until it is free from co-enzyme (Neuberg and
Rosenthal). The zymase of maceration extract is moreover inactivated
in 10 minutes at 50–51°, whereas after this treatment the carboxylase
is still active.
[p084]
The only conclusion that can be legitimately drawn from these
highly interesting facts is that if the decomposition of pyruvic acid
actually be a stage in the alcoholic fermentation of glucose the soluble
co-enzyme is required for some change precedent to this, so that in its
absence the production of pyruvic acid cannot be effected.
CHAPTER VII.
THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION.
[p085]
When pure yeast is allowed to develop in a solution of sugar containing
a suitable nitrogenous diet and the proper mineral salts, the liquid
at the close of the fermentation contains not only alcohol and some
carbon dioxide but also a considerable number of other substances,
some arising from the carbonaceous and others from the nitrogenous
metabolism of the cell. Prominent among the non-nitrogenous substances
which are thus found in fermented sugar solutions are fusel oil,
succinic acid, glycerol, acetic acid, aldehyde, formic acid, esters, and
traces of many other aldehydes and acids. In addition to these substances
which are found in the liquid, there are also the carbonaceous
constituents of the newly formed cells of the organism, comprising the
material of the cell walls, yeast gum, glycogen, complex organic phosphates,
as well as other substances.
The attention of chemists has been directed to these compounds
since Pasteur first emphasised their importance as essential products
of the alcoholic fermentation of sugar, and his example was generally
followed in attributing their origin to the sugar.
The study of cell-free fermentation by means of yeast-juice or
zymin has, however, revealed the facts that certain of these substances
are not formed in the absence of living cells, and that their origin is to
be sought in the metabolic processes which accompany the life of the
cell. Their source, moreover, has been traced not to the sugar but to
the amino-acids, formed by the hydrolysis of the proteins, which occur
in all such liquids as beer wort, grape juice, etc., which are usually
submitted to alcoholic fermentation. This has so far been proved with
certainty for the fusel oil and succinic acid, and rendered highly probable
for all the various aldehydes and acids of which traces have been
detected.
Fusel Oil.
All forms of alcohol prepared by fermentation contain a fraction of
high boiling-point, which is termed fusel oil, and amounts to about
[p086]
0·1 to 0·7 per cent. of the crude spirit obtained by distillation. This
material is not an individual substance, but consists of a mixture of
very varied compounds, all occurring in small amount relatively to the
ethyl alcohol from which they have been separated. The chief constituents
of the mixture are the two amyl alcohols, isoamyl alcohol,
(CH3)2·CH·CH2·CH2·OH,
and d-amyl alcohol,
CH3·CH(C2H5)·CH2·OH,
which contains an asymmetric carbon atom and is optically active. In
addition to these, much smaller amounts of propyl alcohol and isobutyl
alcohol are present, together with traces of fatty acids, aldehydes, and
other substances.
The origin of these purely non-nitrogenous compounds was usually
sought in the sugar of the liquid fermented, from which they were
thought to be formed by the yeast itself or by the agency of bacteria
[Emmerling, ,
;
Pringsheim, ,
,
,
],
whilst others traced their formation to the direct reduction of fatty
acids. Felix Ehrlich has, however, conclusively shown in a series of
masterly researches that the alcohols, and probably also the aldehydes,
contained in fusel oil are in reality derived from the amino-acids
which are formed by the hydrolysis of the proteins.
The close relationship between the composition of leucine,
(CH3)2·CH·CH2·CH(NH2)·COOH,
and isoamyl alcohol,
(CH3)2·CH·CH2·CH2·OH,
had previously led to the surmise that a genetic relation might exist
between these substances, but the idea had not been experimentally
confirmed. In 1903
Ehrlich discovered [;
,
;
;
;
Ehrlich and Wendel, ]
that proteins also yield on
hydrolysis an isomeride of leucine known as isoleucine, which has the
constitution
CH3·CH(C2H5)·CH(NH2)·COOH,
and therefore stands to d-amyl alcohol,
CH3·CH(C2H5)·CH2·OH,
in precisely the same relation as leucine to isoamyl alcohol. This suggestive
fact at once directed his attention to the problem of the origin
of the amyl alcohols in alcoholic fermentation. Using a pure culture
of yeast, and thus excluding the participation of bacteria in the change,
he found that leucine readily yielded isoamyl alcohol, and isoleucine
d-amyl alcohol when these amino-acids were added in the pure state
[p087]
to a solution of sugar and treated with a considerable proportion of yeast
[;
,
;
,
]. The chemical reactions involved are
simple ones and are represented by the following equations:—
(1)
(CH3)2·CH·CH2·CH(NH2)·COOH
Leucine
+ H2O =
(CH3)2CH·CH2CH2·OH
Isoamyl alcohol
+ CO2 + NH3
(2)
CH3·CH(C2H5)·CH(NH2)·COOH
Isoleucine
+ H2O =
CH3·CH(C2H5)·CH2·OH
d-Amyl alcohol
+ CO2 + NH3
The experiments by which these important changes were demonstrated
were of a very simple and convincing character
[Ehrlich, ].
Two hundred grams of sugar and 3 to 10 grams of the nitrogenous
substance to be examined were dissolved in 2 to 2·5 litres of
tap water in a 3 to 4 litre flask, the liquid was sterilised by being
boiled for several hours, and after cooling 40 to 60 grams of fresh
yeast were added and the flask allowed to stand at room temperature
until the whole of the sugar had been decomposed by fermentation.
In the earlier experiments the amyl alcohols were isolated and identified
by conversion into the corresponding valerianic acids, but as a
rule the fusel oil as a whole was quantitatively estimated in the filtrate
by the Röse-Herzfeld method [Lunge, , p. 571].
The following are typical results. (1) An experiment carried
out as above without any addition of leucine gave 97·32 grams of
alcohol containing 0·40 per cent. of fusel oil. (2) When 6 grams
of synthetic, optically inactive leucine were added, 97·26 grams of
alcohol were obtained, containing 2·11 per cent. of fusel oil, which
was also optically inactive; 2·5 grams of leucine were recovered, so
that 87 per cent. of the theoretical yield of isoamyl alcohol was obtained
from the 3·5 grams of leucine decomposed. (3) In the presence
of 2·5 grams of d-isoleucine (prepared from molasses residues),
200 grams of sugar gave 93·99 grams of alcohol, containing 1·44 per
cent. of fusel oil, which was lævo-rotatory. This corresponds with 80
per cent. of the theoretical yield of d-amyl alcohol from the isoleucine
added.
This change, which Ehrlich has termed the alcoholic fermentation
of the amino-acids, although brought about by living yeast, does not
appear to occur at all when zymin [Ehrlich, ;
Pringsheim, ]
or yeast-juice [Buchner and Meisenheimer, ]
is substituted
for the intact organism, nor is it effected even by living yeast in the
absence of a fermentable sugar [Ehrlich, ].
The reaction appears
indeed to be intimately connected with the nitrogenous metabolism of
the cell, and the whole of the ammonia produced is at once assimilated
and does not appear in the fermented liquid. Other amino-acids
[p088]
undergo a corresponding change, and the reaction appears to be a
general one. Thus tyrosine,
OH·C6H4·CH2·CH(NH2)·COOH,
yields p-hydroxyphenylethyl alcohol, or tyrosol
[Ehrlich, ;
Ehrlich and Pistschimucka, ],
OH·C6H4·CH2·CH2OH, a substance of
intensely bitter taste, which was first prepared in this way and is probably
one of the most important factors in determining the flavour of
beers, etc. Phenylalanine,
C6H5·CH2·CH(NH2)·COOH, in a
similar way yields phenylethyl alcohol,
C6H5·CH2·CH2OH, one of
the constituents of oil of roses, whilst tryptophane,
C6H4
╱
│
HN
│
╲
│
H
C═══
C·CH2·CH(NH2)·COOH
yields tryptophol,
C6H4
╱
│
HN
│
╲
│
H
C═══
C·CH2·CH2OH
which was also first prepared in this way
[Ehrlich, ] and has a
very faintly bitter, somewhat biting taste.
The extent to which the amino-acids of a medium in which yeast
is producing fermentation are decomposed in this sense depends on
the amount of the available nitrogen and on the form in which it is
present. Thus the addition of ammonium carbonate to a mixture of
yeast and sugar was found to lower the production of fusel oil from
0·7 to 0·33 per cent. of the alcohol produced. The addition of leucine
alone raised the percentage from 0·7 to 2·78, but the addition of both
leucine and ammonium carbonate resulted in the formation of only
0·78 per cent. of fusel oil, The production of fusel oil therefore and
the character of the constituents of the fusel oil alike depend on the
composition of the medium in which fermentation occurs. This affords
a ready explanation of the fact that molasses, which contains almost
equal amounts of leucine and isoleucine, yields a fusel oil also containing
approximately equal amounts of isoamyl alcohol and d-amyl alcohol
[Marckwald, ], whilst corn and potatoes, in which leucine
preponderates over isoleucine, yield fusel oils containing a relatively
large amount of the inactive alcohol. The subject is, in fact, one of
great interest to the technologist, for as Ehrlich points out "the great
variety of the bouquets of wine and aromas of brandy, cognac, arrak,
rum, etc., may be very simply referred to the manifold variety of the
proteins of the raw materials (grapes, corn, rice, sugar cane, etc.) from
which they are derived".
Yeast can also form fusel oil at the expense of its own protein,
but this only occurs to any considerable extent when the external
[p089]
supply of nitrogen is insufficient. Under these circumstances the
amino-acids formed by autolysis may be decomposed and their
nitrogen employed over again for the construction of the protein of
the cell.
The yield is also influenced by the condition of the yeast employed
with regard to nitrogen, a yeast poor in nitrogen being more efficacious
in decomposing amino-acids than one which is already well supplied
with nitrogenous materials. The nature of the carbonaceous nutriment
and finally the species of yeast are also of great importance [see
Ehrlich, ;
Ehrlich and Jacobsen, ].
A very important characteristic of the action of yeast on the
amino-acids is that the two stereo-isomerides of these optically active
compounds are fermented at different rates. When inactive, racemic
leucine is treated with yeast and sugar, the naturally occurring
component, the l-leucine, is more rapidly attacked, so that if the
experiment be interrupted at the proper moment the other component,
the d-leucine, alone is present and may be isolated in the pure
state. In an actual experiment 3·8 grams of this component were
obtained in the pure state from 10 grams of dl-leucine [Ehrlich, ], so that the whole of the
l-leucine (5 grams) had been decomposed but only 1·2 grams of the
d-leucine. This mode of action has been found to be characteristic
of the alcoholic fermentation of the amino-acids by yeast. In all the
instances so far observed, both components of the inactive amino-acid
are attacked, but usually the naturally occurring isomeride is the
more rapidly decomposed, although in the case of β-aminobutyric acid
both components disappear at the same rate [Ehrlich and Wendel, ]. This reaction therefore
must be classed along with the action of moulds on hydroxy-acids [McKenzie
and Harden, ],
and the action of lipase on inactive esters [Dakin, , ],
in which both isomerides are attacked but at unequal rates, and differs
sharply from the action of yeast itself on sugars [Fischer and Thierfelder,
], and of emulsin,
maltase, etc., which only act on one isomeride and leave the other entirely
untouched [see Bayliss, , pp. 55, 77,
117].
Succinic Acid.
The origin of the succinic acid formed in fermentation has also
been traced by Ehrlich [] to the
alcoholic fermentation of the amino-acids. It was shown by Buchner and by
Kunz [] that succinic acid like fusel oil
is not formed during fermentation by yeast-juice or zymin, and, in the
light of Ehrlich's work on fusel oil, several [p090] modes
of formation appeared possible for this substance [Ehrlich, ]. The dibasic amino-acids might,
for example, undergo simple reduction, the NH2 group being
removed as ammonia and replaced by hydrogen. Aspartic acid would thus pass
into succinic acid:—
COOH·CH2·CH(NH2)·COOH + 2 H = COOH·CH2·CH2·COOH + NH3.
This change can be effected in the laboratory only by heating with
hydriodic acid. Biologically it has been observed [E. and H. Salkowski,
] when aspartic acid is submitted to the action of
putrefactive bacteria, and almost quantitatively when Bacillus coli communis
is cultivated in a mixture of aspartic acid and glucose [Harden,
]. In this case a well-defined source of hydrogen exists in the
glucose, which when acted on by this bacillus yields a large volume
of gaseous hydrogen, which is not evolved in the presence of aspartic
acid. Some such source is also available in the case of yeast, although
it cannot be chemically defined, for this organism is known to produce
many reducing actions, which are usually ascribed to the presence of
reducing ferments or reductases in the cell.
A similar action would convert glutamic acid,
COOH·CH2·CH2·CH(NH)2·COOH,
into glutaric acid,
COOH·CH2·CH2·CH2·COOH,
which also is found among the products of fermentation, whilst the
monamino-acids would pass into the simple fatty acids.
On submitting these ideas to the test of experiment, however,
Erhlich found that the addition of aspartic acid did not in any way increase
the yield of succinic acid, and that of all the amino-acids which
were tried only glutamic acid,
COOH·CH2·CH2·CH(NH2)·COOH,
produced a definite increase in the amount of this substance. Further
experiments showed that glutamic acid was actually the source of the
succinic acid, the relations being quite similar to those which exist for
the production of fusel oil.
Succinic acid is formed whenever sugar is fermented by yeast, even
in the absence of added nitrogenous matter, and amounts to 0·2 to 0·6
per cent. of the weight of the sugar decomposed, its origin in this case
being the glutamic acid formed by the autolysis of the yeast protein.
When some other source of nitrogen is present, such as asparagine or
an ammonium salt, the amount falls to 0·05 to 0·1. If glutamic acid
be added it rises to about 1 to 1·5 per cent. but falls again to about
0·05 to 0·1 when other sources of nitrogen, such as asparagine or ammonium
salts, are simultaneously available, either in the presence or
[p091]
absence of added glutamic acid. As in the case of fusel oil, the production
does not occur in the absence of sugar, and is not effected by
yeast-juice or zymin.
The chemical reaction involved in the production of succinic acid
differs to some extent from that by which fusel oil is formed, inasmuch
as an oxidation is involved:—
COOH·CH2·CH·CH(NH2)·COOH + 2 O = COOH·CH2·CH2·COOH + NH3 + CO2.
From analogy with the production of amyl alcohol from leucine,
glutamic acid would be expected to yield γ-hydroxybutyric acid:—
COOH·CH2·CH2·CH(NH2)·COOH + H2O = NH3 + CO2 + COOH·CH2·CH2·CH2·OH.
As a matter of fact this substance cannot be detected among the
products of fermentation, but succinic acid as already explained is
formed. This acid might, however, possibly be formed by the oxidation
of the γ-hydroxybutyric acid:—
COOH·CH2·CH2·CH2·OH + 2 O = COOH·CH2·CH2·COOH + H2O,
although this change is on biological grounds improbable.
The conversion of the group —CH(NH2)— into the terminal
CH2·OH in fusel oil, or COOH in succinic acid, may possibly be
effected in several different ways, the most probable of which are the
following:—
I. Direct elimination of carbon dioxide, followed by hydrolysis of
the resulting amine:—
(1) R·CH(NH2)·COOH = R·CH2·NH2 + CO2.
(2) R·CH2·NH2 + H2O = R·CH2·OH + NH3.
The reaction (1) is actually effected by many bacteria and has been
employed for the preparation of bases from amino-acids [cf. Barger,
, p. 7], although there is no direct evidence that it can be brought
about by yeast. On the other hand reaction (2) has actually been observed
with some yeasts. Thus it has been found [Ehrlich and Pistschimuka,
] that many "wild" yeasts produce this change with
great readiness in presence of sugar, glycerol or ethyl alcohol as
sources of carbon and grow well in media in which amines, such as p-hydroxyphenylethylamine
or iso-amylamine, form the only source of
nitrogen. Willia anomala (Hansen), a yeast which forms surface
growths, succeeds admirably under these conditions, whereas culture
yeasts are much less active in this way, although they produce a certain
amount of change. It is therefore possible that this mode of decomposition
plays some part in the production of fusel oil, but in the
case of culture yeasts it is entirely subordinated to the mode next to
be discussed.
[p092]
II. Oxidative removal of the –NH2 group with formation of an
α-ketonic acid:—
(1) R·CH(NH2)·COOH + O = R·CO·COOH + NH3
followed by the decomposition of the ketonic acid into carbon dioxide
and an aldehyde and the subsequent reduction or oxidation of the
aldehyde:—
(2) R·CO·COOH = R·CHO + CO2.
(3) (a) R·CHO + 2 H = R·CH2OH.
(b) R·CHO + O = R·COOH.
The evidence for the occurrence of reaction (1) is supplied by the
experiments of Neubauer and Fromherz []. Having previously
found that amino-acids undergo a change of this kind in the animal
body, Neubauer investigated their behaviour towards yeast. Taking
dl-phenylaminoacetic acid,
C6H5·CH(NH2)·COOH,
it was found
that the changes produced were essentially the same as in the animal
body. The l-component of the acid was partly acetylated and partly
unchanged, whereas the d-component of the acid yielded benzyl alcohol,
C6H5·CH2·OH,
phenylglyoxylic acid,
C6H5·CO·COOH, and the
hydroxy-acid
C6H5·CH(OH)·COOH.
Since however this hydroxy-acid
was produced in the l-form it probably arose by the asymmetric
reduction of phenylglyoxylic acid, a reaction which can be effected by
yeast as was also found to be the case in the animal body [see Dakin,
, pp. 52, 78]. Moreover it was shown that when the effects of yeast
on a ketonic acid and the corresponding hydroxy-acid were compared,
the alcohol was formed in much better yield from the ketonic acid (70
per cent.) than from the hydroxy-acid (3–4 per cent.), the actual
example being the production of tyrosol (p-hydroxyphenylethyl
alcohol),
OH·C6H4·CH2·CH2OH,
from p-hydroxyphenylpyruvic acid,
OH·C6H4·CH2·CO·COOH,
and p-hydroxyphenyl-lactic acid,
OH·C6H4·CH2·CH(OH)·COOH
respectively.
Neubauer by these experiments established two extremely important
points. 1. That the amino-acids actually yield the corresponding
α-ketonic acids when treated with yeast and sugar solution. 2. That
the a-ketonic acids under similar conditions give the alcohol containing
one carbon atom less in good yield, whereas the corresponding
hydroxy-acids only give an extremely small amount of these alcohols.
It is therefore probable that at an early stage in the decomposition
of the amino-acids by yeast a ketonic acid is produced, which
then undergoes further change.
The source of the oxygen required for this reaction and the mechanism
of oxidation have not yet been definitely ascertained. It is possible
[p093]
that hydrated imino-acids of the type
O
H
│
R·
C
—COOH
│
N
H2
are first formed [Knoop, ], but these
have not as yet been isolated.
The spontaneous production of ketonic aldehydes from amino-acids
and from hydroxy-acids in aqueous solution, which has been demonstrated
by Dakin and Dudley [], points however to the possibility
that the ketonic acid may be a secondary product derived from the
corresponding ketonic aldehyde [see also
Dakin, ;
Neuberg, ,
].
This itself may either arise directly from the amino-acid or
from a previously formed hydroxy-acid, the latter alternative being,
however, improbable in view of the small yield of alcohol obtained from
hydroxy-acids by the action of yeast in the experiments of Neubauer
and Fromherz.
R·CH(NH2)·COOH
→R·CH(OH)·COOH
⇅
⇅
R·CO·CHO
↓ + Oxygen
R·CO·COOH
(2) Whatever be the exact mode by which the ketonic acid is
formed, it appears most probable that a compound of this nature forms
the starting-point for the next stage in the production of the alcohols.
The researches of Neuberg, which have already been discussed on p.
81, have revealed a mechanism in yeast—the enzyme carboxylase—by
which these α-ketonic acids are rapidly broken up into an aldehyde
and carbon dioxide:
R·CO·COOH = R·CHO + CO2
and it can scarcely be doubted that this is the actual
course of the reaction.
(3) The final conversion of the aldehyde into the corresponding alcohol
is also a change which it has been proved can be effected by yeast [Neuberg
and Rosenthal, ]
probably by the aid of the reductase which is one of the weapons in its
armoury of enzymes.
Yeast is capable of producing many vigorous reducing actions and
rapidly reduces methylene blue and sodium selenite. It is in all
probability due to a reaction of this kind that the iso-amylaldehyde and
isovaleraldehyde were reduced to the alcohols in Neuberg and Steenbock's
experiments [,
], and
that considerable quantities of ethyl alcohol are formed in the
sugar free fermentation of pyruvic acid [Neuberg and Kerb, ] (see later p. for a discussion of this question).
A further possibility exists that in some cases the aldehyde may [p094]
be simultaneously oxidised and reduced or the molecule of one
aldehyde reduced and that of another oxidised with production of the
corresponding acid and alcohol by an "aldehydo-mutase," similar to that
which has been observed by
Parnas []
in many animal tissues. Finally
the aldehyde may simply be converted into the corresponding acid by
oxidation as appears to take place in the formation of succinic acid.
The intermediate production of an aldehyde would thus be consistent both
with the production of alcohols and acids from amino-acids.
Fusel oil would be formed by the reduction of the aldehydes arising
from the simple monobasic amino-acids, succinic acid would be produced by
oxidation of the aldehyde derived from the dibasic glutamic acid.
In favour of this view is to be adduced the fact that
aldehydes such as isobutyraldehyde and valeraldehyde have
been found in crude spirit, whilst acetaldehyde is a regular
product of alcoholic fermentation [see Ashdown and Hewitt, ]. Benzaldehyde,
moreover, has been actually detected as a product of the
alcoholic fermentation of phenylaminoacetic acid, C6H5·CH(NH2)·COOH
[Ehrlich, ]. Further,
the aldehydes so produced would readily pass by oxidation into the
corresponding fatty acids, small quantities of which are invariably
produced in fermentation.
This view of the nature of the alcoholic fermentation of the amino-acids
is undoubtedly to be preferred to that previously suggested by
Ehrlich []
according to which a hydroxy-acid is first formed
and then either directly decomposed into an alcohol and carbon dioxide
or into an aldehyde and formic acid, the aldehyde being reduced and
the formic acid destroyed (see p. ).
R·CH(NH2)·COOH →R·CH(
OH)·COOH
↓ or ↓
R·CH2OH + CO2
R·CHO + H·CO2H
↓
R·CH2OH
The most probable course of the decomposition by which isoamyl
alcohol and succinic acid are produced from leucine and glutamic acid
respectively is therefore the following:—
(a) Isoamyl Alcohol.
(1)
(CH3)2·CH·CH2·CH(NH2)·COOH
Leucine
(2)
(CH3)2·CH·CH2·CO·COOH
α-Ketoisovalerianic acid
(3)
(CH3)2CHCH2·CHO
Isovaleraldehyde
+ CO2
(4)
(CH3)2·CH·CH2·CH2OH
Isoamyl alcohol
(b) Succinic Acid.
(1)
COOH·CH2·CH2·CH(NH2)·COOH
Glutamic acid
(2)
COOH·CH2·CH2·CO·COOH
α-Keto-glutaric acid
(3)
COOH·CH2CH2·CHO
Succinic semialdehyde
+ CO2
(4)
COOH·CH2·CH2·COOH
Succinic acid
Glycerol.
[p095]
Of the three chief by-products of alcoholic fermentation, only
glycerol remains at present referable directly to the sugar. This
substance, as shown by the careful experiments of Buchner and
Meisenheimer [],
is formed by the action both of yeast-juice
and zymin to the extent of 3·8 per cent. of the sugar decomposed, and
no other source for its production has so far been experimentally demonstrated.
If it be true that during the decomposition of sugar
into alcohol and carbon dioxide, substances containing three carbon
atoms are formed as intermediate compounds (see p. ), it is obvious
that these might by reduction be converted into glycerol which would
thus be a true by-product of the alcoholic fermentation of sugar. [See
Oppenheimer, .]
It has, however, been suggested that it may
in reality be a product of decomposition of lipoid substances or of
the nuclein of the cell (Ehrlich).
The effect of Ehrlich's work has been clearly to distinguish the
chemical changes involved in the production of fusel oil and succinic
acid from those concerned in the decomposition of sugar into alcohol
and carbon dioxide, and to bring to light a most important series of
reactions by means of which the yeast-cell is able to supply itself
with nitrogen, one of the indispensable conditions of life.
CHAPTER VIII.
THE CHEMICAL CHANGES INVOLVED IN FERMENTATION.
[p096]
It has long been the opinion of chemists that the remarkable and
almost quantitative conversion of sugar into alcohol and carbon dioxide
during the process of fermentation is most probably the result of a
series of reactions, during which various intermediate products are
momentarily formed and then used up in the succeeding stage of the
process. No very good ground can be adduced for this belief except
the contrast between the chemical complexity of the sugar molecule
and the comparative simplicity of the constitution of the products.
Many attempts have, however, been made to obtain evidence of such a
series of reactions, and numerous suggestions have been made of probable
directions in which such changes might proceed. In making
these suggestions, investigators have been guided mainly by the changes
which are produced in the hexoses by reagents of known composition.
The fermentable hexoses, glucose, fructose, mannose, and galactose,
appear to be relatively stable in the presence of dilute acids at the
ordinary temperature, and are only slowly decomposed at 100°, more
rapidly by concentrated acids, with formation of ketonic acids, such
as levulinic acid, and of coloured substances of complex and unknown
constitution.
In the presence of alkalis, on the other hand, the sugar molecule is
extremely susceptible of change. In the first place, as was discovered
by Lobry de Bruyn [;
Bruyn and Ekenstein, ;
;
,
,
,
],
each of the three hexoses, glucose, fructose, and mannose
is converted by dilute alkalis into an optically almost inactive mixture
containing all three, and probably ultimately of the same composition
whichever hexose is employed as the starting-point.
This interesting phenomenon is most simply explained on the assumption
that in the aqueous solution of any one of these hexoses,
along with the molecules of the hexose itself, there exists a small proportion
of those of an enolic form which is common to all the three
hexoses, as illustrated by the following formulæ, the aldehyde formulæ
[p097]
being employed instead of the γ-oxide formulæ for the sake of
simplicity:—
C
HO
C
HO
C
H2(OH)
C
H(OH)
│
│
│
║
H
C
OH
HO
C
H
C
O
C
OH
│
│
│
│
HO
C
H
HO
C
H
HO
C
H
HO
C
H
│
│
│
│
H
C
OH
H
C
OH
H
C
OH
H
C
OH
│
│
│
│
H
C
OH
H
C
OH
H
C
OH
H
C
OH
│
│
│
│
C
H2(OH)
C
H2(OH)
C
H2(OH)
C
H2(OH)
Glucose
Mannose
Fructose
Enolicform
This enolic form is capable of giving rise to all three hexoses, and
the change by which the enolic form is produced and converted into
an equilibrium mixture of the three corresponding hexoses is catalytically
accelerated by alkalis, or rather by hydroxyl ions. In neutral
solution the change is so slow that it has never been experimentally
observed; in the presence of decinormal caustic soda solution at 70°
the conversion is complete in three hours. Precisely similar effects are
produced with galactose, which yields an equilibrium mixture containing
talose and tagatose, sugars which appear not to be fermentable.
The continued action even of dilute alkaline solutions carries the
change much further and brings about a complex decomposition
which is much more rapidly effected by more concentrated alkalis
and at higher temperatures. This change has been the subject of
very numerous investigations [for an account of these see E. v.
Lippmann, , pp. 328, 713, 835],
but for the present purpose the
results recently obtained by Meisenheimer []
may be quoted as
typical. Using normal solutions of caustic soda and concentrations
of from 2 to 5 grams of hexose per 100 c.c., it was found that at
air temperature in 27 to 139 days from 30 to 54 per cent. of the
hexose was converted into inactive lactic acid, C3H6O3, from 0·5 to
2 per cent. into formic acid, CH2O2, and about 40 per cent. into a
complex mixture of hydroxy-acids, containing six and four carbon
atoms in the molecule. Usually only about 74 to 90 per cent. of the
sugar which had disappeared was accounted for, but in one case the
products amounted to 97 per cent. of the sugar. About 1 per cent.
of the sugar was probably converted into alcohol and carbon dioxide.
No glycollic acid, oxalic acid, glycol, or glycerol was produced.
The fact that alcohol is actually formed by the action of alkalis on
sugar was established by Buchner and Meisenheimer [], who obtained
small quantities of alcohol (1·8 to 2·8 grams from 3 kilos. of cane
sugar) by acting on cane sugar with boiling concentrated caustic soda
[p098]
solution. It is evident that under these conditions an extremely complex
series of reactions occurs, but the formation of alcohol and carbon
dioxide and of a large proportion of lactic acid deserves more particular
attention.
The direct formation of alcohol from sugar by the action
of alkalis appears first to have been observed by Duclaux [], who exposed a solution of glucose
and caustic potash to sunlight and obtained both alcohol and carbon
dioxide. As much as 2·6 per cent. of the sugar was converted into
alcohol in a similar experiment made by Buchner and Meisenheimer []. When the
weaker alkalis, lime water or baryta water, were employed instead
of caustic potash, however, no alcohol was formed, but 50 per cent.
of the sugar was converted into inactive lactic acid [Duclaux, , ].
Duclaux therefore regarded the alcohol and carbon dioxide as secondary
products of the action of a comparatively strong alkali on preformed
lactic acid. Ethyl alcohol can, in fact, be produced from lactic acid
both by the action of bacteria [Fitz, ]
and of moulds [Mazé, ], and also by
chemical means. Thus Duclaux []
found that calcium lactate solution exposed to sunlight underwent
decomposition, yielding alcohol and calcium carbonate and
acetate, whilst Hanriot [, ], by heating calcium lactate with slaked
lime obtained a considerable quantity of a liquid which he regarded
as ethyl alcohol, but which was shown by Buchner and Meisenheimer [] to be a mixture
of ethyl alcohol with isopropyl alcohol.
It appears, therefore, that inactive lactic acid can be quite readily
obtained in large proportion from the sugars by the action of alkalis,
whilst alcohol can only be prepared in comparatively small amount and
probably only as a secondary product of the decomposition of lactic
acid.
The study of the action of alkalis on sugar has, however, yielded
still further information as regards the mechanism of the reaction by
which lactic acid is formed. A considerable body of evidence has
accumulated, tending to show that some intermediate product of the
nature of an aldehyde or ketone containing three carbon atoms is first
formed.
Thus Pinkus [] and subsequently
Nef [, ],
by acting
on glucose with alkali in presence of phenylhydrazine obtained
the osazone of methylglyoxal,
CH3·CO·CHO. This osazone
may be formed either from methylglyoxal itself, from acetol,
CH3·CO·CH2·OH,
or from lactic aldehyde,
CH3·CH(OH)·CHO
[Wohl, ].
Methylglyoxal itself may also be regarded as a secondary
[p099]
product derived from glyceraldehyde,
CH2(OH)·CH(OH)·CHO,
or dihydroxyacetone,
CH2(OH)·CO·CH2(OH),
by a process of intramolecular
dehydration, so that the osazone might also be derived
indirectly from either of these compounds [see also Neuberg and
Oertel, ].
Methylglyoxal itself readily passes into lactic acid
when it is treated with alkalis, a molecule of water being taken up:—
CH3·CO·CHO + H2O = CH3·CH(OH)·COOH.
Further evidence in the same direction is afforded by the interesting
discovery of Windaus and Knoop [],
that glucose is converted by
ammonia in presence of zinc hydroxide into methyliminoazole,
CH3·
C
·NH·
C
H
║
║
,
H
C
────
N
a substance which is a derivative of methylglyoxal.
The idea suggested by Pinkus that acetol is the first product of
the action of alkalis on sugar has been rendered very improbable by
the experiments of Nef, and the prevailing view (Nef, Windaus and
Knoop, Buchner and Meisenheimer) is that the first product is glyceraldehyde,
which then passes into methylglyoxal, and finally into lactic
acid:—
(1) C6H12O6 = 2 CH2(OH)·CH(OH)·CHO.
(2) CH2(OH)·CH(OH)·CHO = CH3·CO·CHO + H2O.
(3) CH3·CO·CHO + H2O = CH3·CH(OH)·COOH.
All these changes may occur at ordinary temperatures in the presence
of a catalyst, and in so far resemble the processes of fermentation by
yeasts and bacteria.
The first attempt to suggest a scheme of chemical reactions by
which the changes brought about by living organisms might be
effected was made in 1870 by Baeyer [],
who pointed out that
these decompositions might be produced by the successive removal
and re-addition of the elements of water. The result of this would
be to cause an accumulation of oxygen atoms towards the centre of
the chain of six carbon atoms, which, in accordance with general experience,
would render the chain more easily broken. Baeyer formulated
the changes characteristic of the alcoholic and lactic fermentations
as follows, the intermediate stages being derived from the
hydrated aldehyde formula of glucose by the successive removal and
addition of the elements of water:
[p100]
I.
C
H2·OH
│
C
H·OH
│
C
H·OH
│
C
H·OH
│
C
H·OH
│
C
H(OH)2
II.
C
H2 . . . OH
│
C
OH . . H
│
C
. . OH . . H
│
C
OH . . . H
│
C
OH . . . H
│
C
H . . . (OH)2
III.
C
H3
│
C
H . OH
│
C
(OH)2
│
C
(OH)2
│
C
(OH)2
│
C
H3
IV.
C
H3
│
C
H(OH)
│
C
O
╱
O
╲
C
O
│
C
H(OH)
│
C
H3
V.
C
H3
│
C
H2
╱
O
╲
C
O
╱
O
╲
C
O
╱
O
╲
C
H2
│
C
H3
The immediate precursor of alcohol and carbon dioxide is here seen to
be the anhydride of ethoxycarboxylic acid (V), whilst that of lactic acid
is lactic anhydride (IV). (Baeyer does not appear, as recently stated by
Meisenheimer [, p. 8], Wohl
[], and Buchner and Meisenheimer
[] to have
suggested that lactic acid was an intermediate product in alcoholic
fermentation, but rather to have represented independently the course of
the two different kinds of fermentation, the alcoholic and the lactic.)
It was subsequently pointed out by Buchner and Meisenheimer [] that Baeyer's
principle of oxygen accumulation might be applied in a different way, so
that a ketonic acid would be produced, the decomposition of which, in a
manner analogous to that of acetoacetic acid, would lead to the formation
of two molecules of lactic acid, from which the final products alcohol
and carbon dioxide might be directly derived, as shown in the following
formulæ:—
C
HO
·
C
H(OH)
·
C
H(OH)
·
C
H(OH)
·
C
H(OH)
·
C
H2(OH)
C
OOH
·
C
H(OH)
·
C
H2
·
C
O
·
C
H(OH)
·
C
H3
C
OOH
·
C
H(OH)
·
C
H3
─
───
C
OOH
·
C
H(OH)
·
C
H3
C
O2
─
───
C
H2·OH
·
C
H3
─
───
C
O2
─
───
C
H2·OH
·
C
H3
A scheme based on somewhat different principles has been propounded
by Wohl [Lippmann, ,
p. 1891], and has been accepted by Buchner
and Meisenheimer []
as more probable than that quoted above.
Wohl and Oesterlin []
were able to trace experimentally the
various stages of the conversion of tartaric acid (I) into oxalacetic
acid (III), which can be carried out by reactions taking place at the
ordinary temperature, and they found that the first stage consisted
in the removal of the elements of water leaving an unsaturated
hydroxy derivative (II) which in the second stage underwent intramolecular
change into the corresponding keto-compound (III):
[p101]
C
OOH
C
OOH
C
OOH
·
·
·
C
H(OH)
H
C
(OH)
C
O
·
−·
=║
⇌·
C
H(OH)
OH
C
H
C
H3
·
·
·
C
OOH
C
OOH
C
OOH
I.Tartaricacid
II.
III.Oxalaceticacid.
This change differs in principle from that assumed by Baeyer, inasmuch
as the second stage is not effected by the re-addition of water,
but by the keto-enol transformation, which is now usually ascribed to
the migration of the hydrogen atom, although the same result can
theoretically be arrived at by the addition and removal of the elements
of water. The analogy of this process to what might be supposed to
occur in the conversion of sugar into carbon dioxide and alcohol was
pointed out by Wohl and Oesterlin, and subsequently Wohl developed
a theoretical scheme of reactions by which the process of alcoholic
fermentation could be represented. In the first place the elements of
water are removed from the α and β carbon atoms of glucose (I) and
the resulting enol (II) undergoes conversion into the corresponding
ketone (III), which has the constitution of a condensation product of
methylglyoxal and glyceraldehyde, and hence is readily resolved by
hydrolysis into these compounds (IV). The glyceraldehyde passes by
a similar series of changes (V, VI) into methylglyoxal, and this is
then converted by addition of water into lactic acid (VII), a reaction
which is common to all ketoaldehydes of this kind. Finally, the lactic
acid is split up into alcohol and carbon dioxide (VIII):—
C
HO
C
HO
C
HO
│
│
│
C
H(OH)
C
(OH)
C
O
│
H
║
│
C
H(OH)
−OH C
H
⇌C
H2
│
│
│
C
H(OH)
C
H(OH)
C
H(OH)
│
│
│
C
H(OH)
C
H(OH)
C
H(OH)
│
│
│
C
H2(OH)
C
H2(OH)
C
H2(OH)
I.Glucose.
II.
III.
Methyl-glyoxal
C
HO
C
OOH
C
O2
│
│
─
───
C
O
+H2O
C
H(OH)
C
H2OH
│
│
│
C
H3
C
H3
C
H3
C
HO
C
HO
C
HO
C
OOH
C
O2
│
│
│
│
─
────
C
H(OH)
H C
(OH)
⇌C
O + H2O C
H(OH) C
H2OH
│
· ║
│
│
│
C
H2(OH)
− HO C
H2
C
H3
C
H3
C
H3
IV.Glyceral-dehyde.
V.
VI.Methyl-glyoxal.
VII.Lacticacid.
VIII.Alcoholandcarbondioxide.
[p102]
This scheme agrees well with the current ideas as to the formation
of lactic acid from glucose under the influence of alkalis (p. ). It postulates the formation as intermediate
products of no less than three compounds containing a chain of three carbon
atoms—glyceraldehyde, methylglyoxal, and lactic acid.
The Lactic Acid Theory of Alcoholic Fermentation.
A practical interest was given to these various schemes by the fact
that Buchner and Meisenheimer adduced experimental evidence in
favour of the view that lactic acid is an intermediate product in the
formation of alcohol and carbon dioxide from sugar by fermentation
[,
,
,
].
These observers proved by a series of very careful analyses
that yeast-juice frequently, but not invariably, contains small quantities
of lactic acid, not exceeding 0·2 per cent. When yeast-juice is incubated
alone or with sugar the amount of lactic acid may either
increase or decrease. Moreover, lactic acid added to the juice is sometimes
diminished and sometimes increased in quantity. On the whole
it appears that the addition of a considerable quantity of sugar or of
some lactic acid favours the disappearance of lactic acid. Juices of
low fermenting power produce a diminution in the lactic acid present,
those of high fermenting power an increase.
In all cases the amounts of lactic acid either produced or destroyed
are very small in relation to the volume of the yeast-juice employed.
Throughout the whole series of experiments the greatest increase
amounted to 0·47 per cent. on the juice employed, and the greatest decrease
to 0·3 per cent. [See also Oppenheimer, .]
Buchner and
Meisenheimer at one time regarded these facts as strong evidence that
lactic acid is an intermediate product of alcoholic fermentation. It was
thought probable that the production of alcohol and carbon dioxide
from sugar occurred in at least two stages and under the influence of
two distinct enzymes. The first stage consisted in the conversion of
sugar into lactic acid, and for the enzyme which brought about this
decomposition was reserved the name zymase or yeast-zymase. The
lactic acid was then broken down into alcohol and carbon dioxide by
the second enzyme, lactacidase.
This theory, which is quite in harmony with the current ideas as to
the mode of decomposition of sugars by alkalis, and is also consistent
with Wohl's scheme of reactions, is open to adverse criticism from
several points of view. In the first place, it is noticeable that the total
amount of lactic acid used up by the juice is extremely small, even
[p103]
in the most favourable cases, relatively to the amount of the juice
[Harden, ],
and it may be added to the sugar-fermenting power
of the juice. Moreover, as pointed out by Buchner and Meisenheimer
themselves [],
no proof is afforded that the lactic acid which
disappears is converted into alcohol and carbon dioxide. It is not
even certain, although doubtless probable, that the lactic acid which
occurs or is produced in the juice is really derived from sugar.
The most weighty criticism of the theory is that of
Slator [,
;
,
],
which is based on the consideration that if lactic acid be an intermediate
product of alcoholic fermentation the reaction by which it is fermented
must proceed at least as rapidly as that by which it is formed, in order
to prevent accumulation of lactic acid. The fermentation of lactic acid
by yeast should therefore proceed at least as rapidly as that of glucose.
So far is that from being the case that it has been experimentally
demonstrated that lactic acid is not fermented at all by living yeast.
This conclusion was rendered extremely probable by Slator, who showed
that lactic acid, even in concentrations insufficient to prevent the
fermentation of glucose, is not fermented to any considerable extent.
The final proof that lactic acid is neither formed nor fermented by
pure yeast has been brought by Buchner and Meisenheimer in a series
of very careful quantitative experiments carried out with a pure
yeast and with strict precautions against bacterial contamination
[, ].
At first sight this fact appears decisive against the validity of the
lactic acid theory, and it is recognised as such by Buchner and Meisenheimer.
Wohl has, however, suggested that the non-fermentability of
lactic acid by yeast is not really conclusive
[;
see also Franzen and Steppuhn, ].
The production of lactic acid from glucose
is attended by the evolution of a considerable amount of heat (22
cal.), and it is possible that at the moment of production the molecule
of the acid is in a condition of activity corresponding with a much
higher temperature than the average temperature of the fermenting
liquid. Under these circumstances the molecule would be much more
susceptible of chemical change than at a later period when temperature
equilibrium had been attained. It has, however, been pointed out
by Tafel [],
that such a decomposition of the lactic acid would
occur at the very instant of formation of the molecule, so that no ground
remains even on this view for assuming the actual existence of lactic
acid as a definite intermediate product. It has also been suggested
by Luther []
that an unknown isomeride of lactic acid is formed
as an intermediate product and fermented, and that traces of lactic
[p104]
acid are formed by a secondary reaction from this, but no satisfactory
evidence for this view is forthcoming. There still remains a doubt
as to whether the living yeast-cell is permeable to lactic acid, a fact
which would of course afford a very simple explanation of the non-fermentability
of the acid. Apart from this, however, it is difficult, in
face of the evidence just quoted, to believe that lactic acid is in reality
an intermediate product in alcoholic fermentation.
Methylglyoxal, Dihydroxyacetone and Glyceraldehyde.
As regards the fermentability by yeast of compounds containing three
carbon atoms, which may possibly appear as intermediate products in the
transformation of sugar into carbon dioxide and alcohol, many experiments
have been carried out, with somewhat uncertain results. Care has to be
taken that the substance to be tested is not added in such quantity as
to inhibit the fermenting power of the yeast or yeast-juice, and further
that the conditions are such that the substance in question, often of a
very unstable nature, is not converted by some chemical change into a
different fermentable compound. It is also possible that the substance to
be tested may accelerate the rate of autofermentation in a similar manner
to arsenates (pp. , )
and many other substances. These are all points which have not up to the
present received sufficient attention. In the case of living yeast the
further question arises of the permeability of the cell.
Methylglyoxal, CH3·CO·CHO, has been tested
by Mayer [] and Wohl
[] with yeast, and
by Buchner and Meisenheimer both with acetone-yeast [] and yeast-juice
[], in every
case with negative results, but it may be noted that the concentration
employed in the last mentioned of these experiments was such as
considerably to diminish the autofermentation of the juice.
Glyceraldehyde, CH2(OH)·CH(OH)·CHO, was also tested
with yeast with negative results by Wohl []
and by Emmerling [], who employed
a number of different yeasts. The same negative result attended the
experiments of Piloty [] and Emmerling
[] with pure dihydroxyacetone.
Fischer and Tafel [, ], however, had previously found
that glycerose, a mixture of glyceraldehyde and dihydroxyacetone prepared
by the oxidation of glycerol, was readily fermented by yeast, agreeing
in this respect with the still older observations of Van Deen and of
Grimaux. The reason for this diversity of result has not been definitely
ascertained, but it has been supposed by Emmerling to lie in the formation
of some fermentable sugar from [p105] glycerose when the latter is
subjected to too high a temperature during its preparation.
On the other hand, Bertrand []
succeeded in fermenting pure dihydroxyacetone by treating a solution of 1
gram in 30 c.c. of liquid with a small quantity of yeast for ten days at
30°, the best result being a fermentation of 25 per cent. of the substance
taken. Moreover, Boysen-Jensen [,
, ] states that he has also observed
both the formation from glucose and the fermentation of this substance
by living yeast, but the amounts of alcohol and carbon dioxide produced
were so minute and the evidence for the production of dihydroxyacetone
so inconclusive that the experiments cannot be regarded as in any way
decisive [see Chick, ; Euler
and Fodor, ; Karauschanoff,
; Buchner and Meisenheimer,
].
A careful investigation by Buchner [] and Buchner and Meisenheimer [] has led them
to the conclusion that both glyceraldehyde and dihydroxyacetone are
fermentable. Glyceraldehyde exerts a powerful inhibiting action both on
yeast and yeast-juice, and was only found to give rise to a very limited
amount of carbon dioxide, quantities of 0·15 to 0·025 gram being treated
with 1 gram of yeast or 5 c.c. of yeast-juice and a production of 4 to 12
c.c. of carbon dioxide being attained.
When 0·1 gram of dihydroxyacetone in 5 c.c. of water was brought
in contact with 1 gram of living yeast, about half was fermented,
17 c.c. of carbon dioxide (at 20° and 600 mm.) being evolved in excess
of the autofermentation of the yeast (13 c.c.). A much greater effect
was obtained by the aid of yeast-juice, and the remarkable observation
was made that whilst yeast-juice alone produced comparatively little
action a mixture of yeast-juice and boiled yeast-juice was much more
effective, quantities of 20 to 50 c.c. of yeast-juice mixed with an equal
volume of boiled juice, which in some experiments was concentrated,
yielding with 0·4, 1, and 2 grams of dihydroxyacetone almost the
theoretical amount of carbon dioxide and alcohol in excess of that
evolved in the absence of this substance. It was further observed that
the fermentation of this substance commenced much more slowly than
that of glucose. No explanation of either of these facts has at present
been offered. The conclusion drawn from their experiments by
Buchner and Meisenheimer that dihydroxyacetone is readily fermentable,
was confirmed by Lebedeff [],
who further made the important
observation that during the fermentation of dihydroxyacetone
the same hexosephosphoric acid is produced as is formed during the
fermentation of the hexoses. Lebedeff accordingly propounded
a scheme of alcoholic fermentation according to which the hexose
[p106]
was first converted into two molecules of triose, the latter being
first esterified to triosephosphoric acid and then condensed to hexosediphosphoric
acid, which then underwent fermentation, after being
hydrolysed to phosphoric acid, and some unidentified substance, probably
an unstable modification of a hexose, much more readily
attacked by an appropriate enzyme than the original glucose or fructose
[, pp. 2941–2].
The idea that the sugar is first converted into triose and this into
triosemonophosphoric acid had been previously suggested by Iwanoff
who postulated the agency of a special enzyme termed synthease
[],
and supposed that this triosemonophosphoric acid was then directly
fermented to alcohol, carbon dioxide and phosphoric acid. According
both to Iwanoff and Lebedeff the phosphoric ester is an intermediate
product and its decomposition provides this sole source of carbon
dioxide and alcohol. This is quite inconsistent with the facts recounted
above (Chap. III), which prove that the formation of the
hexosephosphate is accompanied by an amount of alcoholic fermentation
exactly equivalent to the quantity of hexosephosphate produced, and
that the rate of fermentation rapidly falls as soon as the free phosphate
has disappeared, in spite of the fact that at that moment the concentration
of the hexosephosphate is at its highest, whereas according
to Iwanoff's theory it is precisely under these conditions that the
maximum rate of fermentation should be maintained.
It has also been shown that the arguments adduced by Iwanoff in
favour of the existence of his synthease are not valid [Harden and
Young, ].
The fermentation of dihydroxyacetone was moreover proved by Harden and
Young [] to be effected
by yeast-juice and maceration extract at a much slower rate than that of
the sugars, in spite of the fact that the addition of dihydroxyacetone
did not inhibit the sugar fermentation. The same thing has been shown
for living yeast by Slator []
in agreement with the earlier results of Buchner [] and Buchner and Meisenheimer [].
The logical conclusion from Lebedeff's experiments would appear
rather to be that dihydroxyacetone is slowly condensed to a
hexose and that this is then fermented in the normal manner
[Harden and Young, ;
Buchner and Meisenheimer, ;
Kostytscheff, ].
Buchner and Meisenheimer, however, regard this as improbable
on the ground that dihydroxyacetone, being symmetric in constitution,
would yield an inactive hexose of which only at most 50 per
cent. would be fermentable. Against this it may be urged, however,
[p107]
that enzymic condensation of dihydroxyacetone might very probably
occur asymmetrically yielding an active and completely fermentable
hexose. Buchner and Meisenheimer, however, still support the view
that dihydroxyacetone forms an intermediate stage in the fermentation
of glucose and adduce as confirmatory evidence of the probability of
such a change the observation of Fernbach []
that this compound
is produced from glucose by a bacillus, Tyrothrix tenuis, which effects
the change both when living and after treatment with acetone.
The balance of evidence, however, appears to be in favour of the
opinion that dihydroxyacetone does not fulfil the conditions laid
down by Slator (see p. ) as essential for an intermediate product
in the process of fermentation [see also Löb, ].
Lebedeff subsequently [;
Lebedeff and Griaznoff, ]
extended his experiments to glyceraldehyde and modified his theory
very considerably. Using maceration extract it was found in general
agreement with the results of Buchner and Meisenheimer (p. ) that
20 c.c. of juice were capable of producing about half the theoretical
amount of carbon dioxide from 0·2 gram of glyceraldehyde, whereas
0·4 gram caused coagulation of the extract and a diminished
evolution of carbon dioxide. The addition of phosphate diminished
rather than increased the fermentation. Even in the most favourable
concentration however (0·2 gram per 20 c.c.) the glyceraldehyde is
fermented much more slowly than dihydroxyacetone or saccharose,
as is shown by the following figures:—
20 c.c.Extract +0·2 gram.CO2 in grams in successive periods ofDuration of fer-ment-ationTotal CO2
6 hours.
18 hours.
24 hours.
Cane sugar
0·050
0·000
0·000
60·05
Dihydroxy-acetone
0·042
0·000
0·000
60·042
Glycer-aldehyde
0·008
0·022
0·005
48
0·035
Further, during an experiment in which 0·129 gram of CO2 was evolved
in 22·5 hours from 0·9 gram of glyceraldehyde in presence of phosphate,
no change in free phosphate was observed, whereas in a similar experiment
with glucose a loss of about 0·2 gram of P2O5 would have occurred.
Hence the fermentation takes place without formation of hexosediphosphate.
This was confirmed by the fact that the osazone of hexosephosphoric
acid was readily isolated from the products of fermentation
of dihydroxyacetone (0·259 gram of CO2 having been evolved in
twenty hours) but could not be obtained from those of glyceraldehyde
(0·138 gram CO2 in twenty hours).
[p108]
This result is extremely interesting, although it is not impossible
that the rate of fermentation of the glyceraldehyde is so slow that any
phosphoric ester produced is hydrolysed as rapidly as it is formed.
Lebedeff regards the experiments as proof that phosphate takes no
part in the fermentation of glyceraldehyde and bases on this conclusion
and his other work the following theory of alcoholic fermentation.
1. The sugar is split up into equimolecular proportions of glyceraldehyde
and dihydroxyacetone:—
(a) C6H12O6 = C3H6O3 + C3H6O3.
2. The dihydroxyacetone then passes through the stages previously
postulated (p. ).
(b) 4 C3H6O3 + 4 R2HPO4 = 4 C3H5O2PO4R2 + 4 H2O.
(c) 4 C3H5O2PO4R2 = 2 C6H10O4(R2PO4)2.
(d) 2 C6H10O4(R2PO4)2 + 4 H2O = 2 C6H12O6 + 4 R2HPO4.
After which the hexose, C6H12O6 re-enters the cycle at (a).
3. The fermentation of the glyceraldehyde occurs according to
the scheme developed by Kostytscheff (p. ), pyruvic acid being
formed along with hydrogen and then decomposed into carbon dioxide
and acetaldehyde, which is reduced by the hydrogen. Lebedeff,
however, suggests
[,
] that glyceric acid is first formed (1) and
then converted by an enzyme, which he terms dehydratase into pyruvic
acid (2):—
(1) CH2(OH)·CH(OH)·CHO + H2O → CH2(OH)·CH(OH)·CH(OH)2
→ CH2(OH)·CH(OH)·COOH + 2 H
(2) CH2(OH)·CH(OH)·COOH = CH3·CO·COOH + H2O.
The experimental basis for this idea is the fact that glyceric acid is
fermented by dried yeast and maceration juice [compare Neuberg and
Tir, ].
This scheme has the merit of recognising the fact that the carbon
dioxide does not wholly arise from the products of decomposition of
hexosephosphate, nor from its direct fermentation. The function
assigned to the phosphate is that of removing dihydroxyacetone
and thus preventing it from inhibiting further conversion of hexose
into triose, according to the reversible reaction
C6H12O6 ⇌
2 C3H6O3.
This however appears to be quite inadequate, since, on the one hand,
the fermentation of glucose proceeds quite freely in presence of as
much as 5 grams per 100 c.c. of dihydroxyacetone [Harden and Young, ], and on the other
hand alcoholic fermentation appears not to proceed at all in the absence
of phosphate (see p. ). This forms the chief
objection to the theory in its present form. The slow rate at which [p109]
glyceraldehyde is fermented also affords an argument against the
validity of Lebedeff's view, but this may possibly be accounted for to some
extent by the fact that glyceraldehyde is a strong inhibiting agent so that
it might be more rapidly fermented if added in a more dilute condition.
The unfermented glyceraldehyde cannot be recovered from
the solution and nothing is known as to its fate except
that it readily gives rise both to lactic acid and glycerol
[Oppenheimer, , ]. Evidently the reaction between
glyceraldehyde and yeast-juice is by no means a simple one.
The Pyruvic Acid Theory.
The third stage of Lebedeff's theory postulates the intermediate
formation of pyruvic acid. This idea immediately suggested itself when it
became known that yeast was capable of rapidly decomposing a-ketonic
acids with evolution of carbon dioxide [see Neubauer and Fromherz, , p. 350; Neuberg
and Kerb, ;
Kostytscheff, ].
This scheme has been differently elaborated by different
workers. According to Kostytscheff it involves (1) the production of
pyruvic acid from the hexoses, a process accompanied by loss of hydrogen;
(2) the decomposition of pyruvic acid into acetaldehyde and
carbon dioxide; and (3) the reduction of the acetaldehyde to ethyl
alcohol.
(1) C6H12O6 = 2 CH3·CO·COOH + 4[H].
(2) 2 CH3·CO·COOH = 2 CH3·CHO + 2 CO2.
(3) 2 CH3·CHO + 4 H = 2 CH3·CH2·OH.
1. As regards the production of pyruvic acid from the hexoses by
yeast, the only direct evidence is afforded by the experiments of
Fernbach and Schoen [] who have obtained a calcium salt having
the qualitative properties of a pyruvate by carrying out alcoholic
fermentation by yeast in presence of calcium carbonate, but have not
yet definitely settled either the identity of the acid or its origin from
sugar. Pyruvic acid is, however, very closely related to several substances
which are intimately connected both chemically and biochemically
with the hexoses. Thus lactic acid is its reduction product,
CH3·CO·COOH + 2 H → CH3·CH(OH)·COOH,
glyceraldehyde can readily be converted into it by oxidation to glyceric
acid followed by abstraction of water (Erlenmeyer),
[p110]
CH2(OH)·CH(OH)·CHO + O → CH2(OH)·CH(OH)·COOH
CH2(OH)·CH(OH)·COOH − H2O → CH3·CO·COOH,
and finally methylglyoxal CH3·CO·CHO is its aldehyde.
2. The decomposition of pyruvic acid into acetaldehyde and carbon
dioxide has already been fully discussed (Chapter VI). The universality
of the enzyme carboxylase in yeasts and the rapidity of its
action on pyruvic acid form the strongest evidence at present available
in favour of the pyruvic acid theory. Given the pyruvic acid, there is
no doubt that yeast is provided with a mechanism capable of decomposing
it at the same rate as an equivalent amount of sugar.
3. The final step postulated by the pyruvic acid theory is the
quantitative reduction to ethyl alcohol of the acetaldehyde formed
from the pyruvic acid.
The idea that acetaldehyde is an intermediate product in the
various fermentations of sugar has frequently been entertained
[Magnus Levy, ; Leathes, ; Buchner and Meisenheimer, ; Harden and
Norris, D., ]
although no very definite experimental foundation exists for the belief.
It is, however, a well-known fact that traces of acetaldehyde are
invariably formed during alcoholic fermentation [see Ashdown and Hewitt,
], and this is of
course consistent with the occurrence of acetaldehyde as an intermediate
product. Important evidence as to the specific capability of yeast to
reduce acetaldehyde to alcohol has been obtained by several workers. Thus
Kostytscheff [; Kostytscheff
and Hübbenet, ]
found that pressed yeast, dried yeast and zymin all reduced acetaldehyde
to alcohol, 50 grams of yeast in 10 hours producing from 660 mg.
of aldehyde 265 mg. of alcohol in excess of the amount produced by
autofermentation in absence of added aldehyde. Maceration extract was
found to reduce both in absence and in presence of sugar, whereas Lebedeff
and Griaznoff []
obtained no reduction in presence of sugar, and observed that the power
of reduction was lost by the extract on digestion, a circumstance which
suggests the co-operation of a co-enzyme in the process. Neuberg and
Kerb [; ] have also
been able to show by large scale experiments that alcohol is produced in
considerable quantity by the fermentation of pyruvic acid by living yeast
in absence of sugar and that the yield is increased by the presence of
glycerol. When treated with 22 kilos, of yeast, 1 kilo, of pyruvic acid
yielded 241 grams of alcohol in excess of that given by the yeast alone,
whilst in presence of glycerol the amount was 360 grams, the amount
theoretically obtainable being 523 grams. The function of the glycerol is
not understood but is probably that of lessening the rate of destruction
of the yeast enzymes. [p111]
That yeast possesses powerful reducing properties has long been
known and many investigations have been made as to the relation of
these properties to the process of alcoholic fermentation. Thus Hahn
(Buchner, E. and H., and Hahn, 1903, p. 343) found that the power
of reducing methylene blue was possessed both by yeast and zymin and
on the whole ran parallel to the fermenting power in the process of
alcoholic fermentation. The intervention of a reducing enzyme was suggested
by Grüss [,
,
] and was supported by
Palladin [].
The latter observed that zymin which reduces sodium selenite and
methylene blue in absence of sugar almost ceases to do so in presence of a
fermentable sugar, and concluded that the great diminution of reduction
during fermentation was due to the fact that the reducing enzyme was
largely combined with a different substrate arising from the sugar, the
reduction of which was necessary for alcoholic fermentation. Grüss,
however, found that with living yeast the reduction is greatly increased
in presence of a fermentable sugar, while Harden and Norris, R. V.
[]
confirmed the observation of Grüss, but found that the reducing
power of zymin is not seriously affected by the presence of a fermentable
sugar in concentration less then 20 grams per 100 c.c., whilst its fermenting
power for glucose is inhibited by 1 per cent. sodium selenite.
Hence Palladin's conclusion cannot be regarded as proved.
Interesting attempts have been made by Kostytscheff and later by
Lvoff to obtain evidence of the participation of a reductase in alcoholic
fermentation by adding some substance which would be capable
either of taking up hydrogen and thus preventing the reduction of the
acetaldehyde or of converting the aldehyde into some compound less
liable to reduction.
Kostytscheff [;
,
;
;
Kostytscheff and Hübbenet, ;
Kostytscheff and Scheloumoff, ;
Kostytscheff and Brilliant, ]
has examined the effect of the addition of zinc chloride, chosen
with the idea that it might polymerise the aldehyde and thus remove
it from the sphere of action. As pointed out by Neuberg and Kerb
[]
this action is not very probable, and it was subsequently
found [Kostytscheff and Scheloumoff, ]
that the effect of added
zinc salts was more probably specifically due to the zinc ion. Fermentation
of sugar by dried yeast still proceeds when 0·6 gram of ZnCl2
is added to 10 grams of the yeast and 50 c.c. of water, whereas it ceases
in the presence of 1·2 gram of ZnCl2. Even the addition of 0·075 gram
however greatly diminishes the rate of fermentation and the total
amount of sugar decomposed. The most noteworthy effect is that the
production of acetaldehyde is increased both in autofermentation and
[p112]
in sugar fermentation. The course of the reaction is further modified
in the sense that the percentage of sugar used up which can be accounted
for in the products decreases, in other words the "disappearing
sugar" (p. ) increases. In long continued fermentations moreover
and particularly with high concentrations of zinc chloride less
alcohol is produced than is equivalent to the carbon dioxide evolved.
The interpretation of these results is difficult. Kostytscheff takes
them to mean (1) that the zinc salt modifies one stage of the reaction
so that a higher concentration of intermediate products is obtained, and
(2) that the carbon dioxide and alcohol must be produced at different
stages or their ratio, in the absence of secondary changes, would be
unalterable.
Alternative interpretations are, however, by no means excluded. Thus
Neuberg and Kerb [; ] do
not regard it as conclusively proved that the aldehyde really arises from
the sugar since they have observed its production in maceration extract
free from autofermentation. The method used by Kostytscheff for the
separation of alcohol and aldehyde (treatment with bisulphite) has also
proved unsatisfactory in their hands and the results obtained as to the
reduction of acetaldehyde by yeast, etc., are not accepted. They also
consider that in any case the small amounts produced (less than 0·2 per
cent. of the sugar used) would not afford convincing evidence that the
aldehyde is an intermediate product, although it must be admitted that no
large accumulation of an intermediate product could be reasonably expected.
It may also be pointed out that the increase in "disappearing sugar" may
be simply due to the fact that in the controls the whole of the sugar was
fermented, so that any polysaccharide formed at an earlier stage would have
been hydrolysed and fermented, whereas in the presence of zinc chloride
excess of sugar was present throughout the whole experiment.
Lvoff [, , ]
has made quantitative experiments on the effect of methylene blue both
on the sugar fermentation and autofermentation of dried yeast and
maceration extract. In presence of sugar the methylene blue causes a
decrease in the extent of fermentation, the difference during the time
required for reduction of the methylene blue being represented by an
amount of glucose equimolecular to the latter. In the absence of sugar
on the other hand an excess of carbon dioxide equimolecular to the
methylene blue is evolved but no corresponding increase in the alcohol
production occurs. The effect of methylene blue is evidently complex
and it is impossible at present to say whether Lvoff's contention is
correct that the methylene blue actually [p113]
interferes with the fermentation by taking up hydrogen (2 atoms per
molecule of glucose) destined for the subsequent reduction of some
intermediate product or whether the effect is one of general depression
of the fermenting power which would be presumably proportional to
the concentration of methylene blue and inversely proportional to
that of the fermenting complex [see Harden and Norris, R. V., ]. In any
case it will be noticed that Lvoff s interpretation of the results
is at variance with the requirements of Kostytscheff's theory (p. ) according to which 4 atoms of hydrogen should be
given off by a molecule of glucose.
Kostytscheff [;
Kostytscheff and Scheloumoff, ] has
also observed a depression of the extent of fermentation by methylene
blue without any serious alteration in the ratio of CO2 to alcohol,
although an increase occurs in the production of acetaldehyde.
On the whole it cannot be said that the evidence gathered
from experiments on the reduction of acetaldehyde and methylene
blue is very convincing. All that is established beyond doubt
seems to be that yeast possesses a reducing mechanism for many
aldehydes [see also in this connection Lintner and Luers, ; Lintner and von Liebig, ; as well as Neuberg
and Steenbock, , ] and colouring matters.
This mechanism appears to be capable of activity in the absence of sugar
and it is to be supposed that in accordance with the views of Bach [] the necessary hydrogen is derived from water
and that some acceptor for the oxygen simultaneously liberated is also
present. There seems however at the moment to be no sufficient reason to
suppose that this mode of reduction is in any way altered by the presence
of sugar and until the production of intermediate products equivalent to
the amount of substance reduced is actually demonstrated, the conclusions
of these workers may be regarded as not fully justified.
Neuberg and Kerb [] themselves tentatively propose a
complicated scheme possessing some novel features according to which
methylglyoxal is the starting-point for the later stages of the change.
(a) A small portion of this is converted by a reaction which may
be variously interpreted as a Cannizzaro transformation or a reductase
reaction into glycerol and pyruvic acid.
CH2:C(OH)·CHO + H2O H2
+ │ = O
CH2(OH)·CHOH·CH2(OH)(glycerol)
+
CH2:C(OH)·CHO
CH2:C(OH)·COOH(Pyruvic acid)
(b) The pyruvic acid is then decomposed by carboxylase yielding
aldehyde and carbon dioxide (equation 2, p. ).
[p114]
(c) The aldehyde and a molecule of glyoxal then undergo a Cannizzaro
reaction and yield alcohol and pyruvic acid,
CH3·CO·CHO
O
CH3·CO·COOH
+ │ =+
CH3·CHO
H2
CH3CH2(OH)
and the latter then undergoes reaction (b).
A small amount of glycerol is thus necessarily formed, as is actually
found to be the case.
The experimental foundation for stages (a) and (c) will be awaited
with great interest, as well as the proof that methylglyoxal is readily
fermentable (see p. ).
The Formic Acid Theory.
An interesting interpretation of the phenomena of fermentation
was attempted by Schade [] based upon the conception that
glucose under the influence of catalytic agents readily decomposes into
acetaldehyde and formic acid. It was subsequently found that the experimental
evidence upon which this conclusion was founded had been
wrongly interpreted
[Buchner, Meisenheimer, and Schade, ;
Schade, ],
but Schade has succeeded in devising an interesting series of
reactions by means of which alcohol and carbon dioxide can be obtained
from sugar by the successive action of various catalysts. The following
are the stages of this series: (1) Glucose, fructose, and mannose are
converted by alkalis into lactic acid along with other products. (2)
Lactic acid when heated with dilute sulphuric acid yields a mixture of
acetaldehyde and formic acid:—
CH3·CH(OH)·COOH = CH3·CHO + H·COOH.
(3) It has long been known that formic acid is catalysed by metallic
rhodium at the ordinary temperature into hydrogen and carbon dioxide,
and Schade has found that when a mixture of acetaldehyde and formic
acid is submitted to the action of rhodium the acetaldehyde is reduced
to alcohol at the expense of the hydrogen and the carbon dioxide
is evolved:—
CH3·CHO + H·COOH = CH3·CH2(OH) + CO2.
Schade suggests [] that the fermentation of sugar may proceed by
a similar series of reactions catalysed by enzymes, the acetaldehyde
and formic acid being derived not from the relatively stable lactic acid
but more probably from a labile substance capable of undergoing
change either into lactic acid or into aldehyde and formic acid.
It will be noticed that this theory resembles the pyruvic acid
[p115]
theory in postulating the immediate formation of acetaldehyde but
differs from it by supposing that the reduction is effected at the expense
of formic acid produced at the same time.
The acetaldehyde question has already been discussed. In view of
the fact that formic acid is a regular product of the action of many
bacteria on glucose [see Harden, ],
Schade's theory of alcoholic fermentation may be said to be a possible
interpretation of the facts. Formic acid is known to be present in
small amounts in fermented sugar solutions and the actual behaviour of
yeast towards this substance has been investigated in some detail by
Franzen and Steppuhn [;
, ], who have obtained results
strongly reminiscent of those obtained with lactic acid by Buchner and
Meisenheimer (p. ). Many yeasts when grown in
presence of sodium formate decompose a certain proportion of it, whereas
in absence of formate they actually produce a small amount of formic
acid—the absolute quantities being usually of the order of 0·0005 gram
molecule (0·023 gram) per 100 c.c. of medium in 4 to 5 days. Only in the
case of S. validus did the consumption of formic acid in 5 days reach
0·0017 gram molecule (0·08 gram). Somewhat similar but rather smaller
results were given by yeast-juice, a small consumption of formic acid being
usually observed. The possibility thus exists that formic acid may be an
intermediate product of alcoholic fermentation and Franzen argues strongly
in favour of this view.
Direct experiment, on the other hand, shows that yeast-juice cannot
ferment a mixture of acetaldehyde and formic acid, even when these
are gradually produced in molecular proportions in the liquid by the
slow hydrolysis of a compound of the two, ethylideneoxyformate, OHC·O·CH(CH3)·O·CH(CH3)·O·CHO,
this method being adopted to avoid the inhibiting effect of
free acetaldehyde and formic acid [Buchner and Meisenheimer, ]. Nor is the
reduction of acetaldehyde assisted by the presence of formate [Neuberg
and Kerb, ;
Kostytscheff and Hübbenet, ].
A modified form of Schade's theory has been suggested by Ashdown
and Hewitt [], who have found that when brewer's
yeast is cultivated in presence of sodium formate the yield of aldehyde,
as a rule, becomes less. They regard the aldehyde as derived from
alanine, CH3·CH(NH2)·COOH,
one of the amino-acids formed from
the proteins by hydrolysis, which is known to be attacked by yeast
in the characteristic manner (p. ), forming alcohol, carbon dioxide,
and ammonia. Fermentation is supposed to proceed in such a way
that the sugar is first decomposed into two smaller molecules, C3H6O3
[p116]
(equation i), and that these react with formamide to produce alanine
and formic acid (ii). The alanine then enters into reaction with formic
acid, producing alcohol, carbon dioxide, and formamide (iii):—
(i) C6H12O6 = 2 C3H6O3.
(ii) C3H6O3 + H·CO·NH2 = CH3·CH(NH2)·COOH + H·COOH.
(iii) CH3·CH(NH2)·COOH + H·COOH = CH3·CH2·OH + CO2 + H·CO·NH2.
According to this scheme all the sugar fermented passes through the
form of alanine, and the formic acid acts along with the enzyme as
catalyst, passing into formamide in reaction (iii) and being regenerated
in (ii). The alanine is in the first place derived from the hydrolysis of
proteins, or possibly by the reaction of the C3H6O3 group with one of
the higher amino-acids:—
C3H6O3 + CnH(2n+1)·CH(NH2)·COOH =
CnH(2n+1)·CH2·OH + CO2 + CH(NH2)·COOH.
There is as little positive evidence for this course of events as for
that postulated by Schade, and the theory suffers from the additional
disability that the chemical reactions involved have not been realised
in the laboratory. Direct experiments with yeast-juice, moreover, show
that a mixture of alanine with formic acid or a formate is not fermented,
whilst neither the added mixture nor formamide seriously effects the
action of the juice on glucose.
Other Theories.
Among other suggestions may be mentioned that of Kohl []
who asserts that sodium lactate is readily fermented, whilst Kusseroff
[] holds the view that the glucose is first reduced to sorbitol and
the latter fermented, in spite of the fact that sorbitol itself in the free
state is not fermented by yeast.
The rapid appearance and disappearance of glycogen in the yeast
cell at various stages of fermentation
[see Pavy and Bywaters, ;
Wager and Peniston, ]
has led to the suggestion
[Grüss, ;
Kohl, ]
that this substance is of great importance in fermentation,
and represents a stage through which all the sugar must pass before
being fermented. The fact that the formation of glycogen has been observed
in yeast-juice by Cremer [],
and that complex carbohydrates
are also undoubtedly formed (p. ), are consistent with this theory.
The low rate of autofermentation of living yeast, which is only a few per
cent. of the rate of sugar fermentation, renders this supposition very improbable
(Slator), as does the fact that the fermentation of glycogen by
yeast-juice is usually slower than that of glucose [see also
Euler, ].
An entirely different explanation of the chemical changes attendant
on alcoholic fermentation has been suggested by Löb [;
[p117]
,
;
,
Löb and Pulvermacher, ], founded
on the idea that the various decompositions of the sugar molecule both
by chemical and biological agents are to be explained by a reversal of
the synthesis of sugar from formaldehyde. As the sugar molecule can
be built up by the condensation of formaldehyde, so it tends to break
down again into this substance, and the products observed in any particular
case are formed either by partial depolymerisation in this sense
or by partial re-synthesis following on depolymerisation.
Löb has adduced many striking facts in favour of this view, and
has shown that very dilute alkalis produce no lactic acid but formaldehyde
and a pentose as primary products. These substances represent
the first stage of depolymerisation and are also formed by the electrolysis
of glucose.
Löb has himself been unable to detect definite intermediate products
of fermentation by adding reagents, such as aniline, ammonia,
and phloroglucinol, which would combine with such substances and prevent
their further decomposition [].
The occurrence of traces of formaldehyde as a product of alcoholic
fermentation by yeast-juice [Lebedeff, ] is at least consistent
with this theory, but no decisive evidence has so far been obtained
either for or against it.
In all the foregoing attempts to indicate the probable stages in the
production of alcohol and carbon dioxide from sugar, a single molecule
of the sugar forms the starting-point. The facts recounted in Chapter
III as to the function of phosphates in alcoholic fermentation, which
are summed up in the equation:—
2 C6H12O6 + 2 R2HPO4 = 2 CO2 + 2 C2H6O + 2 H2O + C6H10O4(PO4R2)2,
render it in the highest degree probable that two molecules of the sugar
are concerned. The most reasonable interpretation of this equation
appears to be that in the presence of phosphate and of the complicated
machinery of enzyme and co-enzyme two molecules of the hexose,
or possibly of the enolic form, are each decomposed primarily into
two groups.
Of the four groups thus produced, two go to form alcohol and
carbon dioxide and the other two are synthesised to a new chain of six
carbon atoms, which forms the carbohydrate residue of the hexosephosphate.
The introduction of the phosphoric acid groups may
possibly occur before the rupture of the original molecules, and may
even be the determining factor of this rupture, or again this introduction
may take place during or after the formation of the new carbon
[p118]
chain. Sufficient information is not yet available for the exact formulation
of a scheme for this reaction. Such a scheme, it may be
noted, would not necessarily be inconsistent with the views of Wohl and
of Buchner as to the way in which the carbon chain of a hexose is broken
in the process of fermentation, but would interpret differently the subsequent
changes which are undergone by the simpler groups which are
the result of this rupture. The reaction might thus proceed without
the formation of definite intermediate products, whilst opportunity
would be afforded for the production of a small quantity of by-products
such as formaldehyde, glycerol, lactic acid, acetic acid, etc., by secondary
reactions.
A symmetrical scheme can readily be constructed for such a change,
but much further information is required before any decisive conclusion
can be drawn as to the precise course of the reaction which actually
occurs in alcoholic fermentation.
CHAPTER IX.
THE MECHANISM OF FERMENTATION.
[p119]
The analysis of the process of alcoholic fermentation by yeast-juice
and other preparations from yeast which has been carried out in the
preceding chapters has shown that the phenomenon is one of a very
complex character. The principal substances directly concerned in the
change appear to be the enzyme and co-enzyme of the juice, a second
enzyme, hexosephosphatase, and, in addition, sugar, phosphate, and the
hexosephosphate formed from these. During autofermentation two
other factors are involved, the complex carbohydrates of the juice,
including glycogen and dextrins, and the diastatic ferment by which
these are converted into fermentable sugars. It is also possible that
the supply of free phosphate is partially provided by the action of proteoclastic
ferments on phosphoproteins. Under special circumstances
the rate at which fermentation proceeds may be controlled by the
available amount of any one of these numerous substances.
When the juice from well-washed yeast is incubated, the phenomenon
of autofermentation is observed. The juice contains an abundant
supply of enzyme, co-enzyme, and phosphate or hexosephosphate,
and in this case the controlling factor is usually the supply of sugar,
which is conditioned by the concentration of the diastatic enzyme or of
the complex carbohydrates as the case may be. When this is the case
the measured rate of fermentation is the rate at which sugar is being
produced in the juice, this being the slowest of the various reactions
which are proceeding under these circumstances. If sugar be now
added, an entirely different state of affairs is set up. As soon as any
accumulated phosphate has been converted into hexosephosphate, the
normal rate of fermentation which is usually higher than that of autofermentation
is attained, and, provided that excess of sugar be present,
fermentation continues for a considerable period at a slowly diminishing
rate and finally ceases. During the first part of this fermentation
the rate is controlled entirely by the supply of free phosphate, and this
depends mainly on the concentration of the hexosephosphatase and of
the hexosephosphate, and only in a secondary degree on the decomposition
[p120]
of other phosphorus compounds by other enzymes and on the
concentration of the sugar. The amount of hexosephosphate in yeast-juice
is usually such that an increase in its concentration does not
greatly affect the rate of fermentation, and hence the measured rate
during this period represents the rate at which hexosephosphate is being
decomposed, and this in its turn depends on the concentration of
hexosephosphatase, which is therefore the controlling factor. As
fermentation proceeds, the concentration of both enzyme and co-enzyme
steadily diminishes, as already explained, probably owing to the action
of other enzymes, so that at an advanced stage of the fermentation,
the controlling factor may be the concentration of either of these,
or the product of the two concentrations (see p. ). The hexosephosphatase
appears invariably to outlast the enzyme and co-enzyme.
The condition at any moment could be determined experimentally if
it were possible to add enzyme, co-enzyme and hexosephosphatase at
will and so ascertain which of these produced an acceleration of the
rate.
Unfortunately this can at present be only very imperfectly accomplished,
owing to the impossibility of separating these substances from
each other and from accompanying matter which interferes with the
interpretation of the result.
A third condition can also be established by adding to the fermenting
mixture of the juice and sugar a solution of phosphate. The
supply of phosphate is now almost independent of the action of the
hexosephosphatase, and the measured rate represents the rate at which
reaction (1), p. , can occur between sugar and phosphate in the presence
of the fermenting complex consisting of enzyme and co-enzyme. This
change is controlled, so long as sugar and phosphate are present in
the proper amounts, by the concentration of the fermenting complex
or possibly of either the enzyme or the co-enzyme. If only a single addition
of a small quantity of phosphate be made, the rate falls as soon
as the whole of this has been converted into hexosephosphate and the
reaction then passes into the stage just considered, in which the rate
is controlled by the production of free phosphate.
Although these varying reactions have not yet been exhaustively
studied from the kinetic point of view, owing to the experimental difficulties
to which allusion has already been made, investigations have
nevertheless been carried out on the effect of the variation of concentration
of yeast-juice and zymin as a whole, as well as of the carbohydrate.
Herzog [, ]
has made experiments of this kind
with zymin, and Euler []
with yeast-juice, whilst many of the results
[p121]
obtained by Buchner and by Harden and Young are also available.
The actual observations made by these authors show that the initial
velocity of fermentation is almost independent of the concentration
of sugar within certain limits, but decreases slowly as the concentration
increases. When the velocity constant is calculated on the assumption
that the reaction is monomolecular [see Bayliss, , Chap. VI],
approximate constancy is found for the first period of the fermentation.
This method of dealing with the results is, however, as pointed out by
Slator, misleading, the apparent agreement with the law of monomolecular
reactions being probably due to the gradual destruction of
the fermenting complex.
Experiments with low concentrations of sugar are difficult to interpret,
the influence of the hydrolysis of glycogen and of dextrins on
the one hand, and the synthesis of sugar to more complex carbohydrates
on the other (p. ), having a relatively great effect on the
concentration of the sugar. Unpublished experiments (Harden and
Young) indicate, however, that the velocity of fermentation remains
approximately constant, until a certain very low limit of sugar concentration
is reached, and then falls rapidly. The fall in rate, however,
only continues over a small interval of concentration, after which the
velocity again becomes approximately constant and equal to the rate
of autofermentation. During this last phase, as already indicated, the
velocity is generally controlled by the rate of production of sugar and
no longer by that of phosphate, this substance being now present in
excess. In other words, the rate of fermentation of sugar by yeast-juice
and zymin is not proportional to the concentration of the sugar
present as required by the law of mass, but, after a certain low limit of
sugar concentration, is independent of this and is actually slightly decreased
by increase in the concentration of the sugar.
The relations here are very similar to those shown to exist by
Duclaux [] and Adrian Brown [] for the action of invertase on
cane sugar and are probably to be explained in the manner suggested by
the latter. According to this investigator, the enzyme unites with the
fermentable material, or as it is now termed, the substrate or zymolyte,
forming a compound which only slowly decomposes so that it remains in
existence for a perceptible interval of time. The rate of fermentation
depends on the rate of decomposition of this compound and hence varies
with its concentration. This conception leads to the result that the rate
of fermentation will increase with the concentration of the substrate
up to a certain limit and will then remain [p122] constant,
unless interfered with by secondary actions. This limit of concentration is
that at which there is just sufficient of the material in question present
to combine with practically the whole of the enzyme, so that no further
increase in its amount can cause a corresponding increase in the quantity
of its compound with the enzyme or in the rate of fermentation which
depends on the concentration of that compound.
The curve relating the rate of action of such an enzyme with the
concentration of the zymolyte therefore consists of two portions, one
in which the rate at any moment is proportional to the concentration of
the zymolyte, according to the well-known law of the action of mass, and
a second in which the rate at any moment is almost independent of that
concentration, approximately equal amounts being decomposed in equal times
whatever the concentration of the substrate.
The results of the experiments with yeast-juice therefore indicate
that what is being measured is a typical enzyme action, but afford no
information as to which of the many possible actions is the controlling
one, a fact which must be ascertained for each particular case in the
manner indicated above.
Clowes [], using washed zymin
free from fermenting power and adding various volumes of boiled yeast
extract, found that the velocity of reaction was proportional to the
product of the concentrations of zymin and yeast extract up to a certain
optimum concentration. He interprets these concentrations as representing
the concentrations of zymase and co-enzyme, but they also represent the
concentrations of hexosephosphatase (present in the zymin) and phosphate
(present in the yeast extract), so that at least four factors were being
altered instead of only two.
It has already been mentioned that
Euler and Kullberg []
found the conversion of phosphate into hexosephosphate in presence of
excess of glucose to proceed according to a monomolecular reaction
(p. ).
The rate of fermentation is diminished by dilution of the yeast-juice,
but less rapidly than the concentration of the juice. Herzog
found that when the relation between concentration of enzyme and
the velocity constant of the reaction is expressed by the formula
K1/K2 = (C1/C2)n
where K1 and K2 are the velocity constants corresponding
with the enzyme concentrations C1 and C2, the value for n is
2 for zymin, whilst Euler working with yeast-juice obtained values
varying from 1·29 to 1·67 and decreasing as K increased.
The temperature coefficient of fermentation by zymin was found
[p123]
by Herzog to be
K24·5°/K14·5° = 2·88, which agrees well with the
value found by Slator for yeast-cells (p. ).
When we endeavour to apply the results of the investigations of
the fermentation of sugar by yeast-juice, zymin, etc., to the process
which goes on in the living cell, considerable difficulties present themselves.
A scheme of fermentation in the living cell can, however,
easily be imagined, which is in harmony with these results. According
to the most simple form of this ideal scheme, the sugar which has
diffused into the cell unites with the fermenting complex and undergoes
the characteristic reaction with phosphate, already present in the
cell, yielding carbon dioxide, alcohol, and hexosephosphate. The
latter is then decomposed, just as it is in yeast-juice, but more rapidly,
and the liberated phosphate again enters into reaction, partly with the
sugar formed from the hexosephosphate and partly with fresh sugar
supplied from outside the cell. The main difference between fermentation
by yeast-juice and by the living cell would then consist in the
rate of decomposition of the hexosephosphate, for it has been shown
that yeast-juice in presence of sufficient phosphate can ferment sugar
at a rate of the same order of magnitude (from 30 to 50 per cent.) as that
attained by living yeast.
The difference between the two therefore would appear to lie not
so much in their content of fermenting complex as in their very different
capacity for liberating phosphate from hexosephosphate and thus
supplying the necessary conditions for fermentation.
A simple calculation based on the phosphorus content of living
yeast [Buchner and Haehn, ]
shows that the whole of this
phosphate must pass through the stage of hexosephosphate every five or
six minutes in order to maintain the normal rate of fermentation,
whereas in an average sample of yeast-juice the cycle, calculated in
the same way, would last nearly two hours.
Wherein this difference resides is a difficult question, which cannot
at present be answered with certainty.
In the first place it must be remembered that a very great acceleration
of the action of the hexosephosphatase is produced by arsenates
(p. ), and this suggests the possibility that some substance possessing
a similar accelerating power is present in the yeast-cell and is lost
or destroyed in the various processes involved in rendering the yeast
susceptible to phosphate. The great variety of these processes—extraction
of yeast-juice by grinding and pressing, drying and macerating,
heating, treating with acetone and with toluene—renders this
somewhat improbable, and so far no such substance has been detected.
[p124]
A comparison of living yeast, zymin, and yeast-juice shows that
these are situated on an ascending scale with respect to their response
to phosphate. Taking fructose as the substrate in each case, yeast
does not respond to phosphate at all (Slator), the rate of fermentation
by zymin is approximately doubled (p. ), and that by yeast-juice
increased ten to forty times, whilst the maximum rates are in each case
of the same order of magnitude. Euler and Kullberg, however, have
observed an acceleration of about 25 per cent. in the rate of fermentation
of yeast in presence of a 2 per cent. solution of monosodium phosphate,
NaH2PO4 [,
].
The high rate of fermentation by living yeast and its lack of
response to phosphate may possibly be explained by supposing that
the balance of enzymes in the living cell is such that the supply of
phosphate is maintained at the optimum, and the rate of fermentation
cannot therefore be increased by a further supply.
A further difference lies in the fact that yeast-juice and zymin
respond to phosphate more strongly in presence of fructose than of
glucose, whereas yeast ferments both sugars at the same rate (p. ),
and this property has been shown to be connected with the specific
relations of fructose to the fermenting complex. It seems possible
that these differences are associated with the gradual passage from the
complete living cell of yeast, through the dead and partially disorganised
cell of zymin to yeast-juice in which the last trace of cellular
organisation has disappeared and the contents of the cell are uniformly
diffused throughout the liquid. Living yeast is, moreover, not only
unaffected by phosphate but only decomposes hexosephosphate extremely
slowly (Iwanoff).
Some light is thrown on these interesting problems by the effect
of antiseptics on fermentation by yeast-cells and by yeast-juice. The
action of toluene has hitherto been most completely studied, and this
substance is an extremely suitable one for the purpose since it has
practically no action whatever on fermentation by yeast-juice. The
experiments of Buchner have, in fact, shown that the normal rate of
fermentation and the total fermentation produced, are almost unaffected
by the presence of toluene even in the proportion of 1 c.c. to 20 c.c.
of yeast-juice. What then is the effect of toluene on the living yeast-cell?
When toluene in large excess is agitated with a fermenting
mixture of yeast and sugar, the rate of fermentation falls rapidly at
first and then more slowly until a relatively constant rate is attained
which gradually decreases in a similar manner to the rate of fermentation
by yeast-juice. Thus at air temperature (16°) 10 grams of
[p125]
yeast suspended in 50 c.c. of 6 per cent. glucose solution gave the
following results when agitated with toluene:—
Time afterAddition ofToluene,Minutes
C.c. ofCO2 perMinute.
Time.
C.c. perMinute.
0
4·6
6
1·6
1
4 8
1·2
2
3·3
12
0·85
3
2·6
24
0·8
4
2 32
0·5
5
1·8
constant
Simultaneously with this, the yeast acquires the property of decomposing
and fermenting hexosephosphate and of responding to the addition of
phosphate. This last property is only acquired to a small degree in
this way but it becomes much more strongly developed if the pressed
yeast be washed with toluene on the filter pump. Thus 10 grams of yeast
after this treatment fermented fructose at 1·2 c.c. per three minutes;
after the addition of phosphate (5 c.c. of 0·6 molar phosphate) the rate
rose to 6·9 and then gradually fell in the typical manner [Harden, ; see also Euler and Johansson, ].
The current explanation of the great decrease in rate of fermentation
which attends the action of toluene and other antiseptics on living
yeast, and also follows upon the disintegration of the cell,
appears to be that in living yeast the high rate of fermentation
is maintained by the continued production of relatively large
fresh supplies of fermenting complex, and that when the power of
producing this catalytic agent is destroyed by the poison, the rate
of fermentation falls to a low value, corresponding to the store of
zymase still present in the cell (cf. Buchner, E. and H., and Hahn, , pp.
176, 180).
This explanation implies that the rate of fermentation after the action
of the toluene represents the amount of fermenting complex present, a
supposition which has been shown (p. ) to be
highly improbable.
It further necessitates, as also pointed out independently by
Euler and Ugglas [],
a rapid destruction of the fermenting complex
both in the process of fermentation and by the action of the antiseptic,
as otherwise the store of zymase remaining in the dead cell would be
practically the same as that contained in the living cell at the moment
when it was subjected to the antiseptic, and this store would therefore
suffice to carry out fermentation at the same rate in the dead
as in the living cell. No such rapid destruction, however, occurs
in yeast-juice, as judged by the rate of fermentation, which falls off
[p126]
slowly and to about the same extent in the presence or absence
of toluene. Moreover, as shown above, it is highly probable that the
actual amount of fermenting complex in yeast-juice is a large fraction
of that present at any moment in the cell, and is capable under
suitable conditions of producing fermentation at a rate comparable
with that of the living cell.
This last criticism also applies to the view expressed by Euler
[Euler and Ugglas, ;
Euler and Kullberg, ,
] that in the living
cell the zymase is partly free and partly combined with the protoplasm;
when the vital activity of the cell is interfered with, the combined
portion of the zymase is thrown out of action and only that which was
free remains active.
The suggestion made by Rubner [] that
the action of yeast on sugar is in reality chiefly a vital act, but that a
small proportion of the change is due to enzyme action, is similar in its
consequences to that of Euler and may be met by the same arguments. Buchner
and Skraup [] have
moreover shown that the effects of sodium chloride and toluene on the
fermenting power of yeast which were observed by Rubner, can be explained
in other ways.
Some other explanation must therefore be sought for this phenomenon.
Great significance must be attached in this connection to the relation
noted above between the degree of disintegration and disorganisation
of the cell and the fall in the normal rate of fermentation. It seems
not impossible that fermentation may be associated in the living cell
with some special structure, or carried on in some special portion
of the cell, perhaps the nuclear vacuole described by Janssens
and Leblanc [],
Wager [, ; Wager and Peniston,
]
and others which undergoes remarkable changes both during
fermentation and autofermentation [Harden and Rowland, ]. The
disorganisation of the cell might lead to many modifications of the
conditions, among others to the dilution of the various catalytic agents
by diffusion throughout the whole volume of the cell. As a matter of
observation the dilution of yeast-juice leads to a considerable diminution
of the rate of fermentation of sugar, and it is possible that this is one
of the chief factors concerned. That phenomena of this kind may be involved
is shown by the remarkable effect of toluene on the autofermentation of
yeast. Whereas the fermentation of sugar is greatly diminished by the
action of toluene, the rate of autofermentation, which is carried on
at the expense of the glycogen of the cell, is greatly increased. In a
typical case, for example, the autofermentation of 10 grams of yeast
suspended in 20 c.c. of water amounted to 28 c.c. in 4·8 hours [p127]
at 25°, whereas the same amount of yeast in presence of 2 c.c. of
toluene gave 97·6 c.c. in the same time.
Many salts produce a similar effect on English top yeasts
(in which the autofermentation is large) [Harden and Paine, ], whereas Neuberg and
Karczag in Berlin [] were unable to observe this phenomenon.
A necessary preliminary of the fermentation of glycogen is its
conversion by a diastatic enzyme into a fermentable sugar, and it is
probable that the effect of the disorganisation of the cell by toluene is
that this enzyme finds more ready access to the glycogen, which is
stored in the plasma of the cell. No such acceleration of autofermentation
is effected by the addition of toluene to yeast-juice, and
hence the result is not due to an acceleration of the action of the
diastatic enzyme on the glycogen.
This effect of toluene is similar in character to the action of
anæsthetics on the leaves of many plants containing glucosides and
enzymes, whereby an immediate decomposition of the glucoside is
initiated [see H. E. and E. F. Armstrong, ].
Although as indicated above Euler's theory cannot apply to zymase
itself, if applied to the hexosephosphatase it would afford a consistent
explanation of the facts. According to this modified view it would be
the hexosephosphatase of yeast which existed largely in the combined
form, so that in extracts, in dried yeast and in presence of toluene only
the small fraction which was free would remain active. The zymase
on the other hand would have to be regarded as existing to a large extent
in the free state so that it would pass into extracts comparatively
unimpaired in amount and capable under proper conditions (i.e. when
supplied with sufficient phosphate) of bringing about a very vigorous
fermentation. The theory of combined and free enzymes is undoubtedly
of considerable value, although it cannot be considered as
fully established.
Fermentation by Living Yeast.
Much important information as to the nature of the processes involved
in fermentation has been acquired by the direct experimental
study of the action of living yeast on different sugars.
This phenomenon has formed the subject of several investigations from
the kinetic point of view, and its general features may now be regarded as
well established.
The difficulty, which must as far as possible be avoided in quantitative
experiments of this sort with living yeast, is the alteration [p128]
in the amount or properties of the yeast, due to growth or to
some change in the cells. This has been obviated in the work of Slator
[] by determining in every case the
initial rate of fermentation, so that the process only continues for a very
short period, during which any change in the amount or constitution of the
yeast is negligible. The method has the further advantage that interference
of the products of the reaction is to a large extent avoided. The pressure
apparatus already described (p. ) was employed
by Slator, the rate of production of carbon dioxide being measured by the
increase of pressure in the experimental vessel.
Influence of Concentration of Dextrose on the Rate of
Fermentation.
With regard to this important factor it is found that the action
of living yeast follows the same law as that of most enzymes (p. ); within certain wide limits the rate of
fermentation is almost independent of the concentration of the sugar.
This conclusion has been drawn by many previous investigators from
their experiments [Dumas, ;
Tammann, ; Adrian
Brown, ;
O'Sullivan, , ] and is implicitly contained in the
results of Aberson [], although he himself
regarded the reaction as monomolecular.
Fig. 8.
Slator, working with a suspension of ten to twelve yeast-cells per
1/4000 cubic millimetre at 30°, obtained the results which are embodied
in the curve (Fig. 8).
This shows that, for the amount of yeast in question, the rate of
fermentation is almost constant for concentrations of glucose between
[p129]
1 and 10 grams per 100 c.c., but gradually decreases as the concentration
increases. Below 1 gram per 100 c.c. the rate decreases
very rapidly with the concentration.
It follows from this, in the light of what has already been said
(p. ), that the action of living yeast on sugar follows the same
course as a typical enzyme reaction, although in this case, as in that
of yeast-juice, no information is given as to the exact nature of this
reaction.
Influence of the Concentration of Yeast.
It appears to be well established that, when changes in the quantity
and constitution of the yeast employed are eliminated, the rate of
fermentation is exactly proportional to the number of the yeast-cells
present (Aberson, Slator). This result might be anticipated, as pointed
out by Slator, from the fact that the fermentation takes place within
the cell, each cell acting as an independent individual.
The diffusion of sugar into the yeast-cell which necessarily precedes
the act of fermentation has been shown by
Slator and Sand [] to
occur at such a rate that the supply of sugar is always in excess of the
amount which can be fermented by the cell.
Temperature Coefficient of Alcoholic Fermentation
by Yeast.
The temperature coefficient of fermentation by living yeast has
been carefully determined by Slator by measurements of the initial
rates at a series of temperatures from 5° to 40° C. The coefficient is
found to be of the same order as that for many chemical reactions,
but to vary considerably with the temperature, a rise in temperature
corresponding with a diminution in the coefficient. The following
values were obtained for glucose; they are independent of the concentration
of yeast and glucose, the class of yeast, and presence
or absence of nutrient salts, and remain the same when inhibiting
agents are present. Almost precisely the same ratios are obtained
for fructose and mannose:—
t.
V(t+5)/Vt.
V(t+10)/Vt.
5
2·65
5·6
10
2·11
3·8
15
1·80
2·8
20
1·57
2·25
25
1·43
1·95
30
1·35
1·6
35
1·20
Aberson's result,
K(t+10)/Kt = 2·72, which represents the mean
coefficient for 10° between 12° and 33°, agrees well with this. [p130]
Action of Accelerating Agents on Living Yeast.
Slator [] was unable to find
any agent which greatly accelerated the rate of fermentation of living
yeast. Small concentrations of various inhibiting agents which are often
supposed to act in this way were quite ineffective, and phosphates, which
produce such a striking change in yeast-juice, were almost without action
(cp. p. ).
Euler and Bäckström [],
however, have made the important observation that sodium hexosephosphate
causes a considerable acceleration although it is itself neither fermented
nor hydrolysed under these conditions. The extent of this is evident from
the following numbers:—
20 c.c. of 20 per cent. glucose solution.
0·25 g. yeast [Yeast H of St. Erik's brewery].
Without addition.
+ 0·5 g. Nahexosephosphate.
Time.Min.
CO2.
Time.Min.
CO2.
46
10·5
37
8
76
17·5
73
19
197
45 188
52·5
347
74·5
321
123
488
95 450
193·5
The observation has been confirmed with English top yeast
(Harden and Young, unpublished experiments), but no explanation of
the phenomenon is at present forthcoming.
Euler has also found
[Euler and Cassel, ;
Euler and Berggren, ]
that yeast extract, sodium nucleinate and ammonium formate
also increase the rate of fermentation of glucose by yeast, but these
results have been criticised by
Harden and Young [] on the
ground that the possibility of growth of the yeast during the experiment
has not been excluded.
Fermentation of Different Sugars by Yeast.
Many valuable ideas as to the nature of fermentation have been
obtained by a consideration of the phenomena presented by the
action of yeast on the different hexoses. Of these only glucose, fructose,
mannose, and galactose are susceptible of alcoholic fermentation by
yeast, the stereoisomeric hexoses prepared in the laboratory being unfermentable,
as are also the pentoses, tetroses, and the alcohols corresponding
to all the sugars. The yeast-cell is therefore much more
limited in its power of producing fermentation than such an organism
as, for example, Bacillus coli communis, which attacks substances as
[p131]
diverse as arabinose, glucose, glycerol and mannitol, and yields with
all of them products of the same chemical character, although in varying
proportions.
A careful examination of a number of different genera and species
of the Saccharomycetaceæ and allied organisms by
E. F. Armstrong []
has shown that all yeasts which ferment glucose also ferment
fructose and mannose. Armstrong grew his yeasts in a nutrient solution
containing the sugar to be investigated, and his experiments are
open to the criticism that the organisms were hereby afforded an opportunity
for becoming acclimatised to the sugar. His results, therefore,
only demonstrate the fact that the organisms in question when cultivated
in presence of the sugars examined brought about their fermentation,
and do not exclude the possibility that the same organism when
grown in presence of a different sugar might not be capable of fermenting
the one to which it had in the other type of experiment become
acclimatised.
This has actually been shown to be the case for galactose by Slator
[],
and it is possible that this circumstance explains the
negative results obtained by Lindner []
with S. exiguus and
Schizosaccharomyces Pombe upon mannose, a sugar which, according
to Armstrong, is fermented by both these organisms.
The same problem has been attacked quantitatively by Slator,
who has shown that living yeast of various species and genera ferments
glucose and fructose at approximately the same rate. Moreover,
when the yeast is acted upon by various inhibiting agents, such as
heat, iodine, alcohol, or alkalis, the crippled yeast also ferments glucose
and fructose at the same rate.
With mannose the relations are somewhat different. The relative
rate of fermentation of mannose and glucose by yeast is dependent on
the variety of the yeast and the treatment which it has received.
Fresh samples of yeast ferment mannose more quickly than glucose,
but by older samples the glucose is the more rapidly decomposed.
This is especially the case with yeast, the activity of which has been
partly destroyed by heat, the relative fermenting power to mannose
being sometimes reduced by this treatment from 120 per cent. of that
of glucose to only 12 per cent. (Slator).
A further difference consists in the fact that with certain yeasts the
rate of fermentation of glucose is somewhat increased by monosodium
phosphate whilst that of mannose is unaffected [Euler and Lundeqvist,
].
Mixtures of glucose and fructose are fermented by yeast at the
[p132]
same rate as either the glucose or the fructose contained in the mixture
would be alone. When, however, mannose and glucose are fermented
simultaneously interference between the reactions takes place,
and this is especially evident when the yeast has comparatively little
action on mannose. The following are the results obtained by Slator:—
Yeast.
Relative Rates.
2·5 per cent. Glucose.
2·5 per cent. Mannose.
2·5 per cent. Glucose + 2·5 per cent. Mannose.
S. Thermantitonum
100
105
92
Brewery yeast, 53 per cent. activity destroyed by heat
100
21
33
Brewery yeast, 60 per cent. activity destroyed by heat
100
12
42
The case of galactose merits special attention. Previous investigations
[see Lippmann, , p. 734] have
shown that the fermentation of galactose by yeast differs greatly from
that of the other hexoses. The subject has been re-investigated by E.
F. Armstrong [],
and by Slator []. Armstrong
carried out his experiments in the manner already described (p. ), and found that some yeasts had, and others
had not, the power of fermenting galactose, although all were capable of
fermenting glucose, fructose, and mannose.
Slator made quantitative experiments on the same subject. He
was able to confirm the statement which had previously been made,
that certain yeasts which have the property of fermenting galactose
possess it only after the yeast has become acclimatised by culture in
presence of the sugar. This was shown for brewery yeast and for the
species mentioned below. This phenomenon is one of great interest
and is strictly analogous to the adaptation of bacteria which has now
been quite conclusively established [Neisser, ].
Yeast.
Mode of Culture.Grown in:
Relative Rates.
Glucose.
Galactose.
S. Carlsbergensis
wort
100