CHAPTER XII

FERMENTATION

Ir has been known since very ancient times that sweet juices of fruits, more particularly of the grape, can be made to undergo certain changes, the result of which is, that the juice is no longer a sweet innocuous liquid, but possesses intoxicating properties. During the occurrence of this change the clear fluid becomes turbid, and its surface is covered by bubbles or froth. This latter phenomenon attracting special attention, the name fermentation was given to the process. We now know that the change consists in the transformation of the sweet substance sugar into other materials, of which the most abundant are alcohol, the body possessing the intoxicating properties, and carbonic acid, the evolution of which causes the frothing. The turbidity of the liquid is caused by the presence of numerous unicellular organisms, (torule) which increase rapidly by a process of budding.

This form of fermentation is now usually called the alcoholic fermentation, for it has been found that other chemical changes of the same nature may properly be included under the general term fermentation. As instances of these, the souring of milk, the transformation of urea into ammonium carbonate, &c., may be cited. The complex series of changes which are accompanied by the formation of malodorous gases, and which are known as putrefaction, come also into the same category. In all these instances the transformation is accompanied by the presence of unicellular organisms, which correspond to the torule of the alcoholic fermentation; for instance, in putrefying material, various kinds of bacteria, undergoing rapid growth and multiplication, will be discovered. It was for a long time a matter of doubt, whether these organic growths were the cause, or result, or an accidental concomitant, of the fermentative process. But it is now almost universally acknowledged that the organisms are the cause of the fermentation. It has been shown that the growth of such organisms is accompanied with the fermentation in question, that such fermentation

1 From fervere, to boil.

? A list of a large number of fungi (Saccharomyces cerevisiæ, S. ellipsoideus, Mucor, Mycoderma, &c.) which excite the alcoholic fermentation will be found in a paper by Reess, Bot. Untersuch. ü. d. Alcoholgährungspilze; see also Schunck, J. prakt. Chem. lxii. 222.

occurs only when the organisms are growing, and stops when the organisms are removed or killed.

This vitalistic theory of fermentation becomes especially important to the physiologist and pathologist when applied to disease. The 'germ theory,' as it is termed, explains the infectious or zymotic diseases by considering that the change in the system is of the nature of fermentation, and like the other fermentations we have mentioned, produced by particular forms of bacterium; the transference of the bacteria or their spores from one person to another constituting infection. This theory has not been fully verified for every infectious disease by the discovery of a specific microbe; many able investigators, however, consider it likely that the pathogenic germs of these maladies will be discovered, as in the cases of splenic fever, and relapsing fever, and a few others in which the specific bacterium has been already identified.

There is, however, another class of chemical transformations, which differ very considerably from all to which we have hitherto alluded. They, however, resemble these fermentations in the fact that they occur independently of any apparent change in the agents that produce them. The agents that produce them are not living organisms, but chemical substances, the result of the activity of living cells. As instances of this class of chemical transformations, the following may be taken the change of starch into sugar by the ptyalin of the saliva, the change of proteids into peptones by the pepsin of the gastric juice, the change of fibrinogen into fibrin, when blood is shed, &c. &c. These changes are also included under the term fermentation.1

Fermentations may therefore be divided into two classes: first, those brought about by the organised ferments (torulæ, bacteria, &c.), and, secondly, those brought about by the unorganised ferments (pepsin, diastase, &c.). Each of these classes may be again subdivided, according to the nature of the chemical change produced.

Previous to 1838, the action of yeast was regarded as a catalytic one (Berzelius); that is to say, the influence of its mere presence causes a separation of the constituents of sugar, just as platinum black causes peroxide of hydrogen to give up an atom of its oxygen. A modification of this theory was proposed by Liebig in 1848; he gave the organisms associated with the change a secondary position, holding that they produced substances of a chemical nature which were the true ferments; and he considered that the molecular vibrations of these ferments caused a rearrangement of the atoms of the substance

1 Sheridan Lea suggests the term zymolysis for this variety of fermentation (Journ. of Physiol. 1890, p. 254). Sir W. Roberts suggested the term enzymosis (Proc. Roy. Soc. vol. xxxi. p. 145) many years ago for the same processes.

undergoing fermentation. He compared this action to the decomposition of acetic acid into acetone and carbonic acid produced by heat, or the change of cyanogen dissolved in water into oxamide, produced by the vibrations of a trace of aldehyde. This action is also comparable to the action of the unorganised ferments, in which the living cells, for instance, of the stomach, produce a chemical substance, pepsin, the active agent in producing the fermentative change of albumin into peptone.

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In certain cases this view of Liebig has been justified; soluble ferments have been separated from the organisms, and these have the same action when the organisms are absent as when they are present. Thus yeast cells, in addition to causing the alcoholic fermentation, produce also an inverting ferment, that is, a ferment which transforms cane sugar into glucose; this ferment can be readily separated from the organisms (Barth,' Donath, Lea, &c.). The alkaline fermentation of urine, in which urea is converted into ammonium carbonate, is brought about by an organism very similar to yeast, and to it the name torula urea has been given. Here, again, a soluble ferment with the same power has been separated from the cells (Musculus, Lea). But in the greater number of cases, attempts to separate such soluble chemical ferments have been unsuccessful, and thus attention has been more concentrated on the biological side of the problem. In the case of the alcoholic fermentation, Helmholtz, Mitscherlich, and others, showed that if the yeast cells were prevented from passing into a fermentable liquid by the interposition of an organic membrane, fermentation did not ensue. That the organisms themselves are absolutely necessary, is also shown by experiments with the bacillus anthracis, the specific microbe of anthrax or splenic fever. A cultivation of the bacillus inoculated into an animal causes the death of that animal by splenic fever; but if the bacilli be first carefully filtered off from the cultivation fluid, the filtrate is innocuous.5

If, however, it be freely admitted that the organisms themselves are the cause of the fermentation, the question still remains, how do they act? Do they live on the fermentable matter, and then excrete what we call the products of fermentation? This view is not tenable, because of the immense volume of the substances in which they produce changes; Pasteur considers that of the sugar acted upon by yeast only one per cent. is taken up by the yeast itself. Another view, which

1 Ber. d. deutsch. chem. Gesell. 1878, p. 474. Ibid. 1875, p. 795. 3 Journ. of Physiol. vi. 136. 4 Compt. rend. lxxxii. 333. Pflüger's Archiv, xii. 214. In such experiments the culture fluid employed has been beef-tea or a similar infusion. More recent experiments [by Wooldridge, Hankin, and Martin have shown that if the bacilli be grown in a fluid rich in proteids, they produce a poison, a solution of which causes anthrax (see p. 168).

is probably more correct, is that the organisms produce, very much as Liebig supposed, a soluble ferment, which acts on the fermentable matter. This view, which has received the powerful support of HoppeSeyler, is at once confronted with the difficulties already mentioned, the chief of which is the inability of various observers to separate such soluble ferments from the organisms. It is, however, always unsafe, when results of experiments on any subject are negative, to assume that our knowledge upon that subject is complete and final. The inability of observers in the past to perform an experiment may be from lack of means or of knowledge; and it is possible that the presence of soluble ferments in places where their existence has been hitherto denied, may be demonstrated in the future.

The separation of the inverting ferment from yeast, and of the ureaferment from the torula urea, is a step which may be the first in a series of discoveries. Sheridan Lea in his experiments, indeed, pointed out a possible explanation of the negative results of previous investigators. Both the urea-ferment and the inverting ferment were obtained by precipitation of the cells with alcohol, and subsequent extraction of the alcoholic precipitate with water, but neither is present in the fluid surrounding the cells during the progress of the change which they produce. This is probably due to the fact that ferments, being non-diffusible, are unable to pass from the protoplasm of the torula, through its surrounding investment of cellulose.

It has already been surmised that ferments are of the nature of the living proteids (p. 146); like other proteids they are indiffusible ; this readily accounts for the fact they are not discoverable outside the cell wall; and like all living things their properties during life are different from those after death; this readily accounts for the fact that, with a few exceptions, they are not discoverable inside the cell wall, after the cell has been killed by alcohol. The few exceptions are probably those which are more robust, and withstand the action of alcohol better.

If this hypothesis be admitted, and until it is replaced by a better it must be admitted, the difference between organised and unorganised ferment action is this: an organised ferment is one which does not leave the living cell during the progress of the fermentation; an unorganised ferment is one which is shed out from the cells, and then exerts its activity. Probably the chemical nature of the ferment is in the two cases the same, or nearly the same.

If it be admitted that the ferments are proteid in nature, or something closely akin to proteid, and it be also remembered how imperfect our knowledge of the proteids is, it may seem a task from which one would shrink, to attempt to explain any further how the ferments

act. The ferment actions however consist very largely in the transference of water, or of oxygen; and we happily have in the simpler regions of chemistry, examples of action which seem to be analogous to what we call ferment action in the vaguer regions of organic chemistry. The most striking of the phenomena of fermentation are these :— (1) A small amount of the ferment produces a change in an overwhelmingly large quantity of material. This is even more puzzling in the case of the unorganised than in that of the organised ferments. A needle prick, if the point of that needle is infected with the bacillus anthracis, will cause the animal so inoculated to die of splenic fever. The inoculated bacilli have the power of rapid multiplication, and so rapidly poison the whole of the blood. A minute fragment of rennet will cause curdling throughout a huge volume of milk. There is here, however, no such power of self-multiplication.

(2) The ferment itself takes no apparent part in the change produced, but, after having produced its action, can be used again to produce the same action in another mass of material.

The vibration theory of Liebig is only to a certain extent an explanation of these phenomena; the changes taking place among the atoms composing the molecules of the ferment produce vibrations, which, acting on the molecules of the substance with which the ferment comes in contact, set up there similar molecular vibrations and rearrangements.

This is quite comparable to what is taking place around us every day in a social capacity. An irritable quick-tempered individual enters a room filled with pleasant people. The influence of his presence soon causes the whole assembly to become changed, and bad temper to rule supreme. The analogy to the case of a ferment is completed by the fact that the author of the change is himself unaltered, and is capable of producing the same action on another mass of material.

This homely comparison however helps us very little; it leads us into the regions of psychology, where the problems are even more complicated than in physiology. It will be of greater help to find comparisons in simpler chemical reactions which are well understood.

Take the case of the ordinary way in which oxygen is made. If one heats potassium chlorate (KClO3), the oxygen comes off, and potassium chloride (KCl) is left behind. If, however, a little manganese dioxide be mixed with the chlorate in the first instance, the oxygen

1 A 'contact theory' more recently advanced (Watts' Dictionary, 1889, vol. ii. p. 540) is that the enzymes raise the molecular temperatures of the decomposing molecules to the point at which their molecular equilibrium is destroyed; their decomposition is produced by rearrangement of energy, not by any increase or decrease of the amount present in the system.