Louis Pasteur (1822–95). Scientific Papers.
The Harvard Classics. 1909–14.
II. Fermentation in Saccharine Fruits Immersed in Carbonic Acid Gas
As a consequence of these conclusions it should be an easy matter to show, in the majority of living beings, the manifestation of the phenomena of fermentation; for there are, probably, none in which all chemical action entirely disappears, upon the sudden cessation of life. One day, when we were expressing these views in our laboratory, in the presence of M. Dumas, who seemed inclined to admit their truth, we added: “We should like to make a wager that if we were to plunge a bunch of grapes into carbonic acid gas, there would be immediately produced alcohol and carbonic acid gas, in consequence of a renewed action starting in the interior cells of the grapes, in such a way that these cells would assume the functions of yeast cells. We will make the experiment, and when you come to-morrow”—it was our good fortune to have M. Dumas working in our laboratory at that time—“we will give you an account of the result.” Our predictions were realized. We then endeavoured to find, in the presence of M. Dumas, who assisted us in our endeavour, cells of yeast in the grapes; but it was quite impossible to discover any.
Encouraged by this result, we undertook fresh experiments on grapes, on a melon, on oranges, on plums, and on rhubarb leaves, gathered in the garden of the École Normale, and, in every case, our substance, when immersed in carbonic acid gas, gave rise to the production of alcohol and carbonic acid. We obtained the following surprising results from some prunes de Monsieur: —On July 21, 1872, we placed twenty-four of these plums under a glass bell, which we immediately filled with carbonic acid gas. The plums had been gathered on the previous day. By the side of the bell we placed other twenty-four plums, which were left there uncovered. Eight days afterwards, in the course of which time there had been a considerable evolution of carbonic acid from the bell, we withdrew the plums and compared them with those which had been left exposed to the air. The difference was striking, almost incredible. Whilst the plums which had been surrounded with air (the experiments of Bérard have long since taught us that, under this latter condition, fruits absorb oxygen from the air and emit carbonic acid gas in almost equal volume) had become very soft and watery and sweet, the plums taken from under the jar had remained very firm and hard, the flesh was by no means watery, but they had lost much sugar. Lastly, when submitted to distillation, after crushing, they yielded 6.5 grammes (99.7 grains) of alcohol, more than 1 per cent. of the total weight of the plums. What better proof than these facts could we have of the existence of a considerable chemical action in the interior of fruit, an action which derives the heat necessary for its manifestation from the decomposition of the sugar present in the cells? Moreover, and this circumstance is especially worthy of our attention, in all these experiments we found that there was a liberation of heat, of which the fruits and other organs were the seat, as soon as they were plunged in the carbonic acid gas. This heat is so considerable that it may at times be detected by the hand, if the two sides of the bell, one of which is in contact with the objects, are touched alternately. It also makes itself evident in the formation of little drops on those parts of the bell which are less directly exposed to the influence of the heat resulting from the decomposition of the sugar of the cells.
In short, fermentation is a very general phenomenon. It is life without air, or life without free oxygen, or, more generally still, it is the result of a chemical process accomplished on a fermentable substance capable of producing heat by its decomposition, in which process the entire heat used up is derived from a part of the heat that the decomposition of the fermentable substance sets free. The class of fermentations properly so called, is, however, restricted by the small number of substances capable of decomposing with the production of heat, and at the same time of serving for the nourishment of lower forms of life, when deprived of the presence and action of air. This, again, is a consequence of our theory, which is well worthy of notice.
The facts that we have just mentioned in reference to the formation of alcohol and carbonic acid in the substance of ripe fruits, under special conditions, and apart from the action of ferment, are already known to science. They were discovered in 1869 by M. Lechartier, formerly a pupil in the École Normale Supérieure, and his coadjutor, M. Bellamy. In 1821, in a very remarkable work, especially when we consider the period when it appeared, Bérard demonstrated several important propositions in connection with the maturation of fruits:
I. All fruits, even those that are still green, and likewise even those that are exposed to the sun, absorb oxygen and set free an almost equal volume of carbonic acid gas. This is a condition of their proper ripening.
II. Ripe fruits placed in a limited atmosphere, after having absorbed all the oxygen and set free an almost equal volume of carbonic acid, continue to emit that gas in notable quantity, even when no bruise is to be seen—“as though by a kind of fermentation,” as Bérard actually observes—and lose their saccharine particles, a circumstance which causes the fruits to appear more acid, although the actual weight of their acid may undergo no augmentation whatever.
In this beautiful work, and in all subsequent ones of which the ripening of fruits has been the subject, two facts of great theoretical value have escaped the notice of the authors; these are the two facts which Messrs. Lechartier and Bellamy pointed out for the first time, namely, the production of alcohol and the absence of cells of ferments. It is worthy of remark that these two facts, as we have shown above, were actually fore-shadowed in the theory of fermentation that we advocated as far back as 1861, and we are happy to add that Messrs. Lechartier and Bellamy, who at first had prudently drawn no theoretical conclusions from their work, now entirely agree with the theory we have advanced. Their mode of reasoning is very different from that of the savants with whom we discussed the subject before the Academy, on the occasion when the communication which we addressed to the Academy in October, 1872, attracted attention once more to the remarkable observations of Messrs. Lechartier and Bellamy. M. Fremy, in particular, was desirous of finding in these observations a confirmation of his views on the subject of hemi-organism, and a condemnation of ours, notwithstanding the fact that the preceding explanations, and, more particularly our Note of 1861, quoted word for word in the preceding section, furnish the most conclusive evidence in favor of those ideas which we advocate. Indeed, as far back as 1861 we pointed out very clearly that if we could find plants able to live when deprived of air, in the presence of sugar, they would bring about a fermentation of that substance, in the same manner that yeast does. Such is the case with the fungi already studied; such, too, is the case with the fruits employed in the experiments of Messrs. Lechartier and Bellamy, and in our own experiments, the results of which not only confirm those obtained by these gentlemen, but even extend them, in so far as we have shown that fruits, when surrounded with carbonic acid gas immediately produce alcohol. When surrounded with air, they live in their aërobian state and we have no fermentation; immersed immediately afterwards in carbonic acid gas, they now assume their anaërobian state, and at once begin to act upon the sugar in the manner of ferments, and emit heat. As for seeing in these facts anything like a confirmation of the theory of hemi-organism, imagined by M. Fremy, the idea of such a thing is absurd. The following, for instance, is the theory of the fermentation of the vintage, according to M. Fremy.
“To speak here of alcoholic fermentation alone,” our author says, “I hold that in the production of wine it is the juice of the fruit itself that, in contact with air, produces grains of ferment, by the transformation of the albuminous matter; Pasteur, on the other hand, maintains that the fermentation is produced by germs existing outside of the grapes.”
Now what bearing on this purely imaginary theory can the fact have, that a whole fruit, immersed in carbonic acid gas, immediately produces alcohol and carbonic acid? In the preceding passage which we have borrowed from M. Fremy, an indispensable condition of the transformation of the albuminous matter is the contact with air and the crushing of the grapes. Here, however, we are dealing with uninjured fruits in contact with carbonic acid gas. Our theory, on the other hand, which, we may repeat, we have advocated since 1861, maintains that all cells become fermentative when their vital action is protracted in the absence of air, which are precisely the conditions that hold in the experiments on fruits immersed in carbonic acid gas. The vital energy is not immediately suspended in their cells, and the latter are deprived of air. Consequently, fermentation must result. Moreover, we may add, if we destroy the fruit, or crush it before immersing it in the gas, it no longer produces alcohol or fermentation of any kind, a circumstance that may be attributed to the fact of the destruction of vital action in the crushed fruit. On the other hand, in what way ought this crushing to affect the hypothesis of hemi-organism? The crushed fruit ought to act quite as well, or even better than that which is uncrushed. In short, nothing can be more directly opposed to the theory of the mode of manifestation of that hidden force to which the name of hemi-organism has been given, than the discovery of the production of these phenomena of fermentation in fruits surrounded with carbonic acid gas; whilst the theory, which sees in fermentation a consequence of vital energy in absence of air, finds in these facts the strictest confirmation of an express prediction, which from the first formed an integral part of its statement.
We should not be justified in devoting further time to opinions which are not supported by any serious experiment. Abroad, as well as in France, the theory of the transformation of albuminous substances into organized ferments had been advocated long before it had been taken up by M. Fremy. It no longer commands the slightest credit, nor do any observers of note any longer give it the least attention; it might even be said that it has become a subject of ridicule.
An attempt has also been made to prove that we have contradicted ourselves, inasmuch as in 1860 we published our opinion that alcoholic fermentation can never occur without a simultaneous occurrence of organization, development, and multiplication of globules; or continued life, carried on from globules already formed. Nothing, however, can be truer than that opinion, and at the present moment, after fifteen years of study devoted to the subject since the publication to which we have referred, we need no longer say, “we think,” but instead, “we affirm,” that it is correct. It is, as a matter of fact, to alcoholic fermentation, properly so called, that the charge to which we have referred relates—to that fermentation which yields, besides alcohol, carbonic acid, succinic acid, glycerine, volatile acids, and other products. This fermentation undoubtedly requires the presence of yeast-cells under the conditions that we have named. Those who have contradicted us have fallen into the error of supposing that the fermentation of fruits is an ordinary alcoholic fermentation, identical with that produced by beer yeast, and that, consequently, the cells of that yeast must, according to our own theory, be always present. There is not the least authority for such a supposition. When we come to exact quantitative estimations—and these are to be found in the figures supplied by Messrs. Lechartier and Bellamy—it will be seen that the proportions of alcohol and carbonic acid gas produced in the fermentation of fruits differ widely from those that we find in alcoholic fermentations properly so called, as must necessarily be the case since in the former the fermentation is effected by the cells of a fruit, but in the latter by cells of ordinary alcoholic ferment. Indeed we have a strong conviction that each fruit would be found to give rise to special action, the chemical equation of which would be different from that in the case of other fruits. As for the circumstance that the cells of these fruits cause fermentation without multiplying, this comes under the kind of activity which we have already distinguished by the expression continuous life in cells already formed.
We will conclude this section with a few remarks on the subject of equations of fermentations, which have been suggested to us principally in attempts to explain the results derived from the fermentation of fruits immersed in carbonic acid gas.
Originally, when fermentations were put amongst the class of decompositions by contact-action, it seemed probable, and, in fact, was believed, that every fermentation has its own well-defined equation which never varied. In the present day, on the contrary, it must be borne in mind that the equation of a fermentation varies essentially with the conditions under which that fermentation is accomplished, and that a statement of this equation is a problem no less complicated than that in the case of the nutrition of a living being. To every fermentation may be assigned an equation in a general sort of way, an equation, however, which, in numerous points of detail, is liable to the thousand variations connected with the phenomena of life. Moreover, there will be as many distinct fermentations brought about by one ferment as there are fermentable substances capable of supplying the carbon element of the food of that same ferment, in the same way that the equation of the nutrition of an animal will vary with the nature of the food which it consumes. As regards fermentation producing alcohol, which may be effected by several different ferments, there will be as in the case of a given sugar, as many general equations as there are ferments, whether they be ferment-cells properly so called, or cells of the organs of living beings functioning as ferments. In the same way the equation of nutrition varies in the case of different animals nourished on the same food. And it is from the same reason that ordinary wort produces such a variety of beers when treated with the numerous alcoholic ferments which we have described. These remarks are applicable to all ferments alike; for instance, butyric ferment is capable of producing a host of distinct fermentations, in consequence of its ability to derive the carbonaceous part of its food from very different substances, from sugar, or lactic acid, or glycerine, or mannite, and many others.
When we say that every fermentation has its own peculiar ferment, it must be understood that we are speaking of the fermentation considered as a whole, including all the accessory products. We do not mean to imply that the ferment in question is not capable of acting on some other fermentable substance and giving rise to fermentation of a very different kind. Moreover, it is quite erroneous to suppose that the presence of a single one of the products of a fermentation implies the co-existence of a particular ferment. If, for example, we find alcohol among the products of a fermentation, or even alcohol and carbonic acid gas together, this does not prove that the ferment must be an alcoholic ferment, belonging to alcoholic fermentations, in the strict sense of the term. Nor, again, does the mere presence of lactic acid necessarily imply the presence of lactic ferment. As a matter of fact, different fermentations may give rise to one or even several identical products. We could not say with certainty, from a purely chemical point of view, that we were dealing, for example, with an alcoholic fermentation properly so called, and that the yeast of beer must be present in it, if we had not first determined the presence of all the numerous products of that particular fermentation under conditions similar to those under which the fermentation in question had occurred. In works on fermentation the reader will often find those confusions against which we are now attempting to guard him. It is precisely in consequence of not having had their attention drawn to such observations that some have imagined that the fermentation in fruits immersed in carbonic acid gas is in contradiction to the assertion which we originally made in our Memoir on alcoholic fermentation published in 1860, the exact words of which we may here repeat:—“The chemical phenomena of fermentation are related essentially to a vital activity, beginning and ending with the latter; we believe that alcoholic fermentation never occurs”—we were discussing the question of ordinary alcoholic fermentation produced by the yeast of beer—“without the simultaneous occurrence of organization, development, and multiplication of globules, or continued life, carried on by means of the globules already formed. The general results of the present Memoir seem to us to be in direct opposition to the opinions of MM. Liebig and Berzelius.” These conclusions, we repeat, are as true now as they ever were, and are as applicable to the fermentation of fruits, of which nothing was known in 1860, as they are to the fermentation produced by the means of yeast. Only, in the case of fruits, it is the cells of the parenchyma that function as ferment, by a continuation of their activity in carbonic acid gas whilst in the other case the ferment consists of cells of yeast.
There should be nothing very surprising in the fact that fermentation can originate in fruits and form alcohol without the presence of yeast, if the fermentation of fruits were not confounded completely with alcoholic fermentation yielding the same products and in the same proportions. It is through the misuse of words that the fermentation of fruits has been termed alcoholic, in a way which has misled many persons. In this fermentation, neither alcohol nor carbonic acid gas exists in those proportions in which they are found in fermentation produced by yeast; and, although we may determine in it the presence of succinic acid, glycerine, and a small quantity of volatile acids the relative proportions of these substances will be different from what they are in the case of alcoholic fermentation.