Louis Pasteur (182295). Scientific Papers. The Harvard Classics. 190914.
The Physiological Theory of Fermentation
II. Fermentation in Saccharine Fruits Immersed in Carbonic Acid Gas
THE THEORY which we have, step by step, evolved, on the subject of the cause of the chemical phenomena of fermentation, may claim a character of simplicity and generality that is well worthy of attention. Fermentation is no longer one of those isolated and mysterious phenomena which do not admit of explanation. It is the consequence of a peculiar vital process of nutrition which occurs under certain conditions, differing from those which characterize the life of all ordinary beings, animal or vegetable, but by which the latter may be affected, more or less, in a way which brings them, to some extent within the class of ferments, properly so called. We can even conceive that the fermentative character may belong to every organized form, to every animal or vegetable cell, on the sole condition that the chemico-vital acts of assimilation and excretion must be capable of taking place in that cell for a brief period, longer or shorter it may be, without necessity for recourse to supplies of atmospheric oxygen; in other words, the cell must be able to derive its needful heat from the decomposition of some body which yields a surplus of heat in the process.
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-morrowit was our good fortune to have M. Dumas working in our laboratory at that timewe 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. 1
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:2On 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. 3
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. 4 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 seenas though by a kind of fermentation, as Bérard actually observesand 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. 5 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. 6 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. 7
To speak here of alcoholic fermentation alone, 8 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. 9 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 relatesto 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 estimationsand these are to be found in the figures supplied by Messrs. Lechartier and Bellamyit 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 occurswe were discussing the question of ordinary alcoholic fermentation produced by the yeast of beerwithout 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. 10 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 11 the relative proportions of these substances will be different from what they are in the case of alcoholic fermentation.
Note 1. To determine the absence of cells of ferment in fruits that have been immersed in carbonic acid gas, we must first of all carefully raise the pellicle of the fruit, taking care that the subjacent parenchyma does not touch the surface of the pellicle, since the organized corpuscles existing on the exterior of the fruit might introduce an error into our microscopical observations. Experiments on grapes have given us an explanation of a fact generally known, the cause of which, however, had hitherto escaped our knowledge. We all know that the taste and aroma of the vintage, that is, of the grapes stripped from the bunches and thrown into tubs, where they get soaked in the juice that issues from the wounded specimens, are very different from the taste and aroma of an uninjured bunch. Now grapes that have been immersed in an atmosphere of carbonic acid gas have exactly the flavour and smell of the vintage; the reason is that, in the vintage tub, the grapes are immediately surrounded by an atmosphere of carbonic acid gas, and undergo, in consequence, the fermentation peculiar to grapes that have been plunged in this gas. These facts deserve to be studied from a practical point of view. It would be interesting, for example, to learn what difference there would be in the quality of two wines, the grapes of which, in the one case, had been perfectly crushed, so as to cause as great a separation of the cells of the parenchyma as possible; in the other case, left, for the most part, whole, as in the case in the ordinary vintage. The first wine would be deprived of those fixed and fragrant principles produced by the fermentation of which we have just spoken, when the grapes are immersed in carbonic acid gas. By such a comparison as that which we suggest we should be able to form a priori judgment on the merits of the new system, which has not been carefully studied, although already widely adopted, of milled, cylindrical crushers, for pressing the vintage. [back]
Note 2. We have sometimes found small quantities of alcohol in fruits and other vegetable organs, surrounded with ordinary air, but always in small proportion, and in a manner which suggested its accidental character. It is easy to understand how, in the thickness of certain fruits, certain parts of those fruits might be deprived of air, under which circumstances they would have been acting under conditions similar to those under which fruits act when wholly immersed in carbonic acid gas. Moreover, it would be useful to determine whether alcohol is not a normal product of vegetation. [back]
Note 3. In these studies of plants living immersed in carbonic acid gas, we have come across a fact which corroborates those which we have already given in reference to the facility with which lactic and viscous ferments, and, generally speaking, those which we have termed the disease ferments of beer, develop when deprived of air, and which shows, consequently, how very marked their aërobian character is. If we immerse beet-roots or turnips in carbonic acid gas, we produce well-defined fermentations in those roots. Their whole surface readily permits the escape of the highly acid liquids, and they become filled with lactic, viscous, and other ferments. This shows us the great danger which may result from the use of pits, in which the beet-roots are preserved, when the air is not renewed, and that the original oxygen is expelled by the vital processes of fungi or other deoxidizing chemical actions. We have directed the attention of the manufacturers of beet-root sugar to this point. [back]
Note 4. Lechartier and Bellamy, Comptes rendus de lAcadémie des Sciences, vol., lxix., pp., 366 and 466, 1869. [back]
Note 5. Those gentlemen express themselves thus: In a note presented to the Academy in November, 1872, we published certain experiments which showed that carbonic acid and alcohol may be produced in fruits kept in a closed vessel, out of contact with atmospheric oxygen, without our being able to discover alcoholic ferment in the interior of those fruits. M. Pasteur, as a logical deduction from the principle which he has established in connection with the theory of fermentation, considers that the formation of alcohol may be attributed to the fact that the physical and chemical processes of life in the cells of fruit continue under new conditions, in a manner similar to those of the cells of ferment. Experiments, continued during 1872, 1873, and 1874, on different fruits have furnished results all of which seem to us to harmonize with this proposition, and to establish it on a firm basis of proof.Comptes rendus, vol. lxxix p. 949, 1874. [back]
Note 6. PASTEUR, Faites nouveaux pour servir à la connaissance de la théorie des fermentations proprement dites. (Comptes rendus de lAcadémie des Sciences, vol. lxxv., p. 784.) See in the same volume the discussion that followed; also, PASTEUR, Note sur la production de lalcool par les fruits, same volume, p. 1054, in which we recount the observations anterior to our own, made by Messrs. Lechartier and Bellamy in 1869. [back]
Note 7. Comptes rendus, meeting of January 15th, 1872. [back]
Note 8. As a matter of fact, M. Fremy applies his theory of hemi-organism, not only to the alcoholic fermentation of grape juice, but to all other fermentations. The following passage occurs in one of his notes (Comptes rendus de lAcadémie, vol. lxxv., p. 979, October 28th, 1872): Experiments on Germinated Barley.The object of these was to show that when barley, left to itself in sweetened water, produces in succession alcoholic, lactic, butyric, and acetic fermentations, these modifications are brought about by ferments which are produced inside the grains themselves, and not by atmospheric germs. More than forty different experiments were devoted to this part of my work. Need we add that this assertion is based on no substantial foundation? The cells belonging to the grains of barley, or their albuminous contents, never do produce cells of alcoholic ferment, or of lactic ferment, or butyric vibrios. Whenever those ferments appear, they may be traced to germs of those organisms, diffused throughout the interior of the grains, or adhering to the exterior surface, or existing in the water employed, or on the side of the vessels used. There are many ways of demonstrating this, of which the following is one: Since the results of our experiments have shown that sweetened water, phosphates, and chalk very readily give rise to lactic and butyric fermentations, what reason is there for supposing that if we substitute grains of barley for chalk, the lactic and butyric ferments will spring from those grains, in consequence of a transformation of their cells and albuminous substances? Surely there is no ground for maintaining that they are produced by hemi-organism, since a medium composed of sugar, or chalk, or phosphates of ammonia, potash, or magnesia contains no albuminous substances. This is an indirect but irresistible argument against the hemi-organism theory. [back]
Note 9. PASTEUR, Mémoire sur la fermentation alcoolique, 1860; Annales de Chimie et de Physique. The word globules is here used for cells. In our researches we have always endeavoured to prevent any confusion of ideas. We stated at the beginning of our Memoir of 1860 that: We apply the term alcoholic to that fermentation which sugar undergoes under the influence of the ferment known as beer yeast. This is, the fermentation which produces wine and all alcoholic beverages. This, too, is regarded as the type for a host of similar phenomena designated, by general usage, under the generic name of fermentation, and qualified by the name of one of the essential products of the special phenomenon under observation. Bearing in mind this fact in reference to the nomenclature that we have adopted, it will be seen that the expression alcoholic fermentation cannot be applied to every phenomenon of fermentation in which alcohol is produced, inasmuch as there may be a number of phenomena having this character in common. If we had not at starting defined that particular one amongst the number of very distinct phenomena, which, to the exclusion of the others, should bear the name of alcoholic fermentation, we should inevitably have given rise to a confusion of language that would soon pass from words to ideas, and tend to introduce unnecessary complexity into researches which are already, in themselves, sufficiently complex to necessitate the adoption of scrupulous care to prevent their becoming still more involved. It seems to us that any further doubt as to the meaning of the words alcoholic fermentation, and the sense in which they are employed, is impossible, inasmuch as Lavoisier, Gay-Lussac, and Thénard have applied this term to the fermentation of sugar by means of beer yeast. It would be both dangerous and unprofitable to discard the example set by these illustrious masters, to whom we are indebted for our earliest knowledge of this subject. [back]
Note 10. See, for example, the communications of MM. Colin and Poggiale, and the discussion on them, in the Bulletin de lAcadémie de Médecine, March 2d, 9th, and 30th, and February 16th and 23rd, 1875. [back]
Note 11. We have elsewhere determined the formation of minute quantities of volatile acids in alcoholic fermentation. M. Béchamp, who studied these, recognized several belonging to the series of fatty acids, acetic acid, butyric acid, &c. The presence of succinic acid is not accidental, but constant; if we put aside volatile acids that form in quantities which we may call infinitely small, we may say that succinic acid is the only normal acid of alcoholic fermentation.PASTEUR, Comptes rendus de lAcadémie, vol. xlvii., p. 224, 1858. [back]