Eduard Buchner (1860-1917)

Alcoholic fermentation without yeast cells. Preliminary communication

Berichte des Deutschen chemischen Gesellschaft 30, 117 (1897) [as translated and excerpted in Mikulás Teich, A Documentary History of Biochemistry, 1770-1940 (Rutherford, NJ: Fairleigh Dickinson University Press, 1992)]

A separation of the fermentative action of living yeast cells has not so far been successful; in the following a procedure is described which solves this problem.

1,000 grams of brewers' yeast,[1] purified for the preparation of pressed yeast but still not treated with potato starch, is carefully mixed and then rubbed with an equal weight of quartz sand[2] and 250 grams of kieselguhr, until the mass becomes moist and plastic. The paste is then treated with 100 grams of water and, wrapped in a press cloth, is placed gradually under a pressure of 4 to 500 atmospheres: 300 cubic centimeters of press juice result. The residual mass is then again rubbed, sieved and treated with 100 grams of water. Treated once more in the hydraulic press with the same pressure, it gives another 150 cubic centimeters of press juice. Thus from 1 kilo of yeast 500 cubic centimeters of press juice are gained containing about 300 cubic centimeters of substances which are cell contents. To remove traces of cloudiness, the press juice is finally shaken with 4 grams of kieselguhr and filtered through a paper filter with repeated refiltrations for the first portions.

The press juice thus obtained is a clear, slightly opalescent, yellow liquid with a pleasant yeastlike odour. The specific gravity was once found to be 1.0416 (17°C). When this is boiled, a strong separation of coagulum occurs, so that the liquid almost completely solidifies: the formation of insoluble flakes begins at about 35-40 degrees. Even before this bubbles of gas, demonstrably carbonic acid, are observed rising and consequently saturating the liquid with this gas.[3] The press juice contains over 10 per cent dry substance. In an earlier, less well-prepared press juice there were 6.7 per cent dry substance, 1.15 per cent ash, and according to nitrogen content, 3.7 per cent protein substances.

The most interesting property of the press juice lies in this, that it can promote fermentation of carbohydrates. By mixing it with an equal volume of a concentrated cane sugar solution, there occurs after one quarter to one hour a regular evolution of carbonic acid which lasts for days. Glucose, fructose and maltose behave in the same way, but no appearance of fermentation occurs in mixtures of the press juice with saturated lactose as well as mannite solutions, just as these bodies are also not fermented by living brewers' yeast cells. Mixtures of press juice and sugar solution which have been fermenting for several days, when set in the icebox, usually grow turbid without the appearance of microscopic organisms, but at 700 times magnification they show a rather considerable number of protein curds, whose separation probably depends upon the acids resulting from the fermentation. Saturation of the mixture of press juice and saccharose solution with chloroform does not hinder the fermentation but leads to an early slight precipitation of proteins. Filtration of the press juice through a sterilized Berkefeldt kieselguhr filter, which safely holds back all yeast cells, has just as slight a destructive effect on the fermenting power; the mixture of the entirely clear filtrate with sterilized cane sugar solution even at the temperature of the icebox undergoes fermentation, albeit somewhat delayed, after about a day. If a parchment-paper bag is filled with press juice and hung in a 37 per cent cane sugar solution the surface of the bag after some hours is covered with numerous minute gas bubbles; naturally lively gas evolution was also observed inside the bag, due to diffusion of the sugar solution inward. Further experiments must determine whether the bearer of the fermenting action can actually diffuse through the parchment paper, as it seems. The fermenting power of the press juice is gradually lost with time; press juice kept five days in ice water in a half-filled flask showed itself as inactive towards saccharose. However, it is noteworthy that when treated with cane sugar solution, this fermentatively active press juice retains its fermenting power at least two weeks in the icebox. To begin with, a favorable action must be assumed from this for the action of the carbonic acid formed in the reaction in holding off the oxygen of the air; but the easily assimilable sugar could also contribute to preservation of the agent.

Too few experiments have yet been made to enable a conclusion to be drawn as to the nature of the active substance in the press juice. When the press juice is warmed to 40-50 degrees, carbonic acid evolution first occurs, then general separation of coagulated protein. After an hour this was filtered, with numerous refilterings. The clear filtrate still had weak fermenting power toward cane sugar in one experiment, but in a second not any more; the active substance therefore appears either to lose its action at this strikingly low temperature or to coagulate and precipitate. Further, 20 cubic centimeters of the press juice were put into three times as great volume of absolute alcohol and the precipitate sucked off and dried over sulphuric acid in a vacuum; 2 grams of dry substance resulted, and upon digestion of this with 10 cubic centimeters of water, only the smallest part again dissolved. The filtrate from this had no fermenting action on cane sugar. These experiments must be repeated; especially also, the isolation of the active substance by ammonium sulphate will be attempted.

Up to now, the following conclusions have to be drawn for the theory of fermentation. First, it is proved that to bring about the fermentation process, such a complicated apparatus as represented by the yeast cell is not required. It is considered that the bearer of the fermenting action of the press juice is more truly a dissolved substance, doubtless a protein; this will be designated as zymase. The view that a specially formed protein descended from the yeast cells causes fermentation was expressed as long ago as 1858 by M. Traube as the enzyme or ferment theory, and it was later especially defended by F. Hoppe-Seyler. The separation of such an enzyme from yeast cells, however, had not so far been successful.

It still remains questionable also whether the zymase can be numbered among the enzymes already longer known. As C. v. Nägeli[4] has already stressed, there are important differences between fermenting action and the action of ordinary enzymes. The latter is solely hydrolytic and can be imitated by the simplest chemical means. Even though A. von Baeyer[5] has recently increased our understanding of the chemical processes in alcoholic fermentation when he reduced them to relatively simple principles, the decomposition of sugar to alcohol and carbonic acid still belongs to the more complicated reactions; the loosening of carbon bonds in this manner has not been accomplished by any other method. There is also an important difference in the heat of the reaction.[6]

Invertin can be extracted with water from yeast cells killed by dry heat (heated one hour at 150 degrees), and by precipitation with alcohol it can be isolated as easily water-soluble powder. The substance active in fermentation cannot be obtained in a similar way. It may altogether no longer be present in yeast cells heated so high; alcoholic precipitation changes it to a water-insoluble modification, provided an experiment such as the above permits of a conclusion. One can hardly go wrong in assuming, therefore, that zymase belongs to the true proteins and stands closer to the living protoplasm of the yeast cell than does invertin.

The French bacteriologist, Miguel,[d] has expressed similar views as to urease, the enzyme secreted by bacteria of so-called urea protection of the cell wall acts outside, and which differs from that of the cell contents only in this.[7] Also the experience of E. Fischer and P. Lindner[8] regarding the action of yeast-fungus Monilia candida on cane sugar belong here. This yeast-fungus ferments saccharose; but neither Ch. E. Hansen nor the authors mentioned were successful in extracting from fresh or dried yeast with water an enzyme like invertin which, from what has already been stated, could perform the splitting into glucose and fructose. The experiment went entirely otherwise when Fischer and Lindner used fresh Monilia yeast in which, by careful grinding with glass powder, first a part of the cells was opened. The inverting action was now unmistakable. The inverting agent seems here indeed not to be a stable water-soluble enzyme but to be a part of the living protoplasm.

The fermentation of sugars by zymase can occur within the yeast cells;[9] but more probably the yeast cells secrete this protein into the sugar solution, where it causes fermentation.[10] The process in alcoholic fermentation is, then, perhaps only to be considered a physiological action in so far as it is the living yeast cells which secrete the enzyme. Nägeli[11] and Löw have shown that from yeast cells in an originally weakly alkaline nutrient solution (from K3PO4), which later becomes neutral at 30 degrees, already after 15 hours a considerable amount of protein coagulates by heat has diffused out. Actually, it appears, as the above experiment shows, that zymase can pass through parchment paper.

[1]This is freed from superficially adhering water to the extent that at a pressure of 25 atmospheres no more water is given off. [original note]

[2]Glass powder is less suitable on account of its action as a weak alkali. [original note]

[3]Plant physiologists may decide whether this carbonic acid perhaps originates in the oxidation processes connected with respiration. [original note]

[4]Theorie der Gärung. Munchen 1879. p. 15. [original note]

[5]These Berichte 3, 73. [original note]

[6]The heat development appearing on alcoholic fermentation by yeast has recently been estimated again by A. Bouffard, Compt. rend., 121, 357. [original note]

[d]The name of the French bacteriologist is misspelt and should read Miquel (Pierre, 1850-1922). --Teich

[7]It must be noted however that the so-called urea fermentation, the decomposition of urea into ammonia and carbonic acid, is chemically very different from the proper fermentation processes and therefore many do not regard it at all as fermentation. It is a simple hydrolysis, obtained even with water at 120 degrees. [original note]

[8]These Berichte 28, 3037. [original note] [Apparently the page number is wrong; Teich adds the comment "sic" after it. --CJG]

[9]The diosmotic properties make this seem possible. Cf. v. Nägeli, l.c. p. 39.

[10]This also probably clarifies the experiences of J. de Rey-Pailhade (Compt. rend., 118, 201), who prepared a weak alcoholic extract (22 per cent) from fresh brewers' yeast with the addition of some grape sugar. After being freed from micro-organisms by filtering through a sterile Arsonval candle, this sugar-containing extract spontaneously developed carbonic acid in the absence of oxygen.

[11]loc. cit. p. 94. The experiments were repeated with the same result; only it was shown that they proceeded in the same way in lactose as in saccharose solutions. The diffusion processes are therefore not bound to the carrying on of the fermentative activity, as the named authors assumed.

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