YEASTS AND FERMENTATION.

HISTORICAL AND EXPLANATORY.

It is beyond the scope of this book to treat the development of the science of fermentation elaborately, but it may be desirable to review quite briefly the history of the theory of fermentation.

At different periods the theories of Liebig, Pasteur, Traube, Naegeli and Buchner have served successively to explain the various phenomena attending the process of alcoholic fermentation.

The process of fermentation was undoubtedly practiced in prehistoric times. It was not, however, until the middle of the eighteenth century that science had advanced sufficiently to recognize the gas escaping from fermentation as identical with that produced by the combustion of charcoal, which is now known as carbonic acid gas. Lavoisier, in 1789, was the first to recognize that fermentation was essentially a process of splitting up sugar into two portions, viz., alcohol and carbonic acid, in about equal quantities, "which, if it was possible to reunite, ought to form sugar," while it remained for Pasteur to show that glycerin and succinic acid were regular products of fermentation.

Appert, in the beginning of the nineteenth century, was the first to produce evidence that yeast was necessary for the fermentation of sugar. He preserved beer wort unfermented by simply excluding air from contact with boiled and cooled wort, whereas, if yeast was introduced in such cooled wort fermentation soon set in, though air was excluded. Appert founded a method of preserving perishable articles of food on the principle of heating and excluding air, and thus became the originator of what is now generally termed the process of pasteurization.

The true nature of yeast was not scientifically demonstrated, however, until Cagniard de la Tour and simultaneously Schwann in 1838 described yeast as consisting of numberless living organisms, which multiplied rapidly by budding, and the presence of which in a solution of sugar was absolutely necessary to cause fermentation.

Stahl was the first to formulate a theory which was afterward adopted by Lavoisier for the breaking up of sugar. Long before the nature of yeast was known he advanced the proposition that the ferment communicated its own internal motion to the sugar with the effect of reducing it to new substances. "As chemistry advanced," says Huxley, "facts came to light which put a new phase upon Stahl's hypothesis and gave it a safer foundation than it previously possessed. The general nature of these phenomena may be thus stated: A body A, without giving to, or taking from, another body B, any material particles, causes B to decompose into other substances, C, D, E, the sum of the weights of which is equal to the weight of B, which decomposes.” Some time after Stahl, Thénard, in 1803, explained the decomposition of the sugar by assuming that the ferment combines with a portion of the oxygen of the sugar, thus causing the fermentation to commence; the equilibrium between the principles of the sugar being disturbed, carbonic acid and alcohol is formed. Thus Stahl becomes the forerunner of Liebig, and Thénard of Pasteur.

Schwann undertook his experiments mainly with a view of refuting the doctrine of spontaneous generation, which assumed that living organisms could develop out of lifeless matter without the agency of eggs, germs, seeds, etc. He disposed of that doctrine effectually by showing that air might be admitted in any quantity to solutions which had been boiled in flasks without causing fermentation or putrefaction, provided the germs contained in such air were destroyed.

Liebig was of opinion that fermentation was not dependent upon the vital activity of the yeast plant, that the splitting up of sugar into alcohol and carbonic acid takes place under certain circumstances without growth, development or reproduction of yeast, and that this process is brought about through peculiar chemical changes that take place in certain nitrogenous constituents of the yeast cell which affect the molecules of

sugar sufficiently to bring about decomposition similarly as the inversion of cane-sugar into dextrose in contact with yeast is due to its nitrogenous constituent, invertase.

Pasteur considered the splitting up of sugar into alcohol and carbonic acid a function of the living yeast organism. Fermentation was with him the result of a physiological process. which would set in when the yeast was unable to obtain from its surroundings free oxygen necessary to the exercise of its vital activity. In this case it would extract the required oxygen from the sugar contained in the solution in which the yeast is immersed, resulting in the splitting up of the sugar molecule into alcohol and carbonic acid gas.

A. J. Brown showed that if two fermentations are conducted under the same conditions and so as to arrest entirely the growth of the yeast, the fermentative energy of the yeast in one liquid will be increased if aërated as compared with the other one which is not aërated, thus refuting Pasteur's theory.

Traube, in 1858, explains alcoholic fermentation as being brought about by the influence of ferments (enzymes) contained in the yeast cells, these ferments having a definite chemical composition and being analogous in their action to such substances as diastase, these substances having the power of transferring the oxygen from one group of atoms that constitute a chemical substance, to another group of atoms, thereby causing, as in the case of sugar, a splitting up of complex molecules into simpler ones.

Naegeli claims, unlike Pasteur, that the splitting up of sugar takes place outside and not inside the yeast organism, and is effected by vibrations emanating from the molecules composing the living protoplasm of the yeast cells. The action of the yeast would thus be a purely physical and not a chemical (Liebig), physiological (Pasteur), or enzymatic one (Traube). Fischer showed that the action of yeasts on sugars is a purely chemical function due to enzymes that the yeast contained; that of the sugars, dextrose, levulose, galactose, are directly fermentable, while other sugars, like saccharose and maltose, are fermentable only in case the yeast contains the corresponding enzymes which, like invertase, changes saccharose to dextrose, or maltase, which changes maltose to dextrose. The power of yeast to ferment sugar is dependent upon the conformity of the

geometrical structure or configuration of the sugar molecules with the molecules composing the active agencies or enzymes of the yeast cell, like the construction of a key must conform to the construction of a corresponding lock. The sugars, with a larger molecular weight (saccharose, maltose), are split up or unlocked, yielding sugars with a smaller molecular weight, like dextrose, which then falls apart into alcohol and carbonic acid by the action of yeast.

Will showed that dead yeast may cause fermentation phenomena, that is, decomposition of sugar into alcohol and carbonic acid, and offered in explanation for this and the other fact that watering of yeast lowers its fermentative energy, the suggestion that an enzyme-like substance is contained in yeast which does not lose its power of splitting up sugar with the death of the yeast, and which being soluble in water is extracted from the yeast during the watering process.

It remained for Buchner to obtain a solution of this enzymelike substance from the yeast by rupturing the yeast cell by means of grinding pressed yeast with sand and then subjecting the moistened mass to an immense pressure. After filtration, this clear liquid, when brought together with sugar solutions, induced fermentation just as the living yeast cells would have done. Buchner calls the enzyme contained in this solution from yeast, zymase.

Buchner's theory was not permitted to go entirely unchallenged. It was claimed that the action of the yeast-juice could be explained by assuming that fermentation was due to the particles of living yeast-plasma contained in the juice, and not necessarily to an enzyme-like substance.

This objection was also met by Buchner, who subjected yeast-juice to the action of a centrifugal machine. All particles in suspension, including yeast plasma, were thus collected in one part of the liquid and another part obtained free from yeast-plasma, which latter portion showed the same power of fermentation as the liquid containing the plasma.

Fermentation would thus, if Buchner's theory is correct, appear to be a process similar to the splitting up of starch into maltose and dextrin, which, as we know, is effected by the enzyme diastase contained in the malt. Fermentation, as well as inversion, then would be nothing more than enzymatic

action, and these two interesting phenomena would at last admit of a common explanation.

Although yeast had been observed under the microscope as early as the beginning of the eighteenth century by Lieuwenhoeck, it was not until Cagniard de la Tour, Schwann and Mitscherlich ook up the subject more than a hundred years afterward, that yeast was described and its importance in fermentation recognized. Kützing, about the same time, described the acetic acid ferment, while it was left to Pasteur to discover, study and describe numerous microbes, capable of inciting fermentation, differing from one another, particularly in their products. Pasteur was thus enabled to point out the characteristics of alcoholic, butyric acid, acetic acid, lactic acid fermentations, and it was due to his investigations that the importance was brought home to the winegrower and later to the brewer of excluding from their respective fermentations those microbes, through whose agency the products of wine cellar or brewery are injured. In order to accomplish this end, it was Pasteur's aim, among other precautions, to free the yeasts from undesirable foreign organisms. Although he achieved this object, practically, as far as bacteria were concerned, his methods did not permit of a separation of a mixture of desirable and undesirable types of yeast. This it was left for the master hand of Hansen to accomplish, whose methods are treated elsewhere in detail. (See "Pure Yeast Culture.")

Hansen was thus enabled to point out that the yeasts commonly employed in the brewery, besides often containing wild yeasts, were commonly mixtures of different species of cultivated yeast, each of which, when isolated and used as a pure culture, would give a beer with peculiar properties; that some species of culture yeast found in such mixtures could, under certain circumstances, produce beer diseases. Thus a judicious selection of the type of yeast to be employed becomes an all-important factor in brewing. Hansen's methods and results have been of inestimable value, both for the advancement of purely scientific methods of research and from an economical standpoint.

FERMENTATION OTHER THAN ALCOHOLIC.

Besides alcoholic fermentation there are observable, wherever microbe life finds proper conditions for its sustenance, other