Arthur Harden

“ When we endeavour to apply the results of the investigations of the fermentation of sugar by yeast-juice, zymin, etc., to the process which goes on in the living cell, considerable difficulties present themselves. A scheme of fermentation in the living cell can, however, easily be imagined, which is in harmony with these results. ”

“ Pasteur was therefore led to consider fermentation as a physiological process accompanying the life of the yeast. His conclusions were couched in unmistakable words: "The chemical act of fermentation is essentially a phenomenon correlative with a vital act, commencing and ceasing with the latter. I am of opinion that alcoholic fermentation never occurs without simultaneous organisation, development, multiplication of cells, or the continued life of cells already formed. The results expressed in this memoir seem to me to be completely opposed to the opinions of Liebig and Berzelius. ”

“ History finds man in the possession of alcoholic liquors, and in the earliest chemical writings we find fermentation, as a familiar natural process, invoked to explain and illustrate the changes with which the science of those early days was concerned. ”

“ Glucose itself is not directly esterified, but must first undergo some preliminary change, which is brought about by the action of living yeast. No proof of the existence of a new modification of glucose in this solution has however been advanced, other than its behaviour to extract of Yeast H, so that Euler's conclusion cannot be unreservedly accepted. It is moreover possible and even more probable that some thermostable catalytic substance (perhaps a co-enzyme) passes from the yeast into the glucose solution and enables the yeast extract to attack the glucose and phosphoric acid. ”

“ The action of heat, sulphuric acid, and filtration on the air is to remove, or destroy, not living organisms but particles of decomposing matter, that is to say, ferments which would add their activity to that of the oxygen of the air. Moreover, many ferments, as for example diastase, act without [p011] producing any insoluble deposit whatever which can be regarded as an organism.
"Evidemment," he concludes, "la théorie de M. Liebig explique seule tous les phénomènes de la manière la plus complète et la plus logique; c'est à elle que tous les bons esprits ne peuvent manquer de se rallier." ”

“ When the juice from well-washed yeast is incubated, the phenomenon of autofermentation is observed. The juice contains an abundant supply of enzyme, co-enzyme, and phosphate or hexosephosphate, and in this case the controlling factor is usually the supply of sugar, which is conditioned by the concentration of the diastatic enzyme or of the complex carbohydrates as the case may be. When this is the case the measured rate of fermentation is the rate at which sugar is being produced in the juice, this being the slowest of the various reactions which are proceeding under these circumstances. ”

“ The high rate of fermentation by living yeast and its lack of response to phosphate may possibly be explained by supposing that the balance of enzymes in the living cell is such that the supply of phosphate is maintained at the optimum, and the rate of fermentation cannot therefore be increased by a further supply. ”

“ Alcoholic fermentation depends on the formation of yeast, which is produced when the necessary elements and the proper conditions are present and then propagates itself. The action on the liquid thus increases and the constituents not required to form the organism combine to form unorganised substances, the carbonic acid and alcohol. "It is obvious," says Kützing, in a passage which roused the sarcasm of Berzelius, "that chemists must now strike yeast off the roll of chemical compounds, since it is not a compound but an organised body, an organism." ”

“ When the maximum rate of fermentation of glucose or mannose by yeast-juice in presence of phosphate is greatly lowered by the addition of a large excess of phosphate, the addition of a relatively small amount of fructose (as little as 2·5 per cent. of the weight of the glucose) causes rapid fermentation to occur. This induced activity is not due solely to the selective fermentation of the added fructose, since the amount of gas evolved may be greatly in excess of that obtainable from the quantity added. ”

“ When pure yeast is allowed to develop in a solution of sugar containing a suitable nitrogenous diet and the proper mineral salts, the liquid at the close of the fermentation contains not only alcohol and some carbon dioxide but also a considerable number of other substances, some arising from the carbonaceous and others from the nitrogenous metabolism of the cell. Prominent among the non-nitrogenous substances which are thus found in fermented sugar solutions are fusel oil, succinic acid, glycerol, acetic acid, aldehyde, formic acid, esters, and traces of many other aldehydes and acids. ”

“ Feeding experiments with rabbits and dogs indicate that the ester is capable of hydrolysis in the animal body, a large proportion of the phosphorus being excreted as inorganic phosphate. The ester is also decomposed by Bacillus coli communis.
It is remarkable that the hexosephosphate is not fermented nor hydrolysed by living yeast, a fact observed by Iwanoff, Harden and Young, and Euler, although, according to the experiments of Paine [1911] , the yeast cell is at all events partially permeable to the sodium salt. ”

“ Thus it has been found [Ehrlich and Pistschimuka, 1912, 1] that many "wild" yeasts produce this change with great readiness in presence of sugar, glycerol or ethyl alcohol as sources of carbon and grow well in media in which amines, such as p-hydroxyphenylethylamine or iso-amylamine, form the only source of nitrogen. Willia anomala (Hansen) , a yeast which forms surface growths, succeeds admirably under these conditions, whereas culture yeasts are much less active in this way, although they produce a certain amount of change. ”

“ The study of cell-free fermentation by means of yeast-juice or zymin has, however, revealed the facts that certain of these substances are not formed in the absence of living cells, and that their origin is to be sought in the metabolic processes which accompany the life of the cell. Their source, moreover, has been traced not to the sugar but to the amino-acids, formed by the hydrolysis of the proteins, which occur in all such liquids as beer wort, grape juice, etc., which are usually submitted to alcoholic fermentation. ”

“ When, however, a solution of a hexosephosphate is exposed to the action of either yeast-juice or zymin, entirely or partially freed from co-enzyme, this sugar, being no longer fermented, accumulates and can be examined. It has thus been found [Harden and Young, 1910, 2] that a sugar is in fact produced in this way which can be fermented by living yeast and exhibits the reactions of fructose, although the presence of other hexoses is not excluded. ”

“ In these samples of yeast-juice, therefore, there is present a natural condition of affairs precisely similar to that obtaining in the artificial mixtures of inactive filtration residue and co-enzyme solution made by Harden and Young. The balance of quantities is such that the co-enzyme disappears before the enzyme, leaving a certain amount of enzyme capable of exercising its usual function as soon as sufficient co-enzyme is added. ”

“ If the fermenting power of the juice is to be preserved by repeated regeneration for a long period, it is absolutely necessary to add the co-enzyme solution each time as soon as fermentation has ceased, since the enzyme in the absence of this addition rapidly disappears, even in the presence of sugar. ”

“ A more important difference is that carboxylase decomposes pyruvic acid in the absence of the co-enzyme which is necessary for the fermentation of glucose [Harden, 1913; Neuberg and Rosenthal, 1913] . This can be demonstrated experimentally by washing dried yeast or zymin with water (see p. 63) until it is no longer capable of decomposing glucose (Harden) , or by allowing maceration extract to autolyse or dialyse until it is free from co-enzyme (Neuberg and Rosenthal) . ”

“ When a solution which has been treated in this way is precipitated with lead acetate and kept neutral to litmus, the free phosphate and hexosephosphate are thrown down and the co-enzyme remains in solution. The filtrate can be freed from lead by means of sulphuretted hydrogen and neutralised, and then forms a solution of co-enzyme free from phosphate and hexosephosphate but still containing combined phosphorus. ”

“ If sugar be now added, an entirely different state of affairs is set up. As soon as any accumulated phosphate has been converted into hexosephosphate, the normal rate of fermentation which is usually higher than that of autofermentation is attained, and, provided that excess of sugar be present, fermentation continues for a considerable period at a slowly diminishing rate and finally ceases. ”

“ As this proteoclastic enzyme, like the alcoholic enzyme, cannot be extracted from the living cells, it is termed yeast endotrypsin or endotryptase. Fresh yeast-juice produces a slow fermentation of sugar, which lasts for forty-eight to ninety-six hours at 25° to 30°, about a week at the ordinary temperature, and then ceases, owing, not to exhaustion of the sugar, but to the disappearance of the fermenting agent. ”

“ The problem of alcoholic fermentation, of the origin and nature of that mysterious and apparently spontaneous change which converted the insipid juice of the grape into stimulating wine, seems to have exerted a fascination over the minds of natural philosophers from the very earliest times. ”

“ Since nearly all samples of yeast contain glycogen, yeast-juice and also zymin usually contain this substance as well as the products of its hydrolysis. These provide a source of sugar which enters into alcoholic fermentation, so that a slow spontaneous production of carbon dioxide and alcohol proceeds when yeast-juice is preserved without any addition of sugar. ”

“ Pasteur had at this time firmly established his thesis, no fermentation without life, both for alcoholic fermentation and for those other fermentations which are produced by bacteria, and had put upon a sound and permanent basis the conclusions drawn by Schulze, Cagniard-Latour, Schwann, and Kützing from their early experiments. It became generally recognised that putrefaction and other fermentative changes were due to specific organisms, which produced them in the exercise of their vital functions. ”