ACTION OF DIASTASE AND YEAST ON STARCH-GRANULES. 1085 CXV.-The combined Action o f Diastase and Yeast on Starch-grctnules. By GEORGE HARRIS MORRIS, Ph.D., B.Sc. IT has long been known that when starch conversions, containing high type malto-dextrins or the so-called stable dextrin, are acted on by yeast in the presence of diastase, the action is very greatly in excess of that produced by either yeast or diastase acting separately; in fact, the so-called stable dextrin, which is not acted on either by yeast or by diastase under ordinary conditions, may be completely fermented by yeast in the presence of active diastase. Some experiments described a few years ago by J. Vuylsteke (BUZZ. Acccd. roy. Belgipue, 1892, [iii], 24, 577), in which he showed that certain starches, in the solid state, might be ferment,ed by j e a s t in the presence of malt extract, led me to examine the question whether, in1086 MORRIS: THE COMBINED ACTION OF this case also, the combined action of yeast and diastase exceeded that of either when acting alone.To this end, 100 grams of barley-starch were digested with 50 C.C. of normal cold water malt extract and 250 C.C. of water, with constant agitation in a Dunstan’s laboratory shaking machine. Side by side with this, the same quantities of starch, malt-extract, and water were digested with the addition of 10 grams of washed and pressed yeast, the gas evolved being passed through a small wash-bottle. A t the end of 72 hours the two experiments were stopped, in the first the residual starch was filtered off, and the specific gravity, the optical rotation, and the cupric reducing power of the filtrate determined in the usual way ; in the second, the residual starch and yeast were also removed by filtration, the alcohol in the filtrate determined by distillation, and the specific gravity, the optical rotation, and the cupric reducing power determined in the residue after making up t o the original volume.The results obtained were* : (a) Barley-starch with malt-extract alone. Specific gravity ...................................... 102 1 -6. Optical activity in 100 mm. tube .................. 20.7 divisions. Cupric reducing power on 100 C.C. ............... 6.198 grams CuO. (b) Barley-starch with malt-extract and yeast. Alcohol per 100 C.C. ...............................7-95 grams. Specific gravity of residue Optical activity in 100 mm. tube .................. 2-4 divisions. Cupria reducing power on 100 C.C. .............. 0.8624 gram CuO. Control experiments were made in each case with malt-extract alone, and the above numbers are corrected for the results so obtained. When the excess specific gravity of the filtrate in (a) is divided by 3.934, the divisor for maltose a t this density, it is found that the solid matter in solution per 100 C.C. is 5-490 grams; calculated from the optical activity, the quantity per 100 C.C. is 5.201 grams, and from the cupric reducing power, 4.608 grams per 100 C.C. These numbers are su5ciently close to allow of the conclusion that the substance in solution is maltose, and on the basis of the specific gravity, we find that 16.470 grams have gone into solution in the 92 hours.This corresponds to the dissolution of 15.6 per cent. of the starch employed. I n the second experiment (b) in which diastase (malt-extract) and * For a description of the methods used, see ‘‘ Experimental methods employed in the examination of the products of Starch-hydrolysis by Diastase ” (Brown, Morris, and Millar, Trans., 1897, 71, 72-108). ........................ 1007.27.DIASTASE AND YEAST ON STARCH-GRANULES. 1087 yeast were allowed to act conjointly, the amount of alcohol formed by the fermentation corresponds to 15.437 grams of maltose fermented per 100 c.c., and the cupric reducing power of the residue to a further 0.624 gram of maltose per 109 c.c.; the optical activity required by this amount of maltose is 2-48 divisions, against 2.4 obPerved.The specific gravity of the residue indicates the presence of 1 *85 grams of solid matter per 100 c.c., but as this includes the non-volatile products of fermentation i t does not correctly represent the soluble products directly derived from the starch. The soluble products in this case may therefore be taken as 15,437 + 0.624 = 16.061 grams per 100 c.c., or 48.183 grams in all. This means that 45.6 per cent. of the starch had been decomposed. There W H S therefore a greatly increased action of the diastase in the presence of yeast, the quantity of starch which went into solution being nearly three times the amount dissolved by tho diastase alone. It appeared possible that this increased action might be due to the removal by fermentation of the soluble starch-products first formed by the action of the diastase on the starch-granules, thus allowing the enzyme t o act more freely on the remaining granules.Opposed t o this, however, mas the consideration that the alcohol formed during fermentation would probably exercise as great a retarding influence on the diastase as would the soluble starch-products. I n order t o examine this possibility, 10 grams of barley-starch were shaken with 25 C.C. of cold water malt-extract and 100 C.C. of water, side by side with similar quantities of starch and malt-extract, but with the addition of 100 C.C. of a solution of starch-products in place of the water. After 24 hours, both mixtures were filtered and ex- amined as described above. It was found that there was only a difference of 0.24 gram per 100 C.C.in the quantity of starch dissolved in the two experiments. This point was further tested by taking 20 grams of barley-starch, mixing i t with 50 C.C. of cold water malt- extract and 200 C.C. of water, and solidifying the whole with 3 per cent. of gelatin. The mixture was divided into two portions, one being allowed t o stand for 24 hours, and the other placed in a dialys- ing tube, surrounded with distilled water, for 24 hours. At the end of that time, the two experiments were examined as before, and it was found t h a t the amount of substance dissolved in the first experiment was exactly the same as the sum of the soluble products remaining in the dialysing tube plus the maltose which had diffused into the water surrounding it.In the first, the density of the solution, after making all due corrections, was 1010.0, in the second, 1010.1 (water = 1000). It then appeared desirable to ascertain (a) whether the presence of yeabt, under conditions which prevented the exercise of its fer- mentative power, had a n effect similar t o that which i t exerted when1088 MORRlS: THE COMBINED ACTION OF able t o ferment the soluble products formed by the diastase, and (6) whether any increased activity was conferred on the diastase of the malt-extract by the vital activity of the yeast during fermentation. With this end in view, a series of three experiments was made. In (1) 10 grams of barley-starch were digested with 25 C.C.of malt- extract, 100 C.C. of water, and 2 grams of washed and pressed yeast for 24 hours; in (2) the same quantities of starch, malt-extract, yeast, and water were digested in the shaking machine for 24 hours in the presence of a small quantity of chloroform, in order to check the fermentative activity of the yeast ; and in (3) 25 C.C. of malt-extract were mixed with 100 C.C. of water and 2 grams of yeast and allowed to stand until the fermentation was complete. The liquid was then filtered, and the clear filtrate digested with 10 grams of starch in the shaking machine for 24 hours. After each experiment had proceeded for 24 hours, the residual starch was filtered off, and the filtrates examined as described above. In (1) and (3) the ‘‘ original gravities” * were determined and com- pared with the specific gravity of the solution in (2).The results were as follows : (1) ‘‘ Original gravity ” ......... 1010.34 (2) Specific gravity ............... 1005.29 (3) “ Origiual gravity ” ......... 1006.50 These experiments show that neither the presence of non-fermentative yenst-cells nor the influence of fermentation on the diastase of the malt-extract had the effect of bringing about the dissolution of the Same amount of starch as had the combined action of malt-extract and active yeast. I n all these experiments, the matter in solution, or remaining in solution unfermented, had an optical activity and cupric reducing power corresponding to the presence of maltose only. A furthur experiment wm made to determine whether agitation alone had any influence on the action of diastase on starch-conversion products.For this purpose, a solution of transformation products of high rotatory power was made by converting a starch paste with malt-extract in the usual way. After boiling and filtering the solution, it was divided into two portions, to each of which 5 C.C. of cold water malt-extract per 100 C.C. were added. One portion was shaken for 24 hours, the second was allowed to stand without agitation for the same period. The solutions were then analysed, and found t o be identical * I t is more convenient to express the matter dissolved in this way, since the alcohol, &c., produced by the fermentation of the malt-extract itself can be more easily corrected forDIASTASE AND YEAST ON STARCH-GRANULES.1089 in composition. Agitation, therefore, is without any influence on the action of diastase. Finally, the action of precipitated diastase was compared with that of malt-extract. The experiments were carried out precisely as those first described, with the exception that 50 C.C. of a solution of diastase (containing 0.1 gram of precipitated diastase) mere used in place of cold water malt- extract. I n the experiment with diastase alone, the quantity of matter which went into solution in 24 hours was comparatively small, amounting only to 0,402 gram per 100 c.c., but when the diastase and yeast acted together, the quantity dis5olved was 2.37 grams per 100 c.c., and of this 1.59 grams were fermented. There is therefore a still more marked difference between the action of precipitated diastase alone and that of diastase plus yeast, than in the corre- sponding experiments *where cold water malt-extract mas employed, although in neither instance is the action so considerable or so rapid.The analysis of the matter which went into solution under the action of diastase and of that which remained unfermented points t o it being a mixture of maltose and dextrin, and not maltose only, as is the case when malt-extract is employed. Only those starches, the granules of which are attacked by diastase in the cold, undergo fermentation in the presence of diastase and active yeast; thus potato starch, the granules of which are not attacked or dissolved by malt-extract alone, remains absolutely un- touched by malt-extract and yeast when acting together, The foregoing results are especially interesting in view of the attention which has of late been given to instances of so-called sym- biotic action between moulds and yeasts, the chief action of the former being apparently to secrete diastase and degrade the starch or starch-products to fermentable sugars which can then be attacked by the yeast. It would appear, however, that there exists an action closely analogous to symbiosis between an unorganised and an organised ferment, and i t may be doubted whether the fermentations in which moulds and yeasts are concerned are really instances of true symbiosis, since the former can be replaced by an enzyme with the production of the same result.