carbohydrates as we are considering is quite inapplicable to the determination of molecular weight, and the same probably applies to the colloidal carbohydrates themselves. THE DAVY-FARADAY RESEARCH LABORATORY, XXXII.-The Stable Dextrin of Starch Transformations, and its Relation to the Maltodextrins and Soluble Starch. By HORACE T. BROWN, LL.D., F.R.S., and J. H. MILLAR. III. Is the Stable Dextrin a Reducing Substance? 322 325 VI. Analysis of Calcium Salt of Dextrinic Acid 328 Ir was shown by one of us and Heron in 1879 (Trans., 1879, 35, 596, that when gelatinised starch, or soluble starch, is hydrolysed with a little cold-water malt-extract at temperatures below 60°, the reaction proceeds with great rapidity until the mixed products of transformation show a cupric reduction corresponding to about 80 per cent. of maltose, and a specific rotation very close on [a] 150°.* With an active diastase, this point may be reached in less than 5 minutes, and further change is comparatively very slow indeed. * At ordinary temperatures, the specific rotatory power may be temporarily lower than this owing to the maltose being liberated in the birotatory state. (See Trans., 1895, 67, 309.) This fact has been referred to many times in our papers, and was illustrated in the 1879 paper by a series of time curves, and again in the same manner in a paper on amylodextrin published in 1889 (Trans., 55, 457). These values of [a], 150 and R 80 mark, in fact, a definite resting point in the reaction, beyond which it is difficult to push it, unless a considerable amount of time is allowed. That the cessation of action is due neither to the weakening of the enzyme nor to the action being a reversible one,* may be readily shown in a variety of ways. For instance, (1) the addition of more enzyme has no appreciable effect in hastening the very slow subsequent change after the resting point has been reached; (2) if more gelatinised starch or soluble starch is added to the solution, it is speedily brought to the same condition of optical activity and reducing power; (3) when a complete or partial separation of the maltose is effected by suitable means, as by fermentation or fractionation with alcohol, the residual dextrin shows the same resistance to further hydrolysis with diastase as do the mixed products which have attained the resting stage. In the 1879 paper (loc. cit.), it was also shown that, if we assume the whole of the reducing substance to be maltose, the resting point of the reaction corresponding to [a], 150° and R 80 agrees very closely with the view that the starch has been hydrolysed according to the following empirical equation, which requires 80.8 per cent. of maltose in the products of transformation. This was the so-called 'No. 8 equation,' which at that time was supposed to represent only one of several other halting points in the hydrolysis. We are now of opinion that this well-marked resting stage, corresponding to [a], 150, R 80, is the only one which admits, with certainty, of being expressed by any definite equation. From the solution of the starch products which have been allowed to reach the above-mentioned point, crystallisable maltose can readily be prepared by extraction with alcohol of 80 per cent., or the maltose can be fermented out by yeast. By either of these methods of treatment, by far the greater part of the reducing substance may be removed, and in this respect these complete starch transformations stand in strong contrast with those in which the resting stage has not * By this we do not mean to imply a denial that the action may not be to some extent a reversible one, but only that this particular effect cannot be due to reversion. been fully attained, since, in all incomplete conversions, the reducing constituent is not present wholly as crystallisable, fermentable maltose, but, in part, at any rate, as maltodextrins. Complete and incomplete conversions also differ from one another in their behaviour towards phenylhydrazine. If the transformation products have reached the resting stage corresponding to [a], 150, R 80, the yield and purity of maltosazone is much greater than is obtained from a proportional amount of an incomplete transformation which might be assumed, from its reducing power only, to contain an equal amount of maltose. Indeed, all the available facts bear out the supposition that in a complete conversion which has attained the resting stage, the greater part, if not the whole, of the reducing power is due to free, crystallisable, and readily fermentable maltose. Such a solution, besides maltose, contains an achroodextrin, which is precipitable by alcohol of 80-85 per cent. This dextrin, amounting to about 20 per cent. of the starch originally taken, is attacked only with difficulty by diastase, and differs in this respect from all the intermediate dextrinous products, amongst which is the maltodextrin which has been the subject of a previous paper. We have submitted this stable dextrin to a careful re-examination during the past two years, and the present paper deals with our most recent conclusions with regard to its nature, properties, and constitution. The difficulties of obtaining the stable dextrin in a state of purity are considerable, and even greater than they are in the case of maltodextrin. This arises from the fact that the process of treatment with alcohol necessarily tends to concentrate in the final insoluble product all impurities pre-existent in the starch and the transforming agent, which happen to be insoluble in the lowest strength of alcohol used in the separation. If fermentation has also been employed, there is the additional risk of introducing small amounts of nitrogenous impurities, and also some of the non-volatile products of fermentation, although the latter are, for the most part, removable by the subsequent treatment with alcohol. The actual amount of such impurities, which may amount to 2-3 per cent., can be determined with a close approximation to accuracy by submitting the separated dextrin to the standard methods of acid hydrolysis described in the previous paper (p. 305). II. Separation and Purification of the Stable Dextrin. In these experiments, we always commenced with from 2000 to 3000 grams of well-washed potato starch, which was brought into VOL. LXXV. Y solution with from 16 to 20 litres of water by successive gelatinisations and liquefactions, in the manner described in our previous paper on maltodextrin (this vol., p. 288). We thus obtained the starch solids, for the final conversion, at a concentration of from 12 to 15 per cent. The preliminary conversions at 65-70° were allowed to go sufficiently far to prevent any of the higher starch transformation products coming out of solution when the temperature was reduced to 15-20°. The completion of the conversion down to the 'resting point,' corresponding to [a]» 150°, R 80, was then carried out at the ordinary temperature, with the aid of coldwater malt-extract, made from a very diastasic air-dried malt. This conversion sometimes lasted for two days* or more, the object being to select the best conditions for producing a low conversion, with the employment of the minimum amount of malt-extract. The importance of this condition is rendered evident by what we have already said about the concentration of impurities on the dextrin during the subsequent processes of its extraction. The actual amount of 'normal' malt extract required was from 3 to 6 c.c. of the extract per 100 c.c. of a solution containing from 9 to 15 grams of starch products. This is equal to from 0.3 to 0.4 c.c. of extract per gram of starch converted, and represents about the minimum amount which can be used for these complete conversions. The actual amount of solids introduced with the malt-extract represents about 3.5 per cent. of the starch used, but by far the greater part of these solids are separable by alcohol of from 75-90 per cent., and are removed in the processes of purification to which the dextrin is submitted. After the conversion had been run down to the desired point, the solution, filtered from a certain amount of 'starch-cellulose,' which always separates during starch transformations made in the cold, was evaporated to a thick syrup. The process of separation was then commenced; this consisted of fractionation with alcohol of various degrees of strength with occasional recourse to fermentation. The treatment with alcohol was varied in every conceivable manner. It would be tedious and unprofitable to give anything but a mere sketch of the process, since, at this stage, it did not differ materially from similar methods we have frequently described before, except as regards the number of operations and the length of time required, often amounting to several months for each experiment. * The solution was occasionally sterilised, with the further addition of malt extract. 'Normal' malt extract is obtained by digesting finely divided malt (air dried) with 2.5 times its weight of cold water, and filtering after 6 hours. We will briefly describe the course of one experiment, which is typical of all. A conversion of 2400 grams of starch which had been run down to the 'resting stage' and evaporated to a syrup was treated in the first place with 7 litres of hot alcohol of 90 per cent.,* the strength of the alcohol in the mixture being 75-80 per cent. About 200 grams of dextrin separated. The alcoholic solution, poured off when cold, was distilled, and the syrupy residue poured into 6 litres of boiling spirit of 95.5 per cent., and digested for some time. The second dextrinous residue thus obtained was further digested with 85 per cent. spirit, the insoluble portion being added to the original residue of 200 grams. The alcoholic solutions on distillation yielded a syrup from which maltose readily crystallised. The combined dextrinous portions, amounting to 660 grams, now contained only 42 per cent. of maltose, as against 80 per cent. in the original starch transformation products. This crude dextrin was dissolved in 6 litres of water, and the solution, having a sp. gr. of 1042-6, was set to ferment with 15 grams of washed and pressed yeast. During the progress of the fermentation, which lasted for 10 days, an examination of the solution was made from time to time, and it was shown that, whilst the specific rotatory power of the unfermented matter steadily increased, the optical properties of the portions fermented at any stage corresponded with those of maltose. The fermented solution, which now gave only a trace of insoluble osazone on treatment with phenylhydrazine, was evaporated to remove alcohol. The specific rotatory power of the residue was [ a ], 182.5°. In order to remove any trace of maltodextrin, the solution, made up to sp. gr. 1043, was now once more treated for 2 hours at 50° with 40 c.c. of a very active malt extract. It was then once more heated to the boiling point, cooled, and again set to ferment with 11 grams of washed yeast for 5 days, during which time 14.04 grams of fermentable substance disappeared, which represents about 30 per cent. of the 540 grams of the crude dextrin. The optical properties of this fermentable substance were again found, by the usual process of fractional fermentation, to correspond with those of maltose. Two precipitations were then made with 85 per cent. alcohol, and the dextrin obtained was submitted to nine successive extractions with boiling alcohol, each extraction lasting for a day or more, the mixture being frequently agitated. The first six of these extractions were made with alcohol of 85 per cent., and the other two with alcohol of 80 per cent. The weights and specific rotatory powers of the matter extracted, and the specific rotatory power of the residual dextrin after each extraction, were determined. * Throughout the paper, the percentages of alcohol are expressed by volume. |