S. V. Lebedev and V. V. Tikhomirov.

Determination of the Actual Amount of Undetermined Substances in Dried Brewers’ Malt Sprouts and Study of the Composition of Their Carbohydrate Group

In carrying out ordinary analyses of products of plant origin, discrepancies are unavoidable, expressed in the fact that the sum of the separate analytical groups found by analysis is never equal to 100%. Depending on the nature of the object under investigation, the shortfalls may numerically fluctuate within very wide limits. In analyses of grains, flours, and seeds, along with determinations of moisture, ash, fat, fiber, proteins, and nonprotein nitrogenous substances, starch, dextrins, and pentosans are usually determined directly; as a result, when all these principal groups entering into the composition of normal and mature grain are determined, the sum of the analytical values is generally close to 100%, the shortfall amounting to 1½–4%. These shortfalls are much more considerable in analyses of malted, soaked, immature, and similar grains, where they may be expressed by significantly larger values. Finally, there exist objects of plant origin in which the deficiency reaches 12–28%. Examples are grass and clover hay, as well as brewers’ draff, investigated by König¹. But the quantitative shortfalls of analysis are revealed most clearly in dried brewers’ malt sprouts, in which the generally accepted methods of analysis for similar products find no more than 60% of determinable substances, calculated on the absolutely dry substance. Thus about 40% of an undetermined residue, usually found by difference, remains aside.

In connection with this, in the analytical literature there has become established the concept of the so-called “nitrogen-free extractive substances”—the residue obtained by subtracting from 100 the sum of the directly determined values: ash, fat, fiber, protein, and moisture.

König², examining in detail the concept of “nitrogen-free extractive substances,” considers this designation unfortunate, since the substances responsible for the shortfall do not consist wholly of substances passing into aqueous extraction; on the one hand, the “nitrogen-free extractives” contain soluble carbohydrates and glucosides, organic acids, gummy and mucilaginous substances, and other soluble substances; on the other hand, they are represented by reserve substances insoluble under ordinary conditions in water (inulin, starch), and, finally, they partially enter into the composition of the cell walls in the form of pectin, slime- and gum-like, and similar substances, hemicellulose, lignin, and other incrusting substances.

In addition, the group of “nitrogen-free extractives,” owing to its determination by difference, takes upon itself all the analytical errors of the quantitative accounting of all the other analytically determined—

determined groups. The causes of the inaccuracies lie in the imperfection of the methods for determining the individual analytical groups. Thus, for example, the errors in determining moisture by the usual drying at 100–110°C consist in the inevitable losses of volatile essential oils and volatile acids, and in the oxidation of sugars, dextrins, and fats. In the first case there will be a decrease, in the second case an increase, in weight. In determining “proteins,” the error may also be very considerable. The coefficient 6.25 usually adopted satisfies typical protein substances, corresponding to their accepted (average) nitrogen content of 16%. Meanwhile, the group of so-called “crude protein” includes all possible nitrogenous bases, amides, amino acids, etc., possessing different nitrogen contents—some greater, some smaller than the 16% usually accepted for proteins. Thus, for example, betaine contains 11.96% nitrogen, asparagine 21.2%; therefore for the former the coefficient will be 8.35, and for the latter 4.71. Obviously, the amount of “crude protein” found (by multiplying nitrogen by 6.25) will correspond the less to the actual percentage content of nitrogen-containing substances, the more the plant object contains substances deviating from the conventional coefficient of 6.25. “Crude fat,” determined by extraction with ether, contains as impurities wax-like substances, coloring substances, chlorophyll, etc.—as a result of which the amount found is always higher than the true amount. Finally, “crude fiber” likewise, in addition to true fiber, may contain various incrusting substances.

All that has been said compels one to think that, given a certain diversity of the separate groups entering into the composition of the substance under investigation, the quantitative deficiency of the analysis may be subject to considerable fluctuations. The amount of “nitrogen-free extractives,” determined by difference, may thus be expressed either with a deficiency or with an overstatement relative to the true amount. The diversity of the groups entering into the composition of the “nitrogen-free extractives” makes the very concept of nitrogen-free extractives unclear. At the same time, by determining by difference the group of “nitrogen-free extractives” in studying the composition of little-investigated plant substances, and by avoiding the question of the composition of this group, we to a considerable extent deprive ourselves of the possibility of judging the chemical nature and properties of the very plant object that interests us. Some constituents of the group of “nitrogen-free extractives,” such as, for example, soluble sugars, dextrins, starch, and pentosans, lend themselves quite accurately to direct determination. In certain cases, determinations of these groups by the accepted methods satisfy the requirements of evaluating the material under investigation. But cases are possible when not all of these determinations are applicable, while the concept of “nitrogen-free extractives,” owing to their considerable percentage content, nevertheless requires some differentiation by direct investigation. As an example of the inapplicability of certain methods of determination to some plant objects, let us point to the method of determining starch with gelatinization under pressure (the Reinke method). This method, which is quite satisfactory in analyses of flour and ungerminated seeds, loses much of its accuracy in analyses of malt and spent grain. The reasons for the loss of accuracy of the determination in the latter case lie chiefly in the loss of considerable amounts of monosaccharides, which are largely destroyed under pressure and the influence of high temperature. The quantitative determination of dextrins and sugars suffers in accuracy because of the similarity of the properties of these and other groups. Thus, for example, in the usual method with alcohol it is impos—

one can divide completely on the basis of solubility properties and the ability of dextrins to dissolve in alcohol containing sugar. The drawback common to all direct methods is that they include not only the substances of interest to us, but also neighboring groups. An especially vivid example of a plant object in which the group of “nitrogen-free extractive substances” constitutes a considerable part of its weight and, at the same time, cannot be accurately accounted for by the ordinarily accepted direct determination of such groups as starch, dextrins, and sugars, is furnished by dried brewers’ malt sprouts.

These sprouts have more than once attracted the attention of investigators. Being a waste product of brewing, sprouts, owing to their considerable content of amides and, in general, of lower products of protein substances, are readily used as raw material in yeast production. At the same time, however, the question of accurately accounting for the degree of their utilization in yeast production remains largely unclear. One of the reasons that compelled us in the present case to turn to the study of the composition of sprouts was the difficulty of accounting for the degree of their utilization in yeast production, owing to insufficient knowledge of their composition. Since malt sprouts are used as feed for livestock, their composition has chiefly interested representatives of agriculture; accordingly, the analytical data available in the literature concerning them suffer from one-sidedness and incompleteness.

Table 1.

As is evident from the table, the group of “nitrogen-free extractives” for dried brewers’ malt sprouts fluctuates within the limits of—

¹) Durst. Handbuch der Hefefabrikation. p. 124.
²) Leyser. Die Malzbereitung. 1900, p. 364.
³) Wochenschr. f. Brauerei 1913, 35, p. 471.
⁴) Ibid. 1906, 52, p. 753.
⁵) Chemisches Zentralbl. 1880, p. 224.

41.74% to 48.70% of their weight. It must be supposed that the composition of this group for sprouts must be rather complex. The complexity of the composition of sprouts in general, and in particular of the group of “nitrogen-free extractive substances,” may be thought to be, to a certain extent, also the result of the complexity of the biochemical processes of grain germination and the formation of new tissues¹. The process of grain germination is characterized by the complication of its chemical composition as a result of the breakdown of reserve substances and the formation of new substances with a simplified molecule², Merker³. Prior, Ehrich, and others, in analyzing the enzymatic processes of germination, indicate the presence in malt, in addition to dextrins and maltose, of glucose, cane sugar, and levulose, quantitatively established as early as (1890) by O’Sullivan⁴. The general direction of the processes during germination in the grain is the accumulation of products capable of diffusion. In the young, newly forming tissues, synthetic processes take place. From the simple compounds formed there arise the histological elements of new cells. The accumulated simplest nitrogenous compounds are consumed in the construction of protein substances, and the simplest carbohydrates are consumed in the construction of all carbohydrate compounds. New substances appear—pentosans, which go to build the vegetative organs: roots and stems⁵. Thus, sprouts contain not only the products of breakdown of the germinating grain, but also newly formed substances. Among the compounds obtained as a result of these complex processes there may be intermediate substances, difficult to subject to chemical individualization and determination by ordinary analytical methods. This is indicated by Pfeffer’s experiments⁶, which established that, in the process of regeneration of protein from asparagine, leucine, etc., the addition of nitrogen-free carbohydrate compounds is necessary. Finally, the process of formation of cell walls as a result of the activity of the plasma, still little studied from the chemical side⁷, may also be a source of intermediate compounds. In addition to processes of a synthetic character, in young shoots there occur processes of respiration, expressed in the oxidation of carbohydrates to CO₂, with transition through various organic acids. According to Prior’s investigations, the amount of organic acids in sprouts increases more than twofold in comparison with the grain, calculated on dry matter. Malt sprouts are usually investigated in the form of sprouts of brewer’s dried malt. Without touching on the details of the question of chemical transformations in the drying process, we nevertheless note that, in the presence of acidity and the action of high temperature, a certain shift occurs in the chemical composition of the sprouts. Partial breakdown of unstable intermediate compounds is possible, as is partial formation of new substances, such as, for example, the formation of caramel-like products as a result of caramelization of sugars, and so on. The latter is especially favored by the lack of protection and the tenderness of young cells. From all that has been said it follows that the chemical composition of sprouts, to a considerable degree conditioned by the peculiarities of the processes of malt germination and malt drying, must be in a high

to a degree complex. This position is also confirmed in part by the literary data cited below.

Sprouts, used as a feed material and as a material for yeast production, have been studied most fully with respect to their nitrogenous substances. From the latter histidine, choline, and betaine have been isolated; asparagine has been quantitatively determined (Ioshimura¹, Meissel², and others). The use of sprouts as a medicinal agent prompted the idea of the presence of alkaloids, of which hordenine was isolated—an amine containing a benzene nucleus³. Sprouts, with respect to the extractive non-nitrogenous substances entering into them, have still been little elucidated qualitatively and quantitatively. The latter is to a certain extent explained by the fact that they have been of interest as a nitrogenous feed for livestock.

The very old work of Lermer (1866), the results of which entered into well-known manuals and monographs on brewing, elucidates in part the composition of the “extractive non-nitrogenous” from the qualitative side, without, however, giving any numerical data at all. The cited author names a series of acids: malic, mucic, tartaric, citric, acetic, lactic, oxalic, and propionic; a series of substances of complex composition: gums, resins, wax-like substances, and others. The total amount of sugars was established by the work of Ioshimura⁴; he ascertained the presence of maltose and invert sugar; the question of cane sugar remained open.

Thus, all the foregoing leads to the assumption of the real existence in malt sprouts of the following substances from the group of “extractive non-nitrogenous.” Of the carbohydrates, there are possible various mono- and disaccharides and their anhydrides of the pentosan and hexosan type, caramels, and, finally, intermediate compounds of unknown composition containing the carbohydrate group in a concealed state. In addition, the group of “extractive non-nitrogenous” substances in sprouts includes little-studied wax-like, gum- and resin-like substances of unknown composition and character, and, finally, various plant acids.

Relating these data to the quantitative deficit that interests us in carrying out analyses of plant substances, we see that in sprouts this deficit is composed partly of the underestimation of substances difficult to determine quantitatively (acids, resins, gums, and others), partly is due to inaccuracies of the usual determinations, especially of moisture and proteins. Besides this, the group of substances making up this deficit also includes various carbohydrates that, with the appropriate method, are amenable to quantitative determination. The clarification of the causes of the quantitative deficit in analyses of sprouts, in connection with the above-mentioned industrial significance of this product, is of definite theoretical and technical interest. Therefore, the tasks of the present work include: 1) determination of the actual amount of undetermined substances of the sprouts and 2) accounting for and elucidation of the chemical nature of the individual constituents of the carbohydrate group of extractive non-nitrogenous substances. The complexity of the composition of sprouts, as already stated, does not permit the application of any of the ordinary direct methods for determining the groups of interest to us. For the accurate determination and differentiation of carbohydrates, their separation by groups is required. This can be accomplished on the basis of various

ratios of the individual groups toward particular solvents. It is obvious that, in this connection, the requirement of indifference of the selected solvents with respect to the chemical nature of the substances extracted by them is essential. As applied to the sprouts that interest us, it seems expedient to carry out successive extraction of one and the same weighed portion of these sprouts alternately with ether, alcohol, and water, which was also done in the present work.

Absolute ether, removing fats, fatty acids, and other analogous substances, does not take up the group of carbohydrates which, during extraction, remains in the solid residue. As a result of the subsequent extraction of the solid residue with 90° alcohol, mono- and disaccharides, resinous and wax-like substances, and all free organic acids pass into the alcoholic solution. At the same time, in the solid residue, among the carbohydrate groups of interest to us, there remain anhydro-derivatives of sugars insoluble in alcohol, of the type of dextrins, pentosans, and hexosans. In addition, pectic substances and substances related to them, as well as hemicelluloses, will also enter here.

The solid residue is subjected to aqueous extraction. In this process, some anhydro-derivatives of sugars, of the dextrin type, pass into the aqueous extract. The process of extracting this group takes place in a medium that almost excludes the possibility of hydrolytic processes occurring. Free organic acids have already been extracted by the preceding alcoholic extraction. The action of enzymes remaining after extraction with alcohol can hardly manifest itself, owing to their insignificant amount in dry sprouts and their weakening under the influence of the hot (70°) alcoholic extraction. The solid residue obtained as a result of all these successive extractions thus preserves, unchanged, the higher anhydro-derivatives of sugars, namely cellulose and all groups of carbohydrates related to it: hemicelluloses, hexosans, pentosans, as well as lignin, etc. Analyses of the extracts obtained and of the solid residue make it possible to draw up the balance of the group of so-called “extractive nitrogen-free substances.” In the consolidated balance of analyses, the differentiation of carbohydrates by groups should be revealed, as well as the total amount of actually undetermined substances. Thus, the advantage of analysis with successive extractions lies in separation and, owing to this, in a more exhaustive determination of the individual carbohydrate groups of the sprouts.

Experimental Part

The material used for the investigation consisted of brewers’ sprouts of pale malt from the Tomsk Brewery No. 1, taken from beneath the brushing machine for separating the sprouts. In the course of the work at the plant, the malt is dried for 36 hours in a two-floor kiln, with a gradual change in temperature from 32°—35°C to approximately 70°C. The sprouts taken for investigation were freed from grains, straw, and other impurities and were then roughly ground in a mill. The material was then ground in a Merz mortar; the resulting meal passed entirely through a silk sieve. The air-dry sprouts were stored in a jar with a ground-glass stopper. Their moisture content, determined by repeated analyses, was 6.01%. The customary analysis was carried out according to the directions in the manuals of König¹ and Demyanov², which are generally accepted—

methods. Ash content was determined by ashing in a muffle furnace, fat—by extraction with absolute ether, nitrogen—by Kjeldahl, protein nitrogen—by Barnstein, pentosans—by Tollens. The commonly accepted determination of fiber by Henneberg was replaced by the König method¹. The advantage of this method is that the resulting fiber contains no pentosans, while retaining lignin. In this way a double determination of pentosans is avoided, and the fiber is counted together with the lignins, which, with the Henneberg method, do not remain in the crude fiber. All analyses were carried out in parallel samples. Table II below presents the mean analyses.

Table II.

As % of dry matter

The sprouts under study, as is evident from the table, contain in all 49.9% “nitrogen-free extractives,” of which 14.69% pentosans were determined by the Tollens method. Thus, the undetermined residue obtained by subtracting the sum of all the values found from 100 amounts to 35.23% by weight of the dry sprouts.

Before proceeding along the intended path of successive extractions, for general orientation in the work it was important first to establish the character of this “undetermined residue.” Since it was assumed that a considerable part of it consists of water-soluble sugars and their lower anhydro-derivatives, it was therefore necessary to determine, at least approximately, their total amount. For this purpose a repeated aqueous extraction² was carried out until all substances washable out by water had been completely removed. To weaken the activity of possible enzymes in the sprouts, the extraction temperature was first about 5°C, and then, after five changes of water, was maintained at about 15°C. The weighed portion of sprouts was extracted with a 10-fold amount of water by weight, with stirring and ten successive changes. Since the extraction took about 14 hours, toluene was added as an antiseptic. The separate filtrates and wash waters were combined. In the extract the dry matter was determined by evaporating a definite volume in a platinum dish and drying the residue at 100–103°C. The residue was then ashed. Pentoses were determined by Tollens, nitrogen by Kjeldahl. The total amount of carbohydrates of the hexose type was found by inversion of the extract with hydrochloric acid of specific gravity 1.125.

In view of the uncertainty of the nature of the invertible substances, which might have led to underdetermination, several inversions of different duration were carried out. As a result, the time necessary for inversion was established as 3 hours. Sugars in the neutralized liquid were determined by Bertrand, with a correction for pentoses. For

for precipitating proteins in the inverted liquid, a 3% solution of sodium phosphotungstate is used. The analytical procedure developed for this experiment was also retained in the subsequent aqueous extractions. In the aqueous extract, 40.37% of dry substances was obtained, calculated on the weight of the original dry substances of the sprouts. The extract had the following composition:

Table III.

The numerical data show that the cold aqueous extract consists chiefly of nitrogenous substances—33.93%—and soluble carbohydrates formed upon inversion, sugars of the hexose type—32.53%. The undetermined substances found by difference amount to 15.59%. Pentoses are present in an insignificant quantity in the extract, 4.19%. These numerical data, being recalculated on the weight of the original dry substances of the sprouts, are expressed in the following table.

Table IV.

From these data it is evident that, of the 35.23% insoluble residue of the sprouts, 6.29% passes into the aqueous extract. Aqueous extraction, however, reveals 13.00% of sugars, calculated on the weight of the original sprouts. Therefore, of the 35.23% undetermined residue, there remain undetermined substances amounting in all to \(35.23 - 13.00 = 22.23\%\). Of these, about \(1/3\) passes into the aqueous extract.

Turning to the method of successive extractions, let us consider the conditions under which they were carried out. An accurately weighed sample of sprouts, about 40 g, was placed in a defatted paper thimble (Schleicher & Schüll) of the Soxhlet extraction apparatus and dried at 70°C for 4 hours, until almost all moisture had been removed, so that no more than 0.4–0.5% by weight of the sprouts remained. The thimble was then placed in the Soxhlet extractor, and the sprouts were extracted with absolute ether for 8 h. The criterion for completeness of extraction was the absence of residue on a watch glass after evaporation of a drop of the liquid that had run off. The ether extract was examined for the content of “crude fat” in the usual manner. After extraction, the material was dried at 35–40°C, remaining in the thimble until the ether had been completely removed.

Then this residue, while remaining in the same thimble of the Soxhlet apparatus, was extracted with 90° alcohol for a prolonged period, reaching

up to 30 hours. The extract obtained was transparent, brown in color, and had an acidic reaction to litmus. The residue after extraction with alcohol was quantitatively removed from the thimble and then extracted with five times the amount of water in a beaker with a stirrer for 2 hours. During the first hour the temperature was raised from \(22^\circ\mathrm{C}\) to \(99^\circ\mathrm{C}\), and during the second hour the extraction proceeded at the temperature of a boiling water bath. The extract was filtered off; the residue was washed by decantation twice, also with five times the amount of hot water. The wash waters were combined with the extract. The color of the extract was light yellow, and its reaction to litmus was acidic. The residue after the aqueous extraction was dried at \(65\text{--}70^\circ\mathrm{C}\), which required up to 6 hours, and was then ground into a finely milled powder. Brought to the air-dry state, the solid residue was kept in a jar with a ground-glass stopper. The extracts, each separately made up to exact volumes, were examined for their content of dry matter, ash, nitrogen, pentoses, and hexoses. In each extract, the quantity of undetermined substances was determined by difference. The solid residue after aqueous extraction was examined for the same groups and by the same methods as the sprouts. Taking into account the possibility of certain losses and errors in determining by direct weighing the residue obtained, the weight of the latter was established by subtracting from the weight of the dry matter of the original sprouts the sum of the dry matter of the extracts obtained. Knowing the percentage ratio to the weight of the original sprouts and the composition of the solid residue, it was easy to determine the percentage content in it of the various analytical groups, referred to the weight of the original product.

The experiments carried out with extraction in the thimble did not give sufficiently concordant results, owing to the impossibility of achieving complete extraction with alcohol. The cause was the caking of the entire mass being extracted, which was evidently due to the property of the sprout tissues to contract strongly under the influence of alcohol.

The analyses showed that the dry matter of the alcoholic extract consists, by one third, of undetermined substances. For the rest, the alcoholic extract is characterized by low ash content (about 4% by weight of dry matter), a considerable content of nitrogenous substances (about one third), and also of soluble sugars (about one third). The dry matter of the aqueous extract is likewise distinguished by a considerable content of undetermined substances (about 20% by weight of dry matter). In addition, the aqueous extract contains a considerable amount of mineral substances and sugars of almost exclusively the character of hexoses and their soluble anhydro derivatives. The residue of the substance after all the subsequent extractions carried out is characterized on average by the following composition:

Table V.

As % of the dry matter of the residue

From the data in Table V it is evident that the solid residue obtained after successive extractions contains a considerable amount of undetermined substances, namely 26.32% by weight of the dry matter of this residue. Judging by its composition, one may think that this residue consists of cell walls with proteins and ash that do not pass into solution. The amount of dry matter of the solid residue, recalculated to the weight of the dry matter of the sprouts, is 60.71% of the latter.

Hence, recalculating the obtained data on the composition of the solid residue (Table V) to the weight of the dry matter of the sprouts, we obtain the following figures, characterizing the degree of extraction by extracts of the separate constituent parts of the sprouts.

Table VI.

Content by weight of the dry matter of sprouts, in %

Thus, the solid residue contains 15.94% of undetermined substances, calculated by weight of the dry matter of the original sprouts. The method of determinations and the conventional coefficients (as, for example, 6.25 for nitrogen) are precisely calculated for those groups of compounds by which this solid residue is represented; therefore, it must be thought that the magnitudes of the individual analytically found groups of the solid residue in this case fully correspond to reality. It follows from this that the quantitative deficit, expressed as 26.32% by weight of the solid residue after extraction, constituting 15.94% by weight of the original sprouts, is a really existing value, and one numerically correctly represented. It may be assumed that these are various complex insoluble compounds accompanying the fiber and remaining unaccounted for by the usual methods.

Extraction of the substance with alcohol in a cartridge, for the reasons indicated above, did not satisfy the requirements for completeness of extraction. Therefore, in subsequent experiments, for alcoholic extraction they used not a cartridge, but ordinary extraction with stirring and free washing, eliminating the possibility of caking. As a result, the following procedure for carrying out the method was developed. An accurately weighed sample of sprouts was dried and then, as before, extracted with ether in a Soxhlet cartridge. The residue was quantitatively transferred to an extraction beaker, where it was extracted for 5 hours with a fivefold amount of 90° alcohol, changed three times.

The extract was brought to an exact volume with the same alcohol. In this case the last change of alcohol was neutral to litmus and had a faint yellow color. This indicated the completeness of extraction. The aqueous extraction is divided into 2 fractions: cold and hot. The considerable expediency of this is confirmed by the results given below. First, the residue after alcoholic extraction is extracted at \(t.\ 10^\circ\)—\(12^\circ\)C with an eightfold amount of water, changed 2 times, for 2 hours. Owing to the passage into solution of acid salts and into

properties of \(KH_2PO_4\), sparingly soluble in strong alcohol and readily soluble in cold water; the reaction of the first change of water proves acidic to litmus. The second change of water is only perceptibly acidic, while the washing waters are completely neutral. The residue is washed with hot water into a beaker and extracted at the temperature of a boiling water bath for 3 hours with an eightfold quantity of water, changed twice. Each extract is separately brought to an exact volume, and the residue is treated as before.

The stated extraction conditions satisfy the aims set. The alcohol extraction removes free acids, and the subsequent cold-water extraction proceeds in a medium whose acidity is due chiefly to \(KH_2PO_4\). Since the extraction is carried out cold and since the greater part of the \(KH_2PO_4\) is removed with the first change of water, it must be supposed that the possibility of inversion of hemicelluloses and of other unstable compounds under these conditions should be reduced to a minimum. Owing to this, the following hot-water extraction proceeds in a neutral medium. In carrying out the hot extraction, it was intended to test the behavior of hemicelluloses, and also of various incrusting substances of the cell wall, toward the action of hot water. The composition of all three extracts and of the solid residue after extraction is presented in the following table:

Table VII

The numerical data of the analyses lead to the following conclusions regarding the method of successive extractions:

The alcoholic extract consists almost one-half of compounds containing carbohydrate groups (48.01%). Partly, the carbohydrate group is represented by sugars directly reducing Fehling’s solution; partly by more complex polysaccharides, not directly reducing. The extract contains 28.18% nitrogenous substances and a considerable amount—19.88%—of undetermined substances. It may be thought, from the works of previous authors, that among the latter—

1) Prior, “Chemie u. Physiologie d. Malzes u. Bieres.” 1896.

resinous substances, the water-insoluble part of the alcoholic extract; free organic acids, tannins, wax-like and other substances are also concentrated here.

The cold aqueous extract contains 22.59% ash substances, 27.78% of compounds forming hexoses upon inversion, 18.14% nitrogenous substances, and only 4.68% pentoses. The indeterminate substances in it, by difference, amount to 26.81%. It is impossible to form any clear idea of the composition and nature of the substances constituting this residue. One may only suppose that this includes partly salts of organic acids, partly gums—slime-like substances—and pectin substances. The latter, as is known, always accompany young tissues¹ and are found not only in the composition of the cell walls, but also in the cellular sap. Therefore their partial passage into the cold aqueous extract is possible. As is known, no method has yet been developed for the quantitative determination of pectin substances. Only the part of the complex pectin molecule that is accounted for by furfural is included by us; the unaccounted-for, it may be thought, quite considerable, part of the pectin molecule must inevitably be connected with the appearance of an indeterminate residue.

In the dry substances of the hot aqueous extraction there are contained 17.66% ash substances, 33.9% nitrogenous substances, 14.3% pentoses, and 25.96% carbohydrates forming hexoses after inversion. The indeterminate residue amounts to 8.14%. As is known², pectin substances, upon prolonged boiling, are capable of passing into solution, and therefore their presence in the hot aqueous extract is quite natural. The composition of the extract under consideration confirms this proposition. In comparison with the composition of the preceding extractions, the hot aqueous extract is distinguished by a significant content of groups characteristic of pentoses, more precisely furfural-yielding compounds according to Tollens’ method. The presence, and moreover in considerable quantity, in the hot aqueous extract of such substances under conditions of neutral reaction, after leaching out all substances soluble in alcohol and water, can be explained only by the passage of pectins. And if, in the preceding cold extraction, the appearance of an indeterminate residue may in part be attributed to an undercount of pectin substances, then in the hot extraction it must be thought that the indeterminate residue depends entirely on their presence. The hexoses obtained by inversion of the hot extract likewise belong to complex compounds entering into the composition of the cell walls.

The solid residue obtained after all successive extractions contains 22.12% substances indeterminate by dry weight. Taking into account the work of König, Schulze, and others, as well as the facts established in the present work, it should be assumed that these indeterminate substances, on the one hand, are anhydro-derivatives of hexoses, of the hemicellulose type; on the other hand, the indeterminate residue is explained by the presence of pectin substances not quantitatively accounted for.

The distribution of the dry substances of the extracts by dry weight of the sprouts proceeds in the following order: the maximum quantity of extracted dry substances falls to the alcoholic extraction—19.88%, then to the cold aqueous—15.85%; the hot aqueous contains 4.95%,

¹ Erlich. “Biochem. Zeitschr.” 1926, Heft 4/6, p. 263.
² Ibid.

and ether extract only 1.64%. The solid residue, determined by difference, amounts to 57.68% of the dry matter of the sprouts. Recalculating the obtained data on the composition of the extracts in Table VII to the total dry matter of the sprouts, we obtain the following:

Table VIII.

Considering the data on the composition of the sprouts, obtained on the basis of analyses by the method of successive extractions, the following general conclusions may be drawn with respect to the nitrogen-free extractive substances:

I. The amount of undetermined substances is 20.52%. These undetermined substances are divided into two distinct groups: a) a group extractable with alcohol and cold water and therefore belonging predominantly to the composition of the cell sap; it amounts in total to \((3.7 + 4.25)\% = 7.95\%\) of the weight of the sprouts, and b)—a group of substances insoluble either in alcohol or in cold water, amounting to \((12.17 + 0.40)\% = 12.57\%\), belonging predominantly to the cell walls. Part of these undetermined substances, upon prolonged boiling, passes into the extract.

II. Compounds containing pentose groups total \((12.88 + 1.62) = 14.50\%\). They are divided into: compounds under the heading of pentoses, amounting in all to 0.17% by weight of the sprouts, and more complex compounds, including pentosans. Of these, part is extracted by cold water, making up 0.74% by weight of the sprouts, while the main mass remains in the solid residue. Of them, during 2-hour boiling, only 0.71% of the weight of the sprouts passes into the extract, while the main part, amounting to 12.88%, is present in the form of complex compounds, analytically determined by the adopted methods of investigation, and designated by the term “pentosans.”

III. Compounds containing hexose groups of the nature of sugars and their anhydro-derivatives total 15.30%. Of these, 4.21%—directly

but sugars of the hexose type reducing Fehling’s solution. The remaining carbohydrates of the hexose character, which do not directly reduce Fehling’s solution, constitute in all \((15.30 - 4.21) = 11.09\%\) of the weight of the sprouts.

An attempt was then made to ascertain the nature of the undetermined substances of the solid residue after extraction of the malt sprouts. This solid residue was tested for its content of hexosans by boiling with 3% hydrochloric acid¹. The method was based on the determination of hemicelluloses according to Schulze, who used boiling with a weak acid with a reflux condenser². However, no preliminary cold treatment with alkali to remove proteins was carried out, in view of the possible presence in the solid residue of pectin substances capable of dissolving cold in alkaline solution. An accurately weighed quantity of the residue, containing about 1 gram, was boiled with 100 cm³ of 3% hydrochloric acid, with a reflux condenser. The mixture was transferred to a volumetric flask, treated with phosphotungstic sodium, and almost brought to a neutral reaction with an alkali solution; in the completely colorless, transparent filtrate, the quantity of sugars was determined by Bertrand’s method. A correction was introduced for pentosans. In view of the uncertainty of the nature of the polysaccharides, a series of experiments was carried out with different durations of inversion, within the limits from 2 to 8 hours. As a result it was established that the time necessary for inversion is 6 hours. With an increase in the duration, processes of destruction of monosaccharides evidently proceed. As a result of these experiments it was found that, of the 22.17% of undetermined substances of the solid residue, 11.49% are hexosans, which corresponds to 6.6% by weight of the sprouts. The remaining 10.68% by weight of the residue, or 6.11% by weight of the sprouts, represent an undetermined residue not amenable to accounting by the methods used in the present work. Summing all the actually found residues of undetermined substances of each extract and of the solid residue in the method of successive extraction, we obtain:

From the data obtained it is evident that a considerable proportion of the undetermined substances of the malt sprouts, namely 6.11%, belongs to substances entering into the composition of the cellular tissue of the sprouts.

In order to ascertain the chemical nature of the composition of the incrusting substances of the cell walls, a series of microchemical tests of the original dry malt sprouts was carried out according to the instructions of Molisch’s manual³. These tests were performed with the kind assistance of Lecturer V. P. Chekhov in the cytological laboratory of Tomsk University. A number of sections were prepared on a microtome from the sprouts under investigation. The results of their microchemical treatment showed the following:

1. The cell walls are incrusted.

2. Reactions with phloroglucinol and hydrochloric acid, as well as with hydrochloric-acid aniline, reveal lignin-containing cell walls.

3. Double staining with “Säuregrün und Naphthylenblau,” as well as staining with safranin, indicates the presence of pectic substances.

Conclusions:

I. The amount of undetermined substances in dry malt sprouts is in fact considerably smaller than that usually found by accepted methods from the difference between 100% and the sum of the moisture, ash, fat, crude protein, “crude fiber,” and pentosans found in the sprouts. Under the conditions of the present experiments, the amount of actually undetermined substances decreases from 33.23% to 14.46%.

II. The amount of undetermined substances in ordinary analyses of sprouts is connected with insufficient completeness in the quantitative detection of the group of monosaccharides of the hexose and pentose type.

III. The group of “extractive nitrogen-free” substances is characterized by a significant content of carbohydrates, of which 14.5% are derivatives of pentoses, and 15.37% are derivatives of hexoses. Of the former, the greater part is represented by higher derivatives—14.33%, while of the latter the greater part is occupied by lower representatives (9.71%).

IV. The amount of undetermined substances extracted by water during direct cold-water extraction is 6.29% of the weight of the sprouts. With successive extraction by ether, alcohol, cold water, and then hot water, this amount reaches, by weight of the sprouts, 8.35%; of this, 3.70% falls to the alcoholic extraction, and 4.65% to the cold and subsequent hot aqueous extraction.

1. König, “Zeitschr. f. Unters. Nahr. u. Genussmittel,” 1913, 3, p. 273. ↩↩↩↩↩↩

2. Schulze, “Zeitschr. f. Physiol. Chemie,” 1896, vol. XXI, p. 397. ↩↩↩↩↩↩

3. Molisch, “Mikrochemie der Pflanzen,” 1923. ↩↩↩

4. Ioshimura. Biochem. Zeitschr. 1911, vol. XXXI, p. 221. ↩↩

5. Merker, Rukov. po vinokur. [Manual of Distilling], p. 356, 1909. ↩

6. C. Michel, Lehrbuch d. Bierbrauerei, 1900, p. 230. ↩

7. Czapek, Biochemie d. Pflanzen. 1922, vol. I, p. 708. ↩