Prof. S. V. Lebedev and P. N. Loskutov.

Conditions and Extent of Possible Use of Brewing Malt Sprouts in Yeast Production

In order to increase the yield of yeast in yeast production, various auxiliary nitrogenous materials have long been used, including brewing malt sprouts, which are a waste product of breweries and contain a sufficient quantity of nitrogen in a form assimilable by yeast.

According to Kellner¹), 23–34.9% of the nitrogen contained in malt sprouts is present in the form of amides, which are highly suitable for yeast nutrition. And since amides are readily soluble in water, in yeast production, when malt sprouts are mashed, their amides will pass into the wort and, during fermentation, may be utilized by the yeast. In addition, the yeast will probably also be able, to some extent, to utilize the nitrogen present in the sprouts in other forms as well.

The investigations of Kühn, Hagemann, and Figerling²) give the following composition for air-dry malt sprouts, presented below in Table I.

Table I.

¹) Merker.—Handbook of Distilling. p. 362, 1907.

²) Wochenschrift für Brauerei. 1913, p. 242. Pechtner.—Über die Verwendung von Malzkeimen als Futtermittel und zu Heilzwecken.

Dietrich and König¹) give the following data, averages for 10 analyses:

Table II.

According to Lermer’s²) investigations, 100 parts by weight of malt sprouts contain:

Table III.

In addition, according to investigations by the same author, malt sprouts contain: various organic acids, resin, wax, fatty oils, cholesterol, gummy, bitter, and coloring substances, sugar, and asparagine.

The nitrogenous substances of brewing malt sprouts, according to the investigation of Kiyoschisa, Ioschimura³), are distributed among the separate groups as follows:

¹) Die Malz und Bierbereitung. E. Leiser, p. 364, 1900.

²) Chemie und Phisiologie des Malzes und des Bieres—B. Prior, pp. 190–191, 1896.

³) Biochemische Zeitschrift. 1911, 3 Heft, p. 222.

Table IV.

All these investigations were conducted chiefly with the aim of clarifying the value of malt brewing sprouts as a feedstuff, but these data also show how valuable malt sprouts are for feeding yeast.

The question of the possibility of using malt brewing sprouts for yeast production was first raised as early as 1883, when Uhlik¹ indicated that malt sprouts, owing to their high content of amide nitrogen, are suitable for use as a nitrogenous raw material in obtaining yeast.

This opinion was confirmed by M. Hayduck² in his article “The Use of Malt Sprouts in the Preparation of Pressed Yeast.” In the fermentation experiments carried out by this investigator with an aqueous extract of malt sprouts, with the addition of pure sugar in an amount of 100% by weight of the sprouts, at a temperature of 14°R, up to 50% of the nitrogen that had passed into the extract was assimilated by the yeast, with a yeast yield of 29.7% calculated on the weight of sprouts, without taking into account the amount of sugar added.

From the foregoing it is evident that the use of malt sprouts for obtaining yeast is expedient; however, the conditions of M. Hayduck’s investigation, as is clear from his experiments, do not at all correspond to the modern conditions and requirements of yeast production.

In the literature there are also some other brief data concerning the value of malt brewing sprouts, in the sense of the assimilation of their nitrogen by yeast; factory practice in yeast production has shown that, with the addition of malt sprouts to other grain materials of the mash, the yield of yeast increases; however, in plants that have the opportunity to use malt sprouts, sprouts are added to the mash without any definite established calculation, often simply on the basis of the availability of malt sprouts at a neighboring brewery. There are also no data as to the equivalent of malt sprouts relative to the usual grain materials used in yeast mashes.

In connection with the above, the aim of the work presented below is:

1) to determine the most favorable conditions for the transfer of the nitrogen of malt sprouts into the wort extract of the mash in yeast production;

2) to establish the possible degree to which yeast can utilize the nitrogen of malt brewing sprouts when fermenting their aqueous extract.

For the investigation, malt sprouts from the Tomsk beer brewery, produced in 1927, were taken; all barley grains and other impurities were removed from the sprouts. For analysis, the sprouts were ground to a fine meal. The resulting composition of the sprouts is given in Table V.

Table V.

Analysis of the malt-sprout meal gave the following: water—6,91%, ash—7,34%, C—41,88%, H—5,78%; N—3,94%; P—0,53%, O—33,62%, calorific value—4205 cal. Analysis of the ash of the malt sprouts was as follows: SiO₂—31,24%, Fe₂O₃—2,72%, Al₂O₃—3,83%, CaO—5,36%, MgO—4,18%, SO₃—2,93%, P₂O₆—18,28%, Na₂O—0,85%, K₂O—21,42%, MnO—traces, Li₂O—traces, total—93,81% + water and carbon residues—6,19%.

After determining the chemical composition of the malt sprouts we had taken, experiments were set up on mashing them in order to determine the conditions corresponding to the maximum transfer of nitrogen from the malt sprouts into solution.

The conditions under which the mashing experiments with malt sprouts were set up were as follows: the malt sprouts were ground on a coarse grater, and mashing was carried out in the usual laboratory apparatus for determining extract yield in brewing malt; the mash beakers were equipped with stirrers and thermometers, and there was also a thermometer in the water bath into which the mash beakers were placed.

One of the inconveniences in mashing malt sprouts is that malt sprouts, having a high moisture content, when mashed with a small amount of water greedily absorb it and swell greatly; this hinders the work of the stirrer, as well as the subsequent filtration of the mash. This inconvenience was eliminated by reducing the concentration of the mash made from malt sprouts; the highest concentration at which it was possible satisfactorily to carry out both the mashing itself and the filtration of the mash was determined by the ratio of the weight of sprouts to water as 1 : 8.

The mashing experiments with malt sprouts were carried out at two concentrations: for one concentration, 8 parts by weight of water were taken per 1 part by weight of sprouts; for the other, 20 parts by weight of water were taken per 1 part by weight. In those cases where, in addition to sprouts, the mash included dry pale brewing malt, the sprouts and malt were mashed simultaneously. In this connection, in selecting the temperature and time conditions for mashing malt sprouts, an effort was made to approach the corresponding temperature conditions for preparing an ordinary yeast mash and, moreover, as far as possible, to reduce the duration of mashing.

The first mashing experiments with malt sprouts, conducted at a ratio of the weight of the material being mashed to the weight of water of 1 : 20, i.e., with a mash concentration of

\[ \frac{1}{20}, \]

were carried out under the conditions given below.

Experiment No. 1. Twenty grams of malt sprouts and 400 grams of water were taken. Mashing began at a temperature of 40°C, and mashing proceeded at this temperature for 1 hour; then, over the course of 10 minutes, the temperature was raised to 100°C, and this temperature was maintained for 3 minutes.

Experiment No. 2. The same conditions as in experiment 1, except that the initial temperature was taken as 50°C.

Experiment No. 3. The initial temperature of 60°C was maintained for 1 hour and then raised in 5 minutes to 75°C, and this temperature was maintained for 3 minutes.

After completion of mashing, the mashes were filtered through a dry pleated filter, and in the filtrate the content of nitrogen that had passed into solution was determined by the usual method; its amount was expressed as a percentage of the initial nitrogen content in the malt sprouts.

The results of these experiments are placed in Table VI.

Table VI

From this we see that raising the temperature to 100°C in the first experiment is of no significance, since in experiment No. 3, at a final temperature of 75°C, more nitrogen passes into solution.

The initial mashing temperatures, however, have such an effect that the most favorable temperature proves to be 50°C, as used in experiment No. 2.

This raises the question of what the transition of nitrogen into solution will be if the initial mashing temperature is taken the same as in experiment No. 2, namely about 50°C, while the final temperature is brought close to the conditions of experiment No. 3.

Proceeding from this, in the following experiments the mashing conditions were taken as follows: the initial mashing temperature of 45°C was maintained for 30 minutes; then, in 20 minutes, it was raised to 65°C, and at this temperature mashing proceeded for 30 minutes.

The data from these experiments can be seen in Table VII.

Table VII

The results of these experiments show that, under the latter conditions, the nitrogen content in the solution increased in comparison with the experiments presented in Table VI.

In order to determine how a shortening of the first mashing period affects the transition of nitrogen into solution, an experiment was carried out in which the initial temperature was taken as 45°C and was then immediately raised over 20 minutes to 65°C, after which the latter temperature was maintained for 30 minutes.

Table VIII.

From the results of this experiment, presented in Table VIII, it is clear that, with a reduction in the mashing time, the amount of nitrogen passing into solution decreased; 50 minutes of mashing is insufficient for maximum transfer of nitrogen into solution. Therefore, in the subsequent experiments the mashing conditions indicated in Table VII were adopted.

In yeast production, mashing usually takes place at relatively high mash concentrations, approximately 1 : 4; and since the concentration of the mash affects the transition of soluble substances into the extract, in order to determine to what extent an increase in mash concentration will affect the transition of nitrogen into solution when mashing malt sprouts, the following experiments were set up with a mash concentration of 1 : 8. In addition, it had to be determined how a change in the duration of the final mashing period affects the transition of sprout nitrogen into solution. In connection with this, a series of experiments was carried out, a comparison of the results of which is given in Table IX.

Table IX.

From the results presented in Table IX, it can be seen that increasing the time of the final mashing period from 30 to 45 minutes does not increase the amount of nitrogen passing into solution; with a reduction of this period, however, the amount of dissolving nitrogen decreases.

From the experiments carried out it is evident that the amount of nitrogen passing into solution varies within fairly wide limits, namely: from 39.24% at 5 min. to 43.82% at 30 min. If, however, the mash concentration at the greater dilution of 1 : 20 is taken into account, the limits prove still wider, namely: from 39.24 to 53.74%.

On the basis of the experiments carried out, the following conclusions may be drawn: for the maximum transfer of nitrogen into solution during the mashing of malt sprouts, the concentration of the mash should not be high, and the temperature and time conditions of mashing that favor the transfer of nitrogen into solution should be as follows: the initial mashing temperature is 45°C, then the temperature is raised over 20 min. to 65°C and held at this temperature for 30 min.; thus, the total duration of mashing is 80 minutes.

It is known that protein substances, under the influence of certain factors, can pass from insoluble forms into soluble ones. Thanks to this, it is possible, when mashing sprouts, to increase the amount of nitrogen passing into solution. These factors may be either the proteolytic enzymes of malt or acids.

Experiments with the use of acids under various conditions, both at normal and at elevated pressure, gave very little under conditions suitable for yeast production; therefore, without touching upon this question, as one requiring special consideration and further development, we shall turn to experiments on mashing malt sprouts together with brewing dry malt. In the following experiment on mashing malt sprouts, 20% of crushed brewing malt was added to their mash, in order to determine the action of the proteolytic enzymes of malt on the transfer of nitrogen into solution.

The concentration of the mash was taken as 1:8. In parallel, mashings were carried out of malt sprouts alone, of malt alone, and of sprouts together with malt, the mashing conditions being those that correspond to the best transfer of nitrogen into solution, as determined by the preceding experiments.

In accounting for the nitrogen that passed into solution during the joint mashing of sprouts and malt, the amount of dissolved nitrogen due to the malt alone was excluded.

The results of these experiments are given in Table X.

Table X.

From Table X it is evident that the transfer of nitrogen into solution with the addition of malt increased by 2% of the total amount of nitrogen passing into solution when mashing malt sprouts alone.

Having determined, on the basis of the experiments carried out, what amount of nitrogen can pass into solution under various conditions of mashing malt sprouts, it was subsequently necessary to resolve the question of precisely in what forms this nitrogen occurs in solution, since in the present case only those forms of nitrogen are of interest to us which, during fermentation of wort from malt sprouts, can be assimilated by yeast.

For this purpose, experiments were set up on mashing malt sprouts at various concentrations, namely: \(\frac{1}{8}\) and \(\frac{1}{20}\), and after completion of the mashing, total nitrogen, protein nitrogen, and nitrogen precipitated by phosphotungstic acid were determined in the filtrate. Having determined the nitrogen precipitated by phospho-

with tungstic acid and, subtracting from this quantity the protein nitrogen found by the usual method, we thus find the quantity of nitrogen falling to the albumoses and peptones.

Subtracting, however, from the total amount of all nitrogen that has passed into solution all the nitrogen precipitated by phosphotungstic acid, we regard the remaining nitrogen as the sum of amide and amino nitrogen.

The data placed in Table XI show the quantities of total nitrogen that passed into solution during mashing of malt sprouts. These quantities, expressed in grams and as percentages of the nitrogen content in air-dry sprouts, are given in Table XI.

Table XI.

The distribution of this nitrogen in the wort by individual groups, expressed in grams and as percentages of all the nitrogen that passed into solution during mashing of malt sprouts, is shown in Table XII.

Table XII.

From the results of these experiments, placed in Table XII, it is evident that the amount of nitrogen in the individual groups increases absolutely, only for

of mash of lower concentration, the relative percentage increase of nitrogen, by individual groups, as compared with the high concentration, is observed only for the nitrogen of albumoses and peptones.

The amount of nitrogen that can be assimilated by yeast, i.e. amino and amide nitrogen, ranges from 56.45 to 62.34% at different concentrations.

To determine to what extent the forms of nitrogen passing into solution during the mashing of malt sprouts are utilized by yeast during fermentation, experiments were set up on the fermentation of wort from malt sprouts; and since it is not known whether other substances necessary for yeast nutrition, besides the fermentation of the sprout wort alone, are present in sufficient quantity in wort from malt sprouts, experiments were set up on the fermentation of malt wort alone, and then wort from malt sprouts was added to the malt wort, calculated at 27.4%, reckoned on sprouts—and thus the nitrogen balance during fermentation by yeast of wort from malt sprouts was taken into account.

The wort from sprouts was prepared by introducing 625 g of malt sprouts into 5 liters of water, previously heated to 45°C, and at this temperature the sprouts were kept for 30 min.; then the temperature was raised over the course of 20 min. to 65°C and this temperature was maintained for 30 min.; after this, for the purpose of sterilization, the temperature was raised to 100°C, then the mash was filtered through cloth, after which the wort was sterilized for 3 days for 1 hour each day. Before fermentation the wort was filtered through cotton wool.

For fermentation, 2 liters of wort with a concentration of 5.5° Ball. were taken, its acidity, expressed in Delbrück degrees, corresponding to 0.42°D. As pitching yeast for the experiment, factory pitching non-infected yeast of race XII was taken, checked microscopically for purity, with a nitrogen content in it of 2.24%, and it was pitched in an amount of 10%, reckoned on the dry matter of the wort, i.e. on its extract. Fermentation continued for 8 hours, at a temperature of 25°C, with blowing of air in the amount (converted to factory conditions) of 5 cubic meters of air per 100 liters of liquid.

The percentage yield of yeast was calculated on the dry matter of the sprouts taken into the mash; the amount of them necessary to obtain 2 liters of wort subjected in our experiment to fermentation was 234.2 g.

The amount and distribution of nitrogen by individual groups in these two liters of wort from sprouts fermented in our experiment, as well as the amount of nitrogen utilized by the yeast, are presented in Table XIII.

Table XIII.

The yeast obtained as a result of fermentation was examined for its nitrogen content, and its raising power was also determined by Kusserov’s instrument.

These data are given in Table XIV.

Table XIV.

In the second experiment, one malt wort was fermented, which was prepared in the following manner: 1250 g of dry brewer’s crushed malt was taken and added to 5 liters of water heated to 45°C; at this temperature the malt was mashed for 30 min., after which the temperature was raised over the course of 30 min. to 70°C, and at this temperature mashing proceeded for 1 hour. After this the mash was filtered through cloth and the wort was sterilized. The concentration of the wort obtained was 17.4 Ball.

For fermentation the wort was diluted to 5.5 Ball, and the conditions for fermentation of the malt wort were the same as for the sprout wort, i.e., for the experiment 2 liters of wort were taken; fermentation continued for 8 hours at a temperature of 25°C, with air blown through at 5 cubic meters per 100 liters of liquid, while the acidity of the wort was 0.31D.

The nitrogen balance in the present experiment on fermentation of malt wort is shown in Table XV below.

Table XV.

The yeast obtained in this experiment by fermentation was examined both for nitrogen and for leavening power, as may be seen from Table XVI.

Table XVI.

The third experiment was set up with the addition to the malt wort of sprout wort, calculated on sprouts at 27.4%, the sprout wort being added to the malt wort in such an amount that the total mixture of sprout and malt wort came to 2 liters, with a concentration of 5.5° Balling, as in the preceding experiments; moreover, the acidity of the wort was 0.33D, and the fermentation was carried out under exactly the same conditions as in the first two experiments.

The results obtained in this experiment are placed in Table XVII.

Table XVII.

Examination of the yeast obtained in this experiment gave the following results (see Table XVIII).

Table XVIII.

For convenience in comparing the results of all these experiments, they are brought together in Table XIX.

Table XIX.

From the data presented in Table XIX we see that, when wort made from malt sprouts alone is fermented, the yeast multiplies comparatively weakly, since this medium apparently is not entirely favorable to vigorous yeast multiplication; but the yeast itself proves to be rich in nitrogen. And now comparing the results of this experiment with the experiment on fermenting malt wort alone, which is more favorable for yeast multiplication, we see that although the relative amount of nitrogen utilized from the malt wort is greater than from the sprout wort, the absolute amount of this nitrogen in the given case is much less than was utilized from the sprout wort; consequently, the yeast, multiplying in the malt wort to a greater extent than in the sprout wort, gave a larger yield, namely by 2.7%, but at the same time the nitrogen content in it is very low (only 1.38%), as compared with the nitrogen content in yeast from sprout wort alone, where it is equal to 2.36%. Considering now the data of the experiment with the addition of sprout wort to malt wort, placed in the same Table XIX, we see that, with the addition of sprout wort to malt wort poor in nitrogen content, the yeast yield is appreciably increased, namely by 5%, and at the same time the yeast itself is considerably enriched in its nitrogen content, in accordance with which its lifting power also increased.

If we assume that, during fermentation of the malt wort, all the nitrogen that could be assimilated by the yeast has already been used up completely, then, under the conditions of the experiment performed, with the addition of still more sprout wort to the malt wort, it may be admitted that the increase in the degree of nitrogen utilization occurred only at the expense of the added sprouts. Under these conditions the nitrogen balance may be represented in connection with the calculation given below.

In the sprout wort, in the 300 cubic centimeters added to the malt wort, there is in all 0.691 g of nitrogen; of this amount, according to experiment No. 15, 0.241 g, i.e. 34.8%, should be assimilated by the yeast. In the malt wort, in 1700 cubic centimeters, there is in all 0.907 g of nitrogen; of this amount, according to experiment No. 16, 0.561 g, i.e. 57.8%, should be assimilated by the yeast. In all, according to the data of experiment No. 17, with the addition of malt-sprout wort to malt wort, 1.024 g of nitrogen was assimilated by the yeast, i.e. 62.4%.

Subtracting from this the nitrogen that can be utilized by the yeast from the malt wort, i.e., 0.561 g, we obtain \(1.024 - 0.561 = 0.463\) g. Considering this nitrogen—0.463 g—as having been used by the yeast from 300 cubic centimeters of sprout wort, we find that, from the sprout wort, under the conditions of experiment No. 15, 67.2% of its nitrogen passing into solution during the mashing of malt sprouts can be utilized; or, calculated as percentages of the total nitrogen content in air-dry sprouts, 30%.

Thus, on the basis of all that has been stated above, one may arrive at the following conclusions:

1. For the maximum transfer of nitrogen into solution when mashing malt sprouts, it is necessary, insofar as possible, to reduce the concentration of the mash.

2. The nitrogen contained in the sprouts, by individual groups, passes into solution in absolutely greater quantity at a lower concentration than at a higher concentration of the mash; the relative percentage increase, however, can be attributed only to the nitrogen of albumoses and peptones.

3. When wort from sprouts is added to malt wort, the utilization of the nitrogen of malt sprouts by yeast increases and reaches 67.2% of the amount of nitrogen that passed into solution during mashing.

4. When wort made from malt sprouts alone is fermented, the amount of nitrogen utilized by the yeast falls comparatively, decreasing to 34.8% of the amount of nitrogen that passed into the wort.

5. Under factory conditions, the amount of nitrogen of malt sprouts passing into solution, under the influence of certain favorable factors—such as green malt, souring of the mash, and the absence of repeated sterilization of the wort, which occurred in the present experiments—should undoubtedly increase; and at the same time the utilization of nitrogen by the yeast will also increase.

1. Über die Bedeutung des Eiweisses im Hefenleben.—F. Hayduk. p. 29, 1926. ↩

2. See ibid. ↩