NOTE ON THE FERMENTATION OF MALTOSE AND

GLUCOSE IN ALKALINE SOLUTIONS*

BY IRENE E. STARK AND MICHAEL SOMOGYT
(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis)

(Received for publication, November 4, 1941)

The selective fermentation of glucose in the presence of mal-

tose, in alkaline solutions, furnished the basis of a new approach

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to the analysis of diastatic reaction products (I). While using
this method extensively, we felt that the effect of alkalinity upon
the rate of fermentation of maltose deserved additional study
from a theoretical as well as a practical point of view.
Fermentation in these experiments was carried out at 30” with
washed bakers’ yeast, according to the technique described in
a previous article (2). The rate of fermentation was measured
by the determination of the unfermented sugar in samples period-
ically withdrawn from the fermenting solution; the fermentation
in the samples was stopped by removing the yeast by centrifu-
gation.
The sugar was determined by the copper-iodometric procedure
of Shaffer and Somogyi, with the “high alkalinity” reagent (1).
Because the sugar solutions contained buffers, precaution was
taken to forestall their effect upon the alkalinity of the copper
reagent. Failure to consider this effect causeserrors in the sugar
determination, especially in the acid ranges; the error is as much
as 5 to 6 per cent when the sugar solution is buffered at pH 6.0,
and about 2.5 per cent even at pH 7.0. This source of error
was eliminated by adjusting the buffered sugar solution with
sodium hydroxide to about pH 8.0 as described in a subsequent
paragraph.
The e$ect of pH on the rate of fermentation of maltose was studied
in the range between pH 3.6 and 8.4. (These figures represent
the initial pH values, but in the course of the fermentation they

* This work was aided by the Helen Yonkers Research Fund.

579
580 Fermentation in Alkaline Solutions
were slightly lowered by carbon dioxide derived partly from the
sugar, partly from the “self-fermentation” of the yeast.) From
pH 3.6 to 5.2 acetic acid-sodium acetate and from pH 5.2 to 8.0
phosphates were used as buffers; sodium bicarbonate was em-
ployed to produce pH 8.4. The concentration of the buffers and
of bicarbonate was 0.2 M in all cases.

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0 JO 120
Hinder

FIG. 1. The effect of pH on the rate of maltose fermentation

In Fig. 1 are represented fermentation rates at a few pH values.

As may be seen, the rate was highest at pH 4.8, while at lower
as well as at higher pH values fermentation proceeded at lower
rates. The optimal pH range, according to data not shown in
the graph, lies between 4.8 and 5.2. It may be mentioned that
the rate of fermentation of maltose solutions which contain no
buffers is distinctly higher than the optimal rate in the presence
of buffers, indicating that the electrolytes employed exert a slight
inhibitory effect.
 I. E. Stark and M. Somogyi

Direct Fermentation of Maltose-Willstatter and his collaborators

and later Sobotka and Holzman (3) advanced evidence to the
effect that maltose is fermented by brewers’ yeast directly, with-
out the need of preliminary hydrolysis by maltase. Our results
confirm this view in the instance of bakers’ yeast. Maltase
preparations, extracted from brewers’ yeast in our laboratory,
showed optimal activity in the pH range of 6.0 to 6.8, much the
same as Michaelis and Rona have reported (4). Yet our data
show that the time of half fermentation of maltose at pH 6.4,

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i.e. at the optimum of maltase action, was 93 minutes, whereas
it was only 27 minutes at pH 4.8, at which maltase activity is
almost negligible, and 47 minutes even at pH 3.6, at which mal-
tase is virtually inactive. These figures show that the fermen-
tation of maltose is independent of maltase action.
Application in Analytical Worlc-Our experiments furnish cer-
tain directives for the analysis of diastatic reaction products.
When the non-fermentable reducing substances (dextrins) are to
be determined, both maltose and glucose must be fermented
away. But when diastase is allowed to act in a buffered medium,
as a rule at pH 6.8, complete fermentation of the maltose cannot
be attained even in 4 or 5 hours, unless either the pH is first lowered
to about 5.0 or the phosphates are removed as barium salts. At
the optimal pH or in buffer-free solutions, fermentation is com-
plete in 2.0 to 2.5 hours, provided that the yeast employed is
fresh. The importance of the latter factor is illustrated in Fig. 2.
All three curves show the fermentation rates of unbuffered solu-
tions uniformly containing 40 mg. of maltose per 100 cc., in which
15 gm. (moist weight) of washed bakers’ yeast were suspended.
In the experiment represented by the topmost curve the yeast
was 3 days old (the time necessary to get it from manufacturing
plant to laboratory) ; the other two curves show the marked de-
crease in the fermentation rate when the same yeast was used
10 and 12 days later.
Selective Fermentation of Glucose-Glucose is readily fermentable
at alkaline reactions at which maltose is not affected by bakers’
yeast. Inspection of Fig. 1 reveals that at pH 8.4 no measurable
amount of maltose is fermented in 30 minutes with as much as
15 gm. of yeast suspended in 100 cc. of solution. Table I shows
that at the same alkalinity glucose is almost completely fermented
582 Fermentation in Alkaline Solutions
in 10 minutes with onIy 10 gm. of yeast per 100 cc. of solution.
These facts form the basis of the quantitative determination of
glucose in the presence of maltose.
In our earlier studies of diastatic reactions we used no buffers.
For selective fermentation the reaction products were made alka-
line by the addition of sodium carbonate, and a 30 minute period
was allowed for the complete fermentation of glucose. During

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FIG. 2. The effect of age of yeast on the rate of maltose fermentation

these 30 minutes it was necessary to watch the reaction of the

fermenting fluid lest the pH drop owing to the evolution of carbon
dioxide partly from the sugar, partly from the “self-fermenta-
tion” of yeast. As may be seen in Fig. 1, this was important,
since at pH 8.0 a measurable fraction of the maltose also is fer-
mented in 30 minutes. In the present studies, in which phos-
phate buffers of 0.2 M concentration were used, we found that
 I. E. Stark and M. Somogyi 583
adjustment of the pH to 8.4 is sufficient to safeguard the sharp
separation between glucose and maltose.
Tripartite Analysis-Accordingly, when glucose, maltose, and
reducing dextrins are to be determined in buffered reaction mix-
tures, we proceed as follows: A 20 cc. portion of the reaction
mixture is measured, a drop of phenolphthalein added, and then,
from a burette, sodium hydroxide is run in until a pale but per-
manent pink color is attained; finally the solution is diluted to
25 cc. or to some other convenient volume. Two 5 cc. portions

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of this alkalinized solution are used for the determination of the
aggregate reducing power (A). The remainder is fermented with
washed yeast, approximately 10 gm. (moist weight) being used
per 100 cc. of fluid. After about 20 minutes fermentation the

TABLE I
Rate of Glucose Fermentation at pH 8.4
Unfermented fraction of glucose
Fermentation til me ---___
With :5str cent
With 5 per cent yeast With 10 per cent yeast
~__
min. mg. per cent mg. per cent mg. per cent

0 51.2 51.2 51.2

10 24.8 0.4 0.5
20 11.4 0 0
30 5.7 0 0
I

yeast is removed by centrifugation, and the reducing power (B)

of the solution is determined. A -B = glucose.
To determine the reducing power of the non-fermentable reac-
tion products (dextrins), both glucose and maltose must be com-
pletely fermented away. For this fermentation the reaction of
the solution must be adjusted to a pH between 4.5 and 5.2 by
the addition of hydrochloric acid. Ry titrating 10 cc. of the
reaction mixture with the acid, with methyl red as an indicator,
the amount of acid required is determined. On this basis 20 cc.
of the reaction mixture are acidified to the optimal pH of maltose
fermentation, the volume is brought to 25 cc., and then the mix-
ture is fermented with 15 per cent washed yeast for 2.5 hours.
After the yeast is removed by centrifugation, 5 cc. portions of
the fluid are measured into sugar tubes for the determination
584 Fermentation in Alkaline Solutions

of the reducing power. Before the copper reagent is added, the

reaction of the solution is adjusted to pH 8.0 to 8.4 by adding
sodium hydroxide, with penolphthalein or phenol red as indicator.
The reducing power (C) of this solution represents non-fermentable
substances. B - C = maltose.

SUMMARY

Maltose is fermented by bakers’ yeast without the need of pre-

liminary hydrolysis to glucose by the action of maltase (“direct
fermentation”).

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At pH 8.4 glucose is completely fermented in less than 20 min-
utes, whereas under the same conditions no measurable amount
of maltose is fermented in 30 minutes. This fact is employed
as the basis of an analytical procedure for the determination of
glucose in solutions which contain maltose.

BIBLIOGRAPHY

1. Somogyi, M., J. Biol. Chem., 119, 741 (1937).

2. Somogyi, M., J. Biol. Chem., 78, 117 (1928).
3. Sobotka, H., and Holzman, M., Biochem. J., 28, 734 (1934).
4. Michaelis, L., and Rona, P., Biochem. Z., 67, 70 (1913); Biochem. Z., 68,
148 (1913-14).
 NOTE ON THE FERMENTATION OF
MALTOSE AND GLUCOSE IN
ALKALINE SOLUTIONS
Irene E. Stark and Michael Somogyi
J. Biol. Chem. 1942, 142:579-584.

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