ANALYSIS OF DIASTATIC SPLIT-PRODUCTS OF STARCH

BY MICHAEL SOMOGYI
(From the Laboratory of the Jewish Hospital of St. Louis, St. Louis)

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(Received for publication, March 4, 1938)

In our studies involving the quantitative assay of diastase in

blood, urine, and other biological material, identification of the
enzymatic reaction products appeared desirable as the first step.
Recently improved analytical technique and methods developed
in the past few years became available for application to the prob-
lem, promising information not obtainable with the older methods.
The new methods are concerned first, with the detection and deter-
mination of slight quantities of fermentable sugars in solutions
which contain much non-fermentable reducing matter (l), second,
with the detection and determination of small quantities of glucose
in solutions containing relatively much maltose (l), and third, with
improved procedures for the microdetermination of the copper-
reducing power of sugars (2, 3).

Glucose
Earlier investigators, handicapped by analytical procedure,
were unable to identify glucose in diastatic reaction mixtures,
except after the action of very potent enzyme preparations for
rather long periods of time (4-6). Those workers, who regard
maltose as the end-product of diastase action, ascribe the forma-
tion of glucose in such protracted reactions to a slow process of
hydrolysis, entirely independent of the presence of diastase.
With our method (l), however, it can be demonstrated that
glucose is produced at very early stages of diastase action. We
analyzed numerous diastatic reaction mixtures in which enzyme
preparations derived from a variety of sources, such as blood,
saliva, urine, pancreas, human milk, and barley malt, were em-
ployed. In every instance detectable amounts of glucose were
179
180 Diastatic Split-Products of Starch
formed from starch or from glycogen within the 1st hour of the
reaction. Table I contains examples of such experiments.

TABLE I
Production of Glucose jrom Starch by Diastase in Brief Reaction Periods

Glucose
Total
Experhmt Duration of rzaw;g
Source of diastase reaction (in terms Amount %:gnlt
of glucose) re’ducing
matter

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hrs. ml. mg.
Blood plasma 1 74 4 5.4
Saliva 0.5 882 45 5.1
1 1162 80 6.9
3 1298 94 7.2
Concentrated ex- 16 3470 1425 41.1
tract from
urine
Malt diastase 10 min. 5700 340 6.0

TABLE II

-
Production of Glucose by Pro10 ng
-
red
-
Action of Malt Diastase
-

Total
oarbo-
II- Reducing power in terms
of glucose C:lUCOSe
in pw
“;12- Date 1lydratc After lent of
No. in lkalin, total
eiolutior 1 rmen :1uc0sl :arbo-
;ation :a - b) ydrate
(b)
._ - -
per cent1 per cent er ceni Per cent
1 Aug. 8, 1934, obtained and 10.83 5.81 5.28 0.53 4.9
analyzed
Sept. 29, 1934, 2nd analysis 13.08 8.26 5.97 2.29 17.5
Jan. 25, 1937, 3rd “ 14.14 10.00 2.57 7.43 52.6
2 Oct. 3, 1934, obtained and 13.46 5.70 5.36 0.34 2.5
analyzed
Jan. 26,1937,2nd analysis 13.40 5.83 0.73 5.4
- - -

The experiment presented in Table II shows that the glucose

formed in long drawn out diastatic processes is the product of en-
zymatic hydrolysis and not of side reactions independent of the
diastase. The figures in Table II represent the results of repeated
 M. Somogyi 181

analyses of two samples of mash obtained from a commercial

distillery. The samples were taken at two different occasions
in the course of the routine manufacturing process, in which
starch is subjected to the action of large amounts of barley malt
at about 65”. The duration of this process is confined to exactly
10 minutes; samples for our analysis were taken immediately
at the end of this period. Sample 1, received on August 8, 1934,
was not sterilized, but Sample 2 (October 3, 1934) was heated to
loo’, as soon as withdrawn from the batch, in order to inactivate

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the enzyme.
The samples contained ground grain and malt, but only the
fluid part was analyzed; the solids were removed by filtration
from such portions of the samples as were needed for one analysis.
In 5 cc. of the clear filtrate the total carbohydrate content was
determined as glucose after hydrolysis with H$SOd and subsequent
neutralization with Ba(OH)z. In another portion, after appro-
priate dilution, glucose was determined by means of the selective
fermentation method. As may be seen, the two samples, analyzed
immediately after reaching the laboratory, contained 0.53 and
0.34 gm. of glucose, respectively, per 100 cc. of fluid. (The some-
what higher glucose content of Sample 1 probably resulted from
continued diastase action during the time, a little less than a
full day, that had elapsed between sampling and delivery at the
laboratory.)
The remainder of both samples, with the solid constituents and
fluid unseparated, was preserved with toluene and set aside. A
second analysis of Sample 1 was performed after it had stood at
room temperature for 52 days, and a third when it was nearly
24 years old. In this sample, in which the diastase had not been
inactivated (it was affected only by gradual spontaneous deteriora-
tion), the glucose content increased between the first and second
analysis to more than 4-fold, and in the third analysis it was
found to be 14 times as high as in the fresh sample; at this time it
represented more than one-half of the conversion products. The
increase in the total carbohydrate content of this sample between
consecutive analyses was evidently due to further cleavage of
the starch content of the grain particles that had escaped con-
version in the malting process in the factory. In Sample 2, in
which the enzyme had been inactivated by heat, no change had
182 Diastatic Split-Products of Starch

occurred in the total carbohydrate content; the glucose content

was after 27 months standing only 0.74 per cent, as compared with
7.43 per cent in Sample 1. These experiments show that glucose
was formed in very brief periods of diastase action, and, further-
more, that the large amount of glucose formed in a protracted
diastatic reaction was the product of enzyme action (Sample 1);
the amount of glucose formed without the contribution of the
active enzyme (Sample 2) was trivial in comparison.
Maltose

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After the removal of glucose, maltose is the only fermentable
sugar left among the enzymatic cleavage products of starch.
Reliable detection and determination of it by fermentation,
especially in the presence of large quantities of non-fermentable
reducing substances, are possible only if certain essential facts
are taken into consideration.
For the fermentation of digested starch long periods, such as
24 hours and more, were deemed necessary by previous workers.
This technique opened up two sources of error. First, yeast,
if given time, exudes into the fermenting fluid reducing substances
which are non-fermentable; this is true even when one is working
with pure cultures (7). Second, yeast slowly consumes some
reducing substances which are regarded as non-fermentable.
The latter process commands especial attention when one is
dealing with the fermentation of split-products of starch. Some
of the intermediate polysaccharides contained in such media
are, as evidenced by their rate of dialysis, of small enough
molecular size to permit diffusion through the membrane of the
yeast cell, with ensuing further saccharification by the diastase
located within the cell, and subsequent fermentation. This
assumption is in accord with the known fact that colloidal dextrins
and even starch and glycogen are fermented by pressed juice of
yeast; the process begins with the saccharification of the polysac-
charides by the diastase of the pressed juice and ends with the
fermentation of the maltose produced by the diastase (8). This
may explain why some authors found isomaltose fermentable
(9), while others, including the discoverer (lo), had shown it to be
non-fermentable; and why various workers could report the
existence of fermentable dextrins, whereas generally dextrins are
 M. Somogyi 183

known to be non-fermentable. The conflicts in observations

probably hinge upon the time element; given sufficient time,
yeast acts upon some dextrins (non-fermentable split-products
of starch) of small molecular size.
In our studies we have actually observed a very slow process
of continued fermentation after all of the glucose and maltose
had assuredly been removed from diastatic reaction mixtures.
The rate of this after-fermentation, however, is slow beyond com-
parison with that of the fermentation of maltose and, on the
ground of the foregoing consideration, we regard the polysac-

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charides thus affected by yeast as non-fermentable.
Prolonged fermentation, then, blurs the line of demarcation
between maltose and non-fermentable reducing substances.
The rapid fermentation technique, introduced in biochemical
analysis by Hiller, Linder, and Van Slyke (II), eliminated this
difficulty. With the improvement added by the present writer,
which consists in the use of washed yeast (7), this technique allows
the sharp separation of maltose from non-fermentable polysac-
charides.
Yet, while undue prolongation in the fermentation of maltose
must be avoided, one must be on guard against possible errors
that may arise from incomplete fermentation. In order to avoid
errors of this nature, every new batch of yeast employed must
be tested as to the rate of its action upon maltose. Stale yeast is
especially slow in fermenting maltose. Washed yeast, for instance,
that had stood in a refrigerator under frequently changed water for
several weeks, was found unfit for use with maltose, while still
adequate for the fermentation of glucose.
Non-Fermentable Reducing Substances
With glucose and maltose eliminated by fermentation, one can
determine the reducing power of the non-fermentable residue.
At this step in the analytical procedure again certain facts must
be taken into consideration in order to avoid errors. Earlier
workers determined the reducing power of sugars with strongly
alkaline copper solutions, like Fehling’s reagent, which oxidize
any sugar completely within a few minutes. When the more
sensitive micro copper reagents came into use, in which carbonate
and bicarbonate replace alkali hydroxides, it was found that
 Diastatic Split-Products of Starch

weakening of the alkalinity greatly diminishes the rate of the

reaction, and that the time required for the complete oxidation
of any sugar becomes the longer the less alkaline the copper
solution is. In such protracted reactions it became possible to
observe the fact that various sugars require different lengths of
time for their complete oxidation by one and the same copper
reagent (2). This has considerable bearing on our work. We
found, for example, that Reagent 50 of Shaffer and Somogyi,
which oxidizes glucose in 15 minutes completely, requires for the

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oxidation of the non-fermentable reducing substances in diastatic
reaction mixtures at least 40 minutes and for maltose 25 minutes.
If this fact is ignored (as is the case in many instances encountered
in the literature), and one applies the reaction period of 15 minutes
to mixtures of sugars like those we are dealing with in the present
studies, part of the maltose and a still larger part of the non-
fermentable reducing matter escape oxidation. The conse-
quence is a 2-fold error. First, the aggregate reduction value,
which is presumed to represent quantitatively the saccharogenic
action of diastase, will be too low; second, the quantitative rela-
tionship between glucose, maltose, and non-fermentable reducing
substances will be grossly distorted.
Faulty estimation of copper-reducing power affects still another
result, in that it leads to erroneous values in the determination
of the “reduction quotient.” This is a figure representing the
ratio between the reducing power a polysaccharide attains after
conversion to glucose by acid hydrolysis and the original reducing
power of the polysaccharide before treatment with acid. The
magnitude of the reduction quotient is indicative of the molecular
size of polysaccharides and has been generally employed for
the characterization of diastatic cleavage products. If in the
determination of this figure slowly reacting polysaccharides are
not completely oxidized by the copper reagent employed, while
after their conversion to glucose the oxidation is complete, the
quotient will be too high and hence misleading.
Complete oxidation of slowly reacting polysaccharides with the
available carbonate-copper reagents requires unduly long periods
of heating. To obviate this difficulty we added to the Shaffer-
Somogyi series of copper reagents one sufficiently alkaline (1)
to oxidize completely in 20 minutes all of the reducing substances
 M. Somogyi 185

that occur in diastatic reaction mixtures. We use this heating

period uniformly for fermented reaction mixtures as well as for
unfermented ones which still contain glucose and maltose.
Knowledge of the individual constituents of the non-fermentable
fraction would be of manifest interest from the analytical point
of view. However, all efforts devoted to this problem during
the past 60 years remained fruitless. Various workers described
non-fermentable disaccharides under different names (ptyalose,
isomaltose, dextrinose, amylobiose, etc.) as constituents of dia-

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static reaction products; others claimed to have isolated trisac-
charides; but none of these findings was conclusively substan-

TABLE III
Showing That Non-Fermentable Diastatic Split-Product of Starch, with
Reducing Power of Trisaccharide, Is Not a Homogeneous Substance

Reduction Reduction
(glucose Reduction
Fraction (glucose) equivalent. quotient
No. Mode of fractional precipitation rafter aci$ before
hydrolysis
a
hyd~;Ysls (b)
volumes mg. ml.
Original material, “trisaccharide” 1273 676 2.62
9 alcohol + 3 ether 101 15 6.73
9 ‘I + 6 “ 253 76 3.33
9 ‘I + 10 “ 401 146 2.75
9 “ + 15 “ 270 107 2.52
I‘
9 +20 “ 151 67 2.25
Mother liquor from Fraction 5 53 30 1.77

tiated. As to the dextrins, innumerable fractions belonging to

this group were separated and described as individual compounds.
Some of the fractions barely indicate a reducing power, while
others reduce copper to a considerable extent. None of these
dextrins, however, was proved to represent a homogeneous
chemical entity. Much confusion in this field is due to the fact
that, while the slightest variation in experimental conditions is
apt greatly to change the results, it is true also that careful
observation of some definite scheme of procedure may lead to
rather consistent and reproducible results as regards the reducing
power and optical activity of certain fractions of digested starch.
Thus we repeatedly separated after prolonged incubation of
186 Diastatic Split-Products of Starch

starch with blood plasma a non-fermentable substance that showed

with remarkable consistency a reducing power corresponding to
that of a trisaccharide. Yet, subsequently we partitioned such
“trisaccharide” preparations, either by various methods of frac-
tional precipitation or by fractional dialysis, into numerous
fractions, each differing from the other in reducing power and
optical activity.
An example presented in Table III is illustrative of our findings.
In this instance we divided a “trisaccharide,” which possessed a

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reducing quotient of 2.62, into six fractions. Fraction 1, repre-
senting nearly one-twelfth of the original material, showed a
reduction quotient of 6.73, corresponding to a fairly large average
molecular size. Fraction 6 (the last), the material held in
solution after five successive precipitations with 9 volumes of
alcohol and increasing amounts of ether, had a reduction quotient
of 1.77, which corresponds to that of disaccharides (cellobiose
was used for comparison). The reduction quotients of inter-
mediary Fractions 2 to 5 spread between these two extreme values.
The intricate and unclarified character of the non-fermentable
reducing matter constitutes a limiting factor in the exploration
of the saccharogenic action of diastase. We are constrained by
it to the determination of the aggregate reducing power of this
fraction. Yet, the separate estimation of glucose, maltose, and
the non-fermentable complex opens new possibilities in the study
of diastatic reactions.

SUMMARY

Diastases contained in blood, urine, saliva, pancreas, human

milk, and barley malt produce from starch or from glycogen
maltose and glucose and non-fermentable reducing substances.
It is shown that glucose is formed at very early stages of the
reaction; furthermore that the large amount of glucose, formed
in a protracted diastatic reaction, is the product of enzyme action.
In place of estimating the saccharogenic action of diastase on
the basis of the aggregate reducing power of the reaction products,
methods are offered which permit the separate determination of
glucose, maltose, and non-fermentable reducing matter in the
enzymatic cleavage products of starch.
 M. Somogyi 187

BIBLIOGRAPHY
1. Somogyi, M., J. Biol. Chem., 119, 741 (1937).
2. Shaffer, P. A., and Somogyi, M., J. Biol. Chem., 190, 695 (1933).
3. Somogyi, M., J. Biol. Chem., 117, 771 (1937).
4. Musculus, F., and von Mering, J., 2. physiol. Chem., 2, 403 (1898).
5. Ling, A. R., and Davis, B. F., J. Chem. Sot., 85, 16 (1904).
6. Sherman, H. C., and Punnett, P. W., J. Am. Chem. Sot., 38,1877 (1916).
7. Somogyi, M., J. Biol. Chem., 76,33 (1927); ‘78,117 (1928).
8. Buchner, E., and Rapp, R., Ber. them. Ges., 32, 2086 (1899).
9. Syniewski, V., Ann, Chem., 324, 212 (1902).

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10. Lintner, C. J., and Dull, G., Ber. them. Ges., 26, 2533 (1893).
11. Killer, A., Linder, G. C., and Van Slyke, D. D., J. Biol. Chem., 64,
626 (1925).
 ANALYSIS OF DIASTATIC
SPLIT-PRODUCTS OF STARCH
Michael Somogyi
J. Biol. Chem. 1938, 124:179-187.

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