Simple Method for Detecting Ämylase Inhibitors

in Biological Materials1
KARE POSSUM AND JOHN R. WHITAKER
Department of Microbiology and Immunology, Veterinary College
of Norway, Oslo, Norway, and Department of Food Science and
Technology, University of California, Davis, California 95616

ABSTRACT A simple semiquantitative method for detecting amylase or amylase

inhibitors in biological materials is described. The substrate consists of a 0.25% starch-
1.5% agar gel slab buffered at pH 6.5 with 0.1 M phosphate-0.01 M sodium chloride.
Three millimeters wide cellulose strips saturated with a solution to be examined for in
hibitor are placed parallel on the gel slab for 2 hours at 37°.The strips are removed and
other 3-mm wide cellulose strips saturated with amylase solutions are placed at right
angles across the first strips. The system is incubated for 6 or 18 hours at 37°.After

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flooding the slab with Lugol (an iodine containing) solution, aniylase activity is shown
by clear lysis zones on a deep-purple background. Presence of inhibitors is indicated by
interruption or narrowing of the lysis zone where the inhibitor-containing and amylase-
containing strips crossed. A variation of this method using amylase or amylase-inhibitor
mixtures placed into 7-mm wells cut into the starch—agar gel slab is also described. The
starch—agar gel slab methods were compared with the Bernfeld method of determining
amylase activity. J. Nutr. 104: 930-936, 1974.
INDEXING KEY WORDS amylase •amylase inhibitor

Many raw food materials are known to mangoes (18), colocasia, a tuberous food
contain various types of inhibitors which crop of India (19, 20), acorns (21) and the
affect nutritional quality (1). Some of fermentation liquor from actinomycetes
these inhibitors are proteins which decrease production4 also have been shown to con
specific enzyme activities, as for example tain inhibitors of amylase activity. The
the inhibitors of proteases and amylases amylase inhibitor(s) of wheat is stable to
(2). heat during bread production ( 22 ).
During the past 20 years remarkable The nutritional significance of amylase
progress has been made in methods for inhibitors in foods is not clear. They could
detecting - ( 3 ), purifying ( 4, 5 ) and char be important in celiac disease associated
acterizing (6) the protease protein inhibi with the gluten fraction of wheat (10).
tors. This progress has made possible ex Rats fed raw white kidney beans contain
tensive studies of the structures and mech ing high amounts of amylase inhibitor ex
anisms of action of these inhibitors (6), creted undigested starch in the feces and
including X-ray crystallography of inhib on autopsy the rats were found to have
itor (7) and inhibitor-enzyme complexes
(8). Received for publication January 17, 1974.
Similar progress has not been made with 1The authors are grateful to the Agricultural Re
the amylase inhibitors even though they search Council of Norway for financial support and
to Professor Sandvik and the Department of Micro
appear to be nutritionally important (9, biology and Immunology, Veterinary College of Nor
way for use of facilities.
10). Although the presence of amylase in "Sandvik, O. (1962) Studies on casein precipi
hibitors in beans and wheat was noted as tating enzymes of aerobic and facultatively anaerobic
bacteria. Thesis, Veterinary College of Norway, Oslo.
early as 1945 and 1946, respectively (11, a Schmidt, D. & Puls, W. (1971) Amylase inhibitor.
Ger. Offen 2,003,934; Chem. Abstr. 75: 91290P (1971).
12), they have received very little atten 'Frommer, W., Puls, W., Schaefer. D. & Schmidt,
tion until recently3 (13-15). Rye (10, 16), D. (1972) Glucoside hydrolase inhibitors from ac
tinomycetes. Ger. Offen 2,064,092 ; Chem. Abstr. 77:
rice following microbial infection (17), !iim<¡:tt
(1972).
u:«)

L-
 DETECTION OF AMYLASE INHIBITORS
bloated, white intestines (9). Amylase in
hibitory activity was detected in the feces
of these rats. Oral administration of puri
fied amylase inhibitors from wheat had a
marked effect on the rate of digestion of
starch in tests on dogs, rats and man (23).
We believe that the lack of research,
both nutritionally and biochemically, on
amylase inhibitors is due in part to lack of
simple methods for detecting these inhib
itors. The present paper describes a simple,
semiquantitative method for detecting
amylase inhibitors in biological materials
which is ideal in screening large numbers
of samples. The method can be used
equally well for detecting amylase activity.
It is hoped the availability of this simple
method will stimulate nutritional as well as
other studies on this group of inhibitors.
The quantitative potentialities of the sim
ple method are compared with a more
quantitative, but laborious, method of mea
suring inhibition of amylase activity.
MATERIALS AND METHODS
Materials. The two crude preparations
of tt-amylase ( «-1,4-glucan 4-glucanohy-
drolase; EC 3.2.1.1) used were from swine
pancreas 3 and Bacillus subtiliséInhibiting
antibodies against swine pancreas a-amyl-
ase were elicited by injecting a rabbit with
a suspension consisting of equal amounts
of a 10% a-amylase solution in water and
Freund's adjuvant." Four-milliliter injec
tions were given subcutaneously and intra-
cutaneously at 7-day intervals. On day 28
the rabbit was bled, the blood allowed to
coagulate, and the serum collected after
centrifugation. The blood serum was stored
frozen until used. Lugol solution was pre
pared by dissolving 1.0 gram of Iu. and 2.0
gram of KI in 100 ml of water.
Methods. The starch agar plates con
tained (final concentration) agar7 (1.5%),
starch (0.25%), merthiolate (1:10,000),
0.1 M phosphate buffer, pH 6.5, and 0.01 M
NaCl. The agar and starch were dissolved
separately in 0.1 M phosphate buffer, pH
6.5, containing 0.01 M NaCl. The agar was
melted by heating for 20 to 30 minutes in
an autoclave, mixed immediately with the
starch solution and the mixture poured into
glass trays (12 by IS cm) to a thickness of
2 mm. The thickness was standardized by
931
pouring a fixed volume into each tray. The
plates were allowed to stand until the agar
had solidified.
For detection of inhibitors against
a-amylase, 3-mm wide cellulose strips were
saturated with a solution of the material
expected to contain inhibitor, and placed
in parallel on a starch-agar gel plate. The
plate was covered with a sheet of glass, and
the solution allowed to migrate into the
starch-agar gel for a period of 2 hours at
37°.The paper strips were removed, and
other strips of 3 mm wide cellulose paper,
saturated with enzyme solution to be ex
amined, were placed on the agar gel at
right angles to the first strips. The plates
were again covered with a sheet of glass,
and incubated at 37°for 6 or 18 hours.
The strips were then removed, and the
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surface was flooded with Lugol solution.
Amylase activity was shown by clear lysis
zones. Presence of inhibitors was indicated
by interruption of the lysis zone, or by nar
rowing of the lysis zone depending upon
the strength of both the inhibitor-contain
ing solution and the enzyme solution, at
the point where the two paper strips
crossed. We shall designate this as the
crosswise paper strip method.
A semiquantitative determination of in
hibitory activity by the crosswise paper
strip method was performed on a serial
dilution of the inhibitor-containing mate
rial in order to find the highest dilution
which resulted in inhibition.
Starch-agar plates were also used in a
second way for detection and semiquanti
tative determination of a-amylase and in
hibitor activities. For this purpose, wells 7
mm in diameter were made in the starch-
agar gel (prepared as above) with a cork
borer. A fixed volume (25 /¿I)of an a-amy
lase solution, or a serially diluted solution,
to be tested was introduced into the wells,
then the plate was covered with a tight fit
ting glass plate and incubated for a stan
dard time (6 or 18 hours) at 37°.At the
end of incubation, the starch-agar plate
was flooded with Lugol solution and the
excess solution poured off. The presence of
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imiicn-ns a-innylntip was type VI-A, lot 71C-0210 and
Hni-illHx militili« a-ninylnxe was type III-A, lot
SIM' 117O.
"liltco I,al>.mil.ides, Detroit. Mieli.
; Sec footnote fi. Neutral agar.
932 KARE FOSSUM AND JOHN R. WHITAKER
a-amylase activity was indicated by clear
zones around the wells because of hy
drolysis of starch. The highest dilution of
an enzyme resulting in clear zones is taken
as one diffusion unit. This method has been
extended to permit detection and estima
tion of inhibitors of amylase by adding to
each well a fixed amount of amylase but
varying amounts of inhibitor (total com
bined volume of 25 /J). The diameter of
the lysis zones decreased proportionally as
the inhibitor concentration was increased.
Quantitative determination of amylase
and amylase inhibitory activity was done
by the Bernfeld method (24). Soluble
starch 8 (0.50 g), dispersed in 50 ml of 0.1
M phosphate, pH 6.5, containing 0.01 M
NaCl, was dissolved by heating in a boil
ing water bath with constant stirring for
exactly 10 minutes. This solution was pre
pared fresh each day. The 3,5-dinitrosali-
cylic acid9 reagent was prepared as de
scribed by Bernfeld (24). For activity de
termination, 0.20 ml of starch solution at
25°was added at zero time to 0.20 ml of a
solution, also at 25°,containing amylase in
0.1 M phosphate buffer, pH 6.5, with 0.01
M NaCl. When inhibitor activity was to be
determined, the amylase and inhibitor were
incubated together at 25°for 30 minutes
before adding the starch in order to permit
maximum complex formation as shown by
prior experiments. After adding the starch
to initiate reaction, the solution was incu
bated at 25°for exactly 10 minutes. Re
action was terminated by addition of 0.40
ml of dinitrosalicylic acid reagent. For
color development, the terminated re
actions were heated for exactly 5 minutes
in a vigorously boiling water bath, re
moved, cooled, diluted with 4 ml of water
and the absorbance read at 540 nm against
a blank prepared in an identical fashion as
the reaction except that the enzyme was
added after the dinitrosalicylic acid re
agent. Appropriate controls containing 0.20
ml of starch and various amounts of in
hibitory extract (total volume 0.40 ml)
were also run. The concentration of amy
lase should be low enough to give a linear
activity response with time for at least 15
minutes. Absorbance at 540 nm can be re
lated to concentration of reducing groups
formed by means of a standard curve; pre
pared with maltose. The stock maltose solu
tion should contain 0.20 mg maltose per
milliliter.
RESULTS AND DISCUSSION
Since introduction of the casein-agar
plate method in 1962 for detecting pro-
teolytic activity,2 its use has been extended
to identification of various microorganisms -
(25-27), determination of the heterogene
ity of crude protease preparations (28)
and for detection and estimation of pro
tease inhibitors in biological materials (3).
The principles of this valuable technique
can be extended to include other enzymes
and their inhibitors as shown here for
a-amylase and its inhibitors.
The crosswise paper strip method is par
ticularly useful for detecting amylase ac
tivity and inhibitors of amylase activity.
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Amylase activity is readily observed by
the presence of a clear lysis zone following
treatment of the starch-agar plate with
Lugol solution (fig. 1). In the absence of
amylase activity the starch is stained dark-
purple by the iodine-containing Lugol
solution. Presence of inhibitors is indicated
by a narrowing of the lysis zone at points
where amylase- and amylase inhibitor-con
taining paper strips cross. The extent of
narrowing of the lysis zone is a function of
both the amylase and amylase inhibitor
concentrations. Amylase inhibitor prepared
in rabbit against hog pancreas a-amylase
did not inhibit Bacittus subtilis a-amylase
(fig. 1). Inhibitory activity of as many as
five to eight biological extracts against five
different amylase preparations can be de
termined simultaneously on a single starch-
agar gel plate (12 by 18 cm). Thus, the
method is ideal for screening a large num
ber of samples for amylase or amylase in
hibitor activities.
The crosswise paper strip method can
also be used to estimate the amount of
inhibitor present. For this purpose the
amylase concentration is kept constant but
twofold serial dilutions of the inhibitor-
containing solution are made. Extent of
narrowing of the lysis zone at point of
crossing of the amylase- and amylase in
hibitor-containing strips is proportional to
the concentration of inhibitor (fig. 2). By
definition, the lowest concentration of in-
" MÃŽIIIII
Ki'si'iin-li Lalmnitiirii's, Inr.. NVw York.
* Eastman Kodak Co.. Uocliester, N. V.
 DETECTION OF AMYLASE INHIBITORS 933

SWINE PANCREAS AMYLASE

BACILI US SUBI II IS AMVLASL

Fig. 1 Crosswise paper strip method for dem

onstration of a-amylase inhibitors. From left to
right, the inhibitory materials were: 1, undiluted
and unheated serum from rabbit immunized with
swine pancreas a-amylase; 2, the same serum di
luted 1:10; 3, the same serum heated to 70°for
5 minutes undiluted; and 4, the heated serum
diluted 1:10. The two enzymes used were swine
pancreas o-amylase (top), and a-amylase from
Bacillus subÃ-ais at 0.1 mg per ml. The plate was
incubated for 6 hours at 37°from the time when
Fig. 3 Amylase activity determined on starch-
agar gel plates by the "well" method. The amounts
of swine pancreas a-amylase preparation were:
1, 25.0 Mg; 2, 12.5 Mg; 3, 6.25 Mg; 4, 3.12 Mg;
5, 1.56 Mg; 6, 0.78 Mg; 7, 0.39 Mg; 8, 0.20 Mg;
9, 0.10 Mg; 10, 0.050 Mg; 11, 0.025 Mg; 12, 0.012
Mg. The volume of enzyme solution applied into
each well was 25 pi. The plate was incubated at
37°for 6 hours.

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the enzyme-containing strips were applied.
added to each well. After incubation for
hibitor giving detectable inhibition con 6 hours (or 18 hours) at 37°and treat
tains one unit of inhibitor per milliliter. ment with Lugol solution, amylase activity
This would be the 1:160 dilution (no. 6) is indicated by a lysis zone around the well
in figure 2 since inhibition was not detected (fig. 3). Diameters of the lysis zones are
at a 1:320 dilution (no. 7). Therefore, the
undiluted sample contained 160 units of
inhibitor per milliliter.
Detection and estimation of amylase
and/or amylase inhibitor activities can also
be made by a modification of the cross
wise
"well"paper strip method.
method uniform Insize
this plugs
modified
of
starch-agar gel are removed from the slab
and a fixed volume of solution containing
amylase or amylase-amylase inhibitor is

j iff HOURS INCUBATION

\ 5 HOURS INCUBATION

O 1.0 ¿.O 3.0

Fig. 2 Semiquantitative determination of in
hibitors by the crosswise paper strip method. Dif
ferent dilutions of immune serum against swine
pancreas a-amylase, heated at 70°for 5 minutes,
were used. The serum dilutions were (from left
to right): 1, undiluted; 2, diluted 1:10; 3, 1:20;
4, 1:40; 5, 1:80; 6, 1:160 and 7, 1:320. The
enzyme preparations used were swine pancreas
a-amylase (top, 0.1 mg per ml and bottom, 0.01
mg per ml). The plate was incubated at 37°for
18 hours.
i. OGfo i DIFFUSION UNITS/2Sttlì
Fig. 4 Standard curves for swine pancreas
a-amylase activity by the "well" method, pre
pared from the data of figure 3 as indicated by
the legend. The abscissa units are the logarithms
of the number of diffusion units, and the zone
diameters are shown on the ordinate. One diffusion
unit is the highest dilution of enzyme which
gives a clear zone. This would be well no. 10 con
taining 0.05 Mg enzyme in figure 3.
934 KARE POSSUM AND JOHN R. WHITAKER
proportional to amylase concentration as
well as incubation time (fig. 4). In experi
ments for detection and semiquantitative
estimation of amylase inhibitor activity,
each well contained a fixed amount of amy
lase and variable amounts of inhibitor
(prepared as twofold serial dilutions). The
diameters of the lysis zones were inversely
proportional to the concentration of in
hibitor (data not shown). This modified
method is not as useful as the crosswise
paper strip method for detecting the pres
ence of amylase and amylase inhibitor in
biological materials as it requires much
more work and can lead to misinterpreta
tion (3). Furthermore, it is less sensitive
than the crosswise paper strip method.
However, it is more useful for semiquanti
tative work.
Amylase and amylase inhibitor activities
can be determined quantitatively by ap
propriate modifications of the Bernfeld
procedure (24) as shown by the data in
figures 5 and 6. The change in absorbance
is converted to amount of reducing groups
produced by use of a standard curve pre-
fig H-AMYI.ASE PER REACTION
Fig. 5 Relationship between amylase concen
tration and concentration of reducing groups, as
measured by change in absorbance at 540 nm,
produced from starch as determined by the di-
nitrosalicylic acid method. Various amounts of a
stock solution of swine pancreas a-amylase were
diluted to a total of 0.20 ml with 0.1 M phosphate,
pH 6.5 containing 0.01 M NaCl and brought to
25°. At zero time 0.20 ml of \% starch in the
same buffer equilibrated at 25° was added to
each tube and the tubes incubated at 25° for
exactly 10 minutes. Remainder of procedure as
described in text.
J
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-»-r-
jul BLOOD SERUM PER REACTION
Fig. 6 Relationship between inhibitor concen
tration and percentage inhibition of amylase activ
ity. Each reaction contained 20 fig of swine pan
creas a-amylase, various amounts (0 to 15 n\) of
inhibitor (present in blood serum) and sufficient
buffer to give a volume of 0.20 ml. After incuba
tion at 25°for 30 minutes for complex formation
between enzyme and inhibitor, 0.20 ml of 1%
starch in the same buffer was added. Exactly 10
minutes later the reaction was terminated by addi
tion of 0.40 ml of dinitrosalicylic acid reagent.
Remainder of the procedure was as described in
figure 5 and text.
pared with maltose (Fig. 7). The method
is not ideal for screening large numbers of
samples because of the large amount of
work involved in running all the appropri
ate controls. Proper controls must be run
on the starch and on each concentration of
amylase and amylase inhibitor used so as
to assess the amount of substances capable
of reducing 3,5-dinitrosalicylic acid initially
present. These controls are not needed in
the starch-agar gel methods. The sensi
tivity of the starch-agar gel methods is
approximately 10-fold greater than the
quantitative Bernfeld method primarily be
cause of the longer incubation time.
Regardless of the method selected, de
tection and estimation of amylase inhibitor
in crude biological extracts is complicated
by the presence of endogenous amylase.
This is demonstrated in figure 1 where
amylase activity from the extract showed
up in the sample applied vertically on the
 DETECTION OF AMYLASE INHIBITORS
5.
6.
7.
8.
Fig. 7 Relationship between concentration of
reducing groups and absorbance at 540 run after
reaction with dinitrosalicylic acid reagent. Various
amounts (0 to 0.8 ing) of maltose (2 mg/ml stock 9.
solution) and water to a total of 0.40 ml were
mixed with 0.40 ml dinitrosalicylic acid reagent,
heated for exactly 5 minutes in a boiling water 10.
bath, cooled and diluted with 4.0 ml water. The
absorbance was read at 540 nm against a blank
containing no maltose. Assays were performed in
Oslo (•) and in Davis (O). 11.
12.
left (no. 1). Fortunately, this difficulty
can be overcome by heat denaturation of
the endogenous amylase at 60 to 70°prior
to assay for the inhibitor. In our case, 13.
amylase activity was destroyed in 5 to 20
minutes at 60 to 70°with no detectable
loss in amylase inhibitor activity. The 14.
exact conditions of heat treatment must be
determined for each different extract. Rate
of destruction of amylase activity can be 15.
followed readily by either of the starch-
agar gel methods. 16.
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936 KARE POSSUM AND JOHN R. WHITAKER
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