Amylase inhibitors

An amylase inhibitor consisting essentially of a protein constructed of 248 amino acid residues having two subunits, each identified as SEQ ID NO:1, in which a single band is observed at a mobility of 0.26 by polyacrylamide gel electrophoresis. The amylase inhibitor can be extracted from wheat and purified by absorption on a cation exchange resin. It is useful for inhibiting an increase in blood glucose level, controlling insulin secretion, suppressing appetite, and as a food additive. The new amylase inhibitor can be used in combination with a protein composed of two subunits, each identified as SEQ ID NO:2, the total content of both proteins being not less than 20% by weight.

FIELD OF THE INVENTION 
This invention relates to a new amylase inhibitor comprising specific 
proteins, a process for the preparation of the same from a wheat origin 
material and the application of such amylase inhibitor as a medicine or 
food. 
BACKGROUND OF THE INVENTION 
Intake of excessive nutrients induces secretion of a larger amount of 
insulin to indirectly cause a collapse of metabolic balance, thus leading 
to a reduction of glucose tolerating function (hyperglycemia), diabetes, 
hyperlipemia, arteriosclerosis, etc. Especially in diabetic patients, the 
insulin function is insufficient and the glucose tolerance is lowered. 
Consequently the blood glucose level is remarkably increased after meals, 
thereby causing complications such as damage to blood capillaries and 
arteriosclerosis. For the prevention and treatment of such diseases it is 
effective to ingest foods or materials which can hardly induce an increase 
in blood glucose level. In this respect materials capable of inhibiting or 
preventing hydrolysis of starch into glucose have been desired. Further, 
overeating contributes to diseases of adult people such as adiposity, 
hypertension, diabetes and cardiac diseases. 
From the above aspects, various studies have been made on so-called amylase 
inhibitors which are effective in inhibiting the activity of amylase to 
hydrolyze starch. It is reported that amylase inhibitors are also 
contained in wheat. Since then, amylase inhibitors of wheat origin have 
been investigated [see, for example, U.S. Pat. No. 3,950,319, Japanese 
Patent Kokai 61-171431, Phytochemistry, vol. 20, No. 8, pp. 1781-1784, 
1981; Eur. J. Biochem. 183, 37-40 (1989)]. 
However, it is reported that prior amylase inhibitors of wheat origin as 
mentioned above have exceedingly low or no inhibitory activity against 
human pancreatic .alpha.-amylase, and do not produce an effect as expected 
when orally given to humans, although they have some inhibitory activity 
against amylases of other animals than human. Therefore, there is a need 
for a highly active amylase inhibitor having a strong inhibitory activity 
against human pancreatic .alpha.-amylase and capable of effectively 
inhibiting an increase in blood glucose level and insulin secretion when 
orally administered at a low level, especially capable of effectively 
inhibiting hydrolysis of heated or cooked starch. 
Further it is reported that an agent for inhibiting an increase in blood 
glucose level or insulin secretion brings about an increased level of free 
fatty acids in blood [Puls and Keup, "Diabetologia" 9, 97-101 (1973)]. In 
general, such increased levels bring about the feeling of hunger which 
will lead to overeating. This results in offsetting the effect of 
inhibiting an increase in blood glucose level and the effect of inhibiting 
an insulin secretion, which will make it difficult to effectively treat or 
prevent the diseases such as diabetes.

DETAILED DESCRIPTION OF THE INVENTION 
We have found that a highly active new amylase inhibitor can be produced by 
treating an amylase inhibitor-containing solution extracted from a wheat 
origin material with water or the like, using a specific method 
distinguished from the prior art processes. More particularly, the 
specific method includes treating an extract containing the amylase 
inhibitor with a cation exchanger to adsorb the amylase inhibitor thereon, 
followed by treating the cation exchanger with an alkali solution of a 
specified pH to elute the amylase inhibitor and immediately making the 
amylase inhibitor-containing eluate acid or neutral with an acid. This 
method provides an eluate which contains a material having a higher 
amylase inhibitory activity than that of known amylase inhibitors of wheat 
origin, particularly a material capable of strongly inhibiting the 
activity of human pancreatic .alpha.-amylase. Our further study of the 
material of high amylase inhibitory activity recovered from the eluate 
with regard to molecular weight and primary structure has revealed that it 
is a new protein having the amino acid sequence specified below. 
Thus, the invention relates to a new amylase inhibitor consisting 
essentially of a protein composed of two subunits, each having the 
following amino acid sequence: 
##STR1## 
This sequence is identified as SEQ ID No:1 in the Sequence Listing. 
The invention also relates to a process of preparing an amylase inhibitor 
comprising predominantly a protein composed of two subunits, each 
identified as SEQ ID NO:1, comprises the steps of: 
(a) extracting a wheat origin material with an extracting solution to 
produce a solution containing the amylase inhibitor; 
(b) optionally subjecting the solution to a purification treatment to 
remove contaminants, treating the solution with a cation exchanger to 
adsorb the amylase inhibitor thereon; 
(c) treating the cation exchanger with an alkali solution at pH 9-13 to 
elute the amylase inhibitor from the cation exchanger; 
(d) immediately adjusting the pH of the eluate containing the amylase 
inhibitor within the neutral or acidic range; and 
(e) recovering from the pH adjusted solution a desired amylase inhibitor as 
defined above. 
The new amylase inhibitor of the invention has a molecular weight of 
approximately 25,000 according to the gel filtration chromatography using 
Sephadex G-75 which will be described later. In addition, when the new 
amylase inhibitor of the invention was subjected to polyacrylamide gel 
electrophoresis according to the method of Davis described in "Annals New 
York Academy of Sciences", 121, pp.404-427 (1964), a single band was 
observed at a mobility of 0.26. By SDS-polyacrylamide electrophoresis 
according to the method of Orth et al described in "Cereal Chem.", vol. 
50, pp. 190-197 (1973), a single band was observed at a position 
corresponding to a molecular weight of 12,500. These data reveal that the 
new amylase inhibitor of the invention is a protein composed of two 
subunits, each of which has a molecular weight of 12,500. 
The amino acid sequence of each subunit was determined using a peptide 
sequencer PSQ-1 (manufactured by Shimazu Co., Ltd.), by which the subunit 
was found to have a structure having 124 amino acid residues identified as 
SEQ ID No:1. Accordingly, the amylase inhibitor of the invention is a 
protein constructed of 248 amino acid residues having two subunits, each 
identified as SEQ ID NO:1. The protein of the invention has S--S bonds. 
The new amylase inhibitor of the invention is called hereafter "0.26 AI" 
for convenience, which has a very high inhibitory activity against an 
amylase, especially a human pancreatic .alpha.-amylase. The inhibitory 
activity of 0.26 AI is about 5-30 times higher than other amylase 
inhibitors of wheat origin such as 0.53 AI composed of two subunits, each 
identified as SEQ ID NO:3 in the Sequence Listing (Biochim. Biophys. Acta. 
743, 52-57 (1983)) and 0.28 AI identified as SEQ ID NO:4 in the Sequence 
Listing [Phytochemistry, vol. 20, No. 8, pp. 1781-1784 (1981)]. 
0.26 AI of the invention includes any protein having an amylase inhibitory 
activity composed of two subunits, each identified as SEQ ID NO:1, 
regardless of the process for the preparation including those by chemical 
synthesis. However, 0.26 AI of the invention can be surely prepared by the 
process of the invention which comprises extracting a wheat origin 
material with water, etc., and subjecting the extract to a cation 
exchanger treatment, which process will be detailed below. 
Process Step (a) 
A wheat origin material is extracted with an extracting solution to prepare 
a solution containing the amylase inhibitor. 
The wheat origin material includes, e.g., wheat, wheat flour and wheat 
gluten, any of which can be used regardless of the species, place of the 
production, season of the harvest, crop year, size of the particles, etc. 
Among them is preferably employed wheat flour (including wheat semolina) 
or wheat gluten because of its good extraction efficiency. 0.26 AI can be 
obtained in high yield from Durum wheat among various species of wheat. 
Therefore, Durum wheat or gluten produced therefrom are especially 
preferred for the wheat origin material. 
The extracting solution used in step (a) includes water, an acid, an 
aqueous acid solution, a dilute alkali, an aqueous alcohol, a dilute salt 
solution, a buffer solution and the combination thereof. 
For the extracting solution is preferred the aqueous acid solution at pH 
2-5, water or a dilute aqueous alkali solution. An aqueous alcohol may be 
used. As the aqueous acid solution is preferably employed an aqueous 
solution adjusted to pH 2-5 with an inorganic acid such as hydrochloric or 
phosphoric acid or an organic acid such as acetic acid. As the dilute 
aqueous alkali solution is preferably employed an aqueous alkaline 
solution adjusted to pH 8-10 with ammonia, sodium hydroxide or the like. 
As the aqueous alcohol is preferably used an aqueous solution of an 
alcohol such as methanol, ethanol or isopropyl alcohol in an alcohol 
concentration of approximately 1-50%. The dilute salt solution includes 
e.g. the solution of a neutral salt such as sodium chloride and potassium 
chloride. As the buffer solution is preferably employed a phosphate 
buffer, an acetate buffer and a tris buffer, which are adjusted to pH 
4-8.5. It is preferable that the extracting solution is maintained at a 
constant pH with a tris buffer or an acetate buffer. 
In step (a), a process may be employed in which a sufficient amount of the 
extracting solution (usually about 3 to 50 times amount) is added to the 
wheat origin material and the extraction treatment is accomplished by 
stirring the mixture usually at a temperature of about 
10.degree.-40.degree. C. followed by removal of solids by any of the means 
such as centrifugal separation, filtration or settlement to give an 
extract solution containing the amylase inhibitor. However, the process is 
not restricted to the above, and may optionally use as the extract 
solution a waste liquid or water washings of the dough or batter 
discharged in the recovery of starch or gluten from wheat flour. In this 
case the water washings of dough or batter which have been troubled in 
handling can effectively be utilized. 
Process Step (b) 
The amylase inhibitor-containing solution extracted in step (a) can be 
treated with a cation exchanger. Before the above treatment, said solution 
may be optionally subjected to any purification treatment to remove 
contaminants. In either case, 0.26 AI of the invention can be produced, 
but the purification is preferable for the preparation of 0.26 AI in a 
higher purity with better efficiency. 
Purification before the treatment with a cation exchanger, if applied may 
be conducted by any purification process used in the production of amylase 
inhibitors from wheat. Examples of the purification processes include: 
Process (1) which comprises adding to the amylase inhibitor-containing 
extract solution obtained in step (a) an agent such as ammonium sulfate or 
ethanol to form precipitates containing the amylase inhibitor, collecting 
the precipitates and suspending them in a small amount of water, 
subjecting the suspension to a desalting treatment using a dialyzing 
membrane, adding a phosphate buffer to a desalted solution to remove the 
precipitate, treating the resultant supernatant with an anion exchanger 
such as DEAE cellulose, using a solution that has passed through the 
exchanger as an amylase inhibitor-containing solution either directly 
without further treatment or after drying to powders and dissolution of 
the powders in a buffer, or further hydrophobic chromatography to collect 
a fraction having an amylase inhibitory activity; process (2) which 
comprises heating the amylase inhibitor-containing extract solution 
obtained in step (a), for example, to 70.degree.-90.degree. C. to denature 
heat unstable protein still remaining in the solution, separating and 
removing the denatured products, to use the resultant solution as an 
amylase inhibitor-containing solution; process (3) which comprises further 
treating the solution obtained in process (2) by means of an 
ultrafiltration membrane (preferably, that having a fractionation 
molecular weight of 20,000 cut off) or gel filtration chromatography to 
remove excess salts and other low-molecular contaminants and if necessary 
subjecting to concentration for use as an amylase inhibitor-containing 
solution; and process (4) which comprises adjusting the amylase 
inhibitor-containing extract solution obtained in step (a) to the pH of 
about 3 and again adjusting the pH to neutrality to separate and remove 
the occurred precipitates and using the solution as an amylase 
inhibitor-containing solution. In particular, the above process (1) is 
preferred. 
As the cation exchangers can be employed those such as polymeric cation 
exchanging resin, silicic acid and aluminum silicate. Specifically, 
polymeric cation exchanging resins such as CM-Toyopal (trade name, Toso 
Co., Ltd.) and Diaion HPK-55 (trade name, Mitsubishi Kasei Kogyo) are 
preferably used. The treatment with the cation exchanger may be carried 
out either by a batch process in which a cation exchanger is added to an 
amylase inhibitor-containing solution and the mixture is stirred to effect 
an ion exchange or by a continuous process in which an amylase 
inhibitor-containing solution is passed through a column filled with a 
cation exchanger. The continuous process is preferred. In the process it 
is preferred to treat the amylase inhibitor-containing solution adjusted 
to pH 3-5 with a cation exchanger which has been equilibrated with a 
buffer at pH 3-5. Then the amylase inhibitor in the solution is adsorbed 
on the cation exchanger. 
Process Step (c) 
The cation exchanger is treated with an alkali solution at pH 9-13 or with 
an alkali buffer at pH 9-10 to elute 0.26 AI adsorbed on the cation 
exchanger. 
Before the above treatment, the cation exchanger may be optionally treated 
with a neutral solution containing sodium chloride or the like to remove 
by elution the impurities from the cation exchanger. The alkaline solution 
used in this step can include an aqueous solution of any alkali compounds 
such as sodium, potassium, calcium, magnesium and ammonium hydroxides with 
no particular restriction to the species of the alkali compound provided 
that the pH is 9-13. An aqueous solution of sodium hydroxide is preferred. 
With an aqueous solution of sodium hydroxide used, the concentration is 
preferably 0.05-1.0N. With an alkali buffer, the concentration is 
preferably 20 mM -1M. If the pH of the alkali solution is below 9, smooth 
elution of the 0.26 AI amylase inhibitor from the cation exchanger cannot 
be achieved. If the pH of the alkali solution is above 13, 0.26 AI is 
denatured during elution so that a desired amylase inhibitor cannot be 
obtained. 
Process Step (d) 
An acid is immediately added to 0.26 AI-containing solution obtained in 
step (c), thereby adjusting the pH of the solution to 2-5 in order to 
prevent a modification of the amylase inhibitor contained in the alkaline 
eluate. The pH adjustment of the alkaline eluate with an acid may be 
conducted by adding an acid to the alkaline eluate reserved once in a 
reservoir such as a vessel, or by continuously adding an acid to a vessel 
while introducing the alkaline eluate into the vessel, or by introducing 
the alkaline solution into a vessel containing an acid solution. In any 
case it is required that the pH of the 0.26 AI-containing alkaline 
solution should be adjusted to 2-5 with an acid as soon as possible. The 
acid used for the pH adjustment includes an inorganic acid such as 
hydrochloric, sulfuric or phosphoric acid, and an organic acid such as 
acetic acid. Hydrochloric acid is preferable. 
Process Step (e) 
The 0.26 AI-containing pH adjusted solution obtained in step (d) is dried 
by any means such as lyophilization, drying under reduced pressure, spray 
drying and ball drying to obtain the desired 0.26 AI. Before the above 
treatment, the pH adjusted solution may be optionally subjected to 
dialysis or other purification treatment. The 0.26 AI possesses very high 
inhibitory activity against human pancreatic .alpha.-amylase and can 
effectively inhibit digestion of heated or cooked starch such as cooked 
rice, that is, hydrolysis of heated or cooked starch even when used in a 
small amount. 
In general, physiologically functional proteins such as enzymes are 
susceptible to denature under a strong alkaline condition at a pH above 9. 
Therefore, an alkaline solution at pH 9 or higher has not been used in 
prior art for the preparation of the amylase inhibitors from the wheat 
origin material. On the contrary, the present invention enables one to use 
in the treatment of the cation exchanger the alkaline solution at pH 9-13 
which has not been employed in the art, by which there can be produced new 
0.26 AI of the invention having much higher amylase inhibitory activity 
than the prior amylase inhibitors of wheat origin. Quite unexpectedly, 
such treating operation according to the present invention can produce new 
0.26 AI with superior properties. 
The present invention further includes an amylase inhibitor consisting 
essentially of a material comprising a protein composed of two subunits, 
each identified as SEQ ID NO:1 (0.26 AI) in combination with a protein 
composed of two subunits, each identified as SEQ ID NO:2 (0.19 AI), the 
total content of both proteins in the material being not less than 20% by 
weight. 
If the total content of 0.26 AI and 0.19 AI in the material is less than 
20%, the amylase inhibitor provides low amylase inhibitory activity even 
when administered in a high level, which results in reducing the 
activities of inhibiting an increase in blood glucose level and 
controlling an insulin secretion, thus making impossible an effective use 
as an appetite controlling agent. 
There is no upper limit too the total content of 0.26 AI and 0.19 AI in the 
material. Higher content of both proteins in the material provides higher 
amylase inhibitory activity and higher activities of inhibiting an 
increase in blood glucose level, controlling an insulin secretion and 
inhibiting an appetite. However, the upper limit for the total content of 
0.26 AI and 0.19 AI in the material is preferably about 80% by weight, 
from the viewpoint of productivity and economy. 
The material used in the invention may contain, in addition to 0.26 AI and 
0.19 AI, less than 80% by weight proteins of wheat origin, peptides and 
other materials (e.g., starch, edible fibers, vitamins, minerals, etc). In 
general, it is preferable that the material including 0.26 AI and 0.19 AI 
constitutes not less than 70% by weight of the total weight of the wheat 
protein used in the invention. 
0.19 AI used in combination with 0.26 AI of the invention is known and 
commercially available. 0.19 AI can be prepared by any method, for 
example, the process disclosed in European Patent Publication No. 0 567 
088 A2. 
The new amylase inhibitor of the invention (0.26 AI) and the wheat protein 
comprising 0.26 AI and 0.19 AI can be used alone or in combination with 
conventional carriers or adjuvants for pharmaceutical preparation in the 
form of a liquid preparation or a solid preparation such as granules and 
tablets as an agent for inhibiting an increase in blood glucose level or 
an agent for controlling an insulin secretion and/or an agent for 
inhibiting an appetite. In addition, they may be used as food additives, 
particularly for carbohydrate foods rich in starch such as bread and 
cookie or as additives for tea, soup, seasoned fish meal and spread such 
as butter and jam. 
Thus, the present invention relates to an agent for inhibiting an increase 
in blood glucose level or for controlling an insulin secretion or for 
suppressing an appetite, which comprises, as an active ingredient, an 
amylase inhibitor consisting essentially of a protein composed of two 
subunits, each identified as SEQ ID NO:1 or a material comprising a 
protein composed of two subunits, each identified as SEQ ID NO:1, in 
combination with a protein composed of two subunits, each identified as 
SEQ ID No:2, the total content of both proteins in the material being not 
less than 20% by weight. 
Further, the present invention relates to a food additive, which comprises 
a protein composed of two subunits, each identified as SEQ ID NO:1 or a 
material comprising a protein composed of two subunits, each identified as 
SEQ ID NO:1 in combination with a protein composed of two subunits, each 
identified as SEQ ID NO:2. 
The amount of the 0.26 AI or the material comprising 0.26 AI and 0.19 AI 
administered to the humans and added to foods may adequately be controlled 
depending upon conditions and symptoms of the subject to be administered 
or nature and quantity of foods to be ingested. With the agents of the 
present invention, an administration in a small level can achieve high 
amylase inhibitory activity, high activities of inhibiting an increase in 
blood glucose level and controlling an insulin secretion as well as 
duration in feeling of satiety. 
The agent for inhibiting an increase in blood glucose level, for example, 
with the content of 0.26 AI or the total content of 0.26 AI and 0.19 AI 
being 30% by weight, exhibits an activity of inhibiting an increase in 
blood glucose level even when administered to a healthy person at a dose 
of 100 mg once a day. The administration at a unit dose of 500 to 2500 mg, 
3 times a day can very well inhibit an increase in blood glucose level. 
For diabetic, the administration at a small dose (usually, about 300-1500 
mg/day) can effectively inhibit an increase in blood glucose level. 
The appetite controlling agent of the invention, for example, with the 
content of 0.26 AI or the total content of 0.26 AI and 0.19 AI being 30% 
by weight, exhibits an activity of inhibiting an increase in blood glucose 
level even when administered to a healthy person at a dose of 100 mg once 
a day. The administration at a unit dose of 500 to 2500 mg, 3 times a day 
can very well inhibit an increase in blood glucose level. For diabetic, 
the administration at a small dose (usually, about 300-1500 mg/day) can 
effectively inhibit an increase in blood glucose level. 
The appetite controlling agent of the invention, for example, with the 
content of 0.26 AI or the total Content of 0.26 AI and 0.19 AI being 30% 
by weight, can inhibit appetite by suppressing the feeling of hunger with 
no increase in the content of free fatty acid in blood, when given with a 
meal at a dose of 1000 to 5000 mg once. 
Preferably, the agents of the present invention are formulated into the 
preparations (e.g., capsules, tablets, granules, powders, solutions, etc) 
so that 100 to 5000 mg, preferably 500 to 2500 mg of the active ingredient 
are contained in the agents when administered once. For example, a unit 
dose of 100 to 5000 mg can achieve sufficient and sure ingestion. 
The invention is further illustrated by the following examples in which the 
amylase inhibitors were determined by the following procedure. 
Determination of Molecular Weight by Gel Filtration Chromatography Using 
Sephadex G-75 
A sample solution was prepared by adding 4 ml of a buffer (20 mM tris HCl, 
200 mM NaCl, pH 8.0) to the amylase inhibitor. The solution was subjected 
to gel filtration chromatography at a flow rate of 0.5 ml/hr on a Sephadex 
G-75 gel filtration column (1.6 cm.times.90 cm) which had been 
equilibrated with the same buffer to determine the molecular weight. 
Determination of Amino Acid Sequence 
The amylase inhibitor was pyridylethylated and then hydrolyzed with V8 
protease and lysyl endopeptidase (manufactured by Wako Jun-yaku). The 
hydrolyzed sample was subjected to HPLC to separate a hydrolyzed peptide 
fragment. The separated peptide fragments were analyzed using a peptide 
sequencer PSQ-1 (manufactured by Shimazu) for the structure starting from 
the peptide N terminus to determine the entire primary structure. The C 
terminus was determined by analysis of the amino acid liberated by 
hydrolysis with carboxypeptidase. 
Determination of Inhibitory Activity Against Human Pancreatic 
.alpha.-amylase 
An aqueous sample solution and human pancreatic .alpha.-amylase were added 
to 20 mM piperazine-N,N'-bis(2-ethanesulfonate) buffer (pH 6.9) containing 
50 mM NaCl, 5 mM CaCl.sub.2 and 0.02% egg white albumin. The mixture was 
allowed to stand at 37.degree. C. for 30 min. and then mixed with 0.5 ml 
of a 1.5% soluble starch solution at pH 6.9. The resulting solution was 
allowed to react by maintaining at 37.degree. C. for 10 min. followed by 
addition of 2.5 ml of a reaction terminator solution (0.08 M HCl and 0.4M 
acetic acid). To 0.2 ml of the reaction mixture was added 2.5 ml of an 
iodine solution (0.05% KI and 0.005% iodine), and the mixture was measured 
for absorbancy at 660 nm. The amylase was used in an amount sufficient to 
reduce the absorbancy by 80% when no sample solution was contained and the 
amount of the amylase inhibitor sufficient to inhibit the amylase activity 
by 50% was taken as 1 amylase inhibitory unit (U). 
Determination of Total Protein Content 
It was determined by the Kjeldahl's method using a KJELTEC AUTO 1030 
analyzer (manufactured by Tecator, Sweden). A nitrogen-protein conversion 
factor of 5.70 was adopted. 
Determination of 0.26 AI or 0.19 AI Content 
A test sample was dissolved in a 0.1% aqueous solution of trifluoroacetic 
acid, and the solution was subjected to high performance liquid 
chromatography under the conditions shown below, to determine the peak 
area for 0.26 AI or 0.19 AI in the chromatogram. Separately, an authentic 
sample of 0.26 AI or 0.19 AI (purity 100%) was subjected to high 
performance liquid chromatography under the same condition as above to 
measure the peak area for 0.26 AI or 0.19 AI in the chromatogram. The 0.19 
AI or 0.26 AI content in the same was calculated according to the 
following equation: 
EQU 0.26 AI or 0.19 AI content in the test sample (%) =(Sa/St).times.100 
wherein 
Sa=Peak area for 0.26 AI or 0.19 AI in the test sample 
St=Peak area for 0.26 AI or 0.19 AI in the authentic sample 
Column 
Packing material: CAPCELL PAK C18 SG120A (particle size 5 .mu.m) 
(manufactured by Shiseido) 
Size: 4.6 mm o.times.250 mm 
Temperature: 50.degree. C. 
Flow rate: 1 ml/min 
Detection: Absorbance at 280 nm 
Mobile phase: 
High pressure linear gradient elution with a time/concentration gradient 
shown below, consisting of 
Solution A: 0.1% aqueous solution of trifluoroacetic acid; and 
Solution B: aqueous solution of 80% acetonitrile and 0.1% trifluoroacetic 
acid 
Determination of Increase in Blood Glucose Level 
Blood glucose level was determined by the glucose oxidase method 
immediately after blood drawing from the forearm vein of a test subject. 
Increase in blood glucose level was determined by substracting the value 
in fasting from the value found. In the measurement by the glucose oxidase 
method was used Glucose-B-test Wako (manufactured by Wako Jun-yaku Kogyo). 
Determination of Insulin Level 
Blood was drawn from the forearm vein of a test subject and immediately 
centrifuged to prepare serum. Insulin level in the serum was measured by 
enzyme immunoassay. For the enzyme immunoassay was used Glazyme 
Insulin-EIA test (manufactured by Wako Jun-yaku Kogyo). 
Evaluation of Duration in Feeling of Satiety 
The test subjects were allowed to choose one of seven feelings shown in the 
ratings below: 
1. Hungry and hard 
2. Hungry 
3. Slightly hungry 
4. Feeling of neither hunger nor satiety 
5. Comfortable feeling of satiety 
6. Slightly heavy on the stomach 
7. Heavy and hard on the stomach 
EXAMPLE 1 
To 500 g of Durum wheat flour was added 2 liters of water and the mixture 
stirred at room temperature for 3 hours. The precipitates were then 
removed by centrifugation, and to the supernatant was added hydrochloric 
acid to adjust to pH 3. The mixture was allowed to stand for 1 hour. To 
the supernatant was added an aqueous sodium hydroxide solution to adjust 
to pH 6. After allowing to stand for 1 hour, precipitates were removed by 
centrifugation. To the supernatant was added ammonium sulfate while 
stirring until saturation of 45% was achieved. After allowing to stand for 
2 hours, the solution was centrifuged to recover precipitates. 
To the precipitates was added 50 ml of water to prepare a suspension and 
the suspension was subjected overnight to desalting using a dialysis 
membrane (manufactured by Visking Co., Ltd.). To the desalted solution was 
added a phosphate buffer (pH 7.6) so as to give a concentration of 20 mM. 
The mixture was thoroughly stirred and then centrifuged to remove 
precipitates. The supernatant was passed through an anion exchange resin 
column ("DEAE-Toyopal Column", manufactured by Toso Co., Ltd.) which had 
been equilibrated with a 20 mM phosphate buffer solution. The column was 
thoroughly washed with the same buffer solution and fractions that had 
passed through the column were collected. The collected fractions were 
dialyzed against water and then against a 20 mM tris buffer solution 
containing 200 mM NaCl. After completion of the dialysis, the resulting 
solution was concentrated to 4 ml using a minimodule (manufactured by 
Asahi Kasei). The concentrate was subjected to a gel filtration column 
("Sephacryl S-200", manufactured by Pharmacia) which had been equilibrated 
with the same buffer solution to collect the fractions with an amylase 
inhibitory activity. The collected fractions were dialyzed against water 
and acetate buffer (pH 4.0) was added at a concentration of 20 mM. The 
mixture was subjected to a cation exchange resin column ("CM-Toyopal 
Column", manufactured by Toso) which had been equilibrated with the same 
acetate buffer. 
Then, an acetate buffer solution containing 100 mM NaCl was passed through 
cation exchange resin column to elute impurities. A 0.1N aqueous sodium 
hydroxide solution was then passed through the column to elute the 0.26 AI 
from the column. Immediately, hydrochloric acid was added to the eluate to 
adjust to pH 3.0. The solution was dialyzed against water and lyophilized 
to give 60 mg of a white product. 
The molecular weight of the product obtained above was measured by gel 
filtration chromatography using Sephadex G-75. It was found approximately 
25,000. The product gave a single band at a position corresponding to a 
molecular weight of 12,500 when subjected to SDS-polyacrylamide 
electrophoresis mentioned above. Accordingly, it was found that the above 
product was constructed of two subunits having a molecular weight of 
12,500. Amino acid sequence of each subunit was determined by the 
above-mentioned method to find that it had a structure of 124 amino acid 
residues identified by sequence No. 1. 
The total protein content and the 0.26 AI content in the product were 
measured by the method as described above. It was found 95% and 91%, 
respectively. In addition to the protein 0.26 AI, the product contained 4% 
protein contaminants and 5% moisture. The product obtained in Example 1 
was determined for human pancreatic .alpha.-amylase inhibitory activity by 
the method as described above. The result is shown in Table 1 below. 
REFERENCE EXAMPLE 1 
Gluten was added to the product obtained in Example 1 to prepare a protein 
containing 15% of 0.26 AI, which was determined for human pancreatic 
.alpha.-amylase inhibitory activity. The result is shown in Table 1 below. 
REFERENCE EXAMPLE 2 
To 800 kg of wheat flour was added 410 lit. of water and the mixture was 
kneaded to form a dough. The dough was washed with 7600 lit. of water to 
recover 410 kg of gluten and 505 kg of wheat starch. At this stage, 6200 
lit. of a waste liquid were produced. The pH of the waste liquid (aqueous 
extract) was adjusted with hydrochloric acid to 3, and after allowing to 
stand for 30 min., adjusted with ammonia to 6.5, by which insoluble 
matters were precipitated. The precipitates were removed to recover 5200 
lit. of supernatant (I). 
To the supernatant (I) was added sodium alginate so as to give a 
concentration of 300 ppm. The mixture was adjusted to pH 4.2 and stirred 
for 30 min., thus forming water-insoluble matters. They were recovered by 
means of a De Laval centrifuge. The recovered mass was dispersed in 10 
times amount of water. The dispersion was mixed with 4.7 kg of calcium 
chloride, thoroughly stirred, adjusted with ammonia to pH 8.5 and allowed 
to stand for one hour. The solid matters were separated off by means of a 
De Laval centrifuge to recover 600 lit. of a supernatant. 
The supernatant recovered above was neutralized with hydrochloric acid, and 
the neutralized solution was heated at 80.degree. C. for 30 min. Insoluble 
matters thus formed were separated by means of a De Laval centrifuge to 
recover a supernatant. The supernatant was concentrated by means of a 
ultrafiltration membrane [manufactured by Nitto Denko K.K.; NTU-3250CIR 
(20000 Dalton cut off)], while removing excess calcium salt to give a 
concentrate solution (II). 
140 lit. of the concentrate solution (II) were adjusted with ammonia to pH 
7.5 and passed through a column (900 mm in length, 200 mm in inner 
diameter) in which 28 lit. of a cation exchange resin (Diaion HPK-55, 
manufactured by Mitsubishi Kasei K.K.) has been packed, at a flow rate of 
1 lit./min. Fractions not adsorbed on and eluted from the cation exchange 
resin were collected. The eluted fractions were filtered through a ceramic 
filter for elimination of microbials and then lyophilized to give 1400 g 
of dry powder product. 
The dry powder product was determined for the total protein content and the 
0.19. AI content. It was found 84% and 21%, respectively. The human 
pancreatic .alpha.-amylase inhibitory activity is shown in Table 1 below. 
REFERENCE EXAMPLE 3 
The concentrate solution (II) obtained in Reference Example 2 was dried to 
powders. The total protein content and 0.19 AI content in the powders were 
measured. It was found 60% and 15%, respectively. The human pancreatic 
.alpha.-amylase inhibitory activity is shown in Table 1 below. 
COMATIVE EXAMPLES 1 & 2 
For comparison, known amylase inhibitors, 0.53 AI identified by sequence 
No. 3 and 0.28 AI identified by Sequence No. 4 were measured for human 
.alpha.-amylase inhibitory activity. The result is shown in Table 1 below. 
TABLE 1 
______________________________________ 
Content of 
Total amylase Amylase 
protein 
inhibitor inhibitory 
content 
in protein activity 
(%) (%) (U/mg) 
______________________________________ 
Product of Example 1 
95 0.26 AI 91 23,300 
Product of Reference 
99 0.26 AI 15 1,350 
Example 1 
Product of Reference 
84 0.19 AI 21 3,550 
Example 2 
Product of Reference 
60 0.19 AI 15 2,160 
Example 3 
Comparative Example 1 
100 0.53 AI 100 
3,940 
Comparative Example 2 
100 0.28 AI 100 
840 
______________________________________ 
Table 1 shows that 0.26 AI of the invention has a very high amylase 
inhibitory activity as compared with known amylase inhibitors 0.53 AI and 
0.28 AI. 
EXAMPLE 2 
Two non-diabetic healthy males and one female were each given 250 g of 
cooked rice and 200 ml of sugarless tea. Blood was drawn at 30 min. 
intervals after meal, and an increase in blood glucose level was measured 
by the above-mentioned method using a kit ("Glucose-B-test Wako", 
manufactured by Wako Jun-yaku). One week later, a test was conducted using 
the same persons in the same way as above except that the cooked rice 
contained 350 mg of 0.26 AI of the invention. The results are shown in 
Table 2. 
TABLE 2 
______________________________________ 
Increase in blood glucose level (mg/dl) 
Control Inventive 
(0.26 AI not added) 
(0.26 AI added) 
______________________________________ 
Male 1 
After 0 min. 0 0 
30 min. 74 55 
60 min. 90 52 
90 min. 54 29 
Male 2 
After 0 min. 0 0 
30 min. 40 24 
60 min. 30 29 
90 min. 22 3 
Female 
After 0 min. 0 0 
30 min. 49 49 
60 min. 75 41 
90 min. 58 39 
______________________________________ 
Table 2 shows that 0.26 AI of the invention is also effective in inhibiting 
digestion of heated or cooked starch and inhibits hydrolysis of the starch 
by inhibition of human pancreatic .alpha.-amylase, thus inhibiting 
increase in blood glucose level. 
EXAMPLE 3 
The white product obtained in Example 1 was purified by conventional means 
to prepare a purified 0.26 AI. The dry powder product obtained in 
Reference Example 2 was purified by conventional means to prepare a 
purified 0.19 AI amylase inhibitor. The total protein content (A), content 
of the amylase inhibitor in protein (B) and amylase inhibitory activity 
(C) of the purified product were determined with the following result. 
______________________________________ 
A B C 
(%) (%) (U/mg) 
______________________________________ 
0.26 AI 100 100 26100 
0.19 AI 100 100 20300 
______________________________________ 
The purified 0.26 AI and the purified 0.19 AI were mixed in the ratio shown 
in Table 3 to evaluate the amylase inhibitory activity of the mixture. The 
result is shown in Table 3 below. 
TABLE 3 
______________________________________ 
Mixed ratio Amylase inhibitory activity 
0.19 AI (%) 
0.26 AI (%) 
(U/mg) 
______________________________________ 
5 20 6300 
10 15 5600 
20 5 5100 
5 35 9800 
20 20 8900 
30 10 8400 
______________________________________ 
EXAMPLE 4 
Two non-diabetic healthy males and one female, after fasted for 10 hours, 
were each given 300 g of cooked rice and 200 ml of sugarless tea. Blood 
was drawn at 30 min. intervals after meal to determine blood glucose level 
and insulin level, and to evaluate the duration in feeling of satiety. The 
test was run five times in total at one week interval for each subject, by 
giving to the subject sugarless tea containing 350 mg each the product of 
Example 1 for the first run, the product of Reference Example 1 for the 
second run, the product of Example 2 (20/20 mixture of 0.26 AI/0.19 AI) 
for the third run and the product of Reference Example 3 for the fourth 
run. 
In the fifth run, the subject received the cooked rice and the sugarless 
tea free of the product (Control). The results are shown in Table 4 below. 
TABLE 4 
__________________________________________________________________________ 
Reference Reference 
Example 
Example 
Example 
Example 
1 1 2 3 Control 
__________________________________________________________________________ 
Blood glucose level (mg/dl): 
Before meal 
101 103 101 99 102 
After meal 
30 min. 128 158 141 167 162 
60 min. 150 187 155 178 183 
90 min. 145 151 148 140 148 
120 min. 116 130 123 127 124 
Insulin level (.mu.U/ml): 
Before meal 
9 12 8 10 8 
After meal 
30 min. 14 27 15 25 23 
60 min. 26 38 30 35 32 
90 min. 22 30 24 36 33 
120 min. 20 33 21 33 30 
Feeling of Satiety: 
Before meal 
1.9 1.7 2.0 2.1 2.1 
After meal 
30 min. 5.3 4.9 5.0 4.9 4.8 
60 min. 4.3 4.0 4.4 4.0 4.1 
90 min. 4.9 3.3 4.8 3.7 3.5 
120 min. 4.1 3.1 3.7 3.2 3.0 
__________________________________________________________________________ 
Table 4 shows that the amylase inhibitor of the present invention obtained 
in Examples 1 and 2 containing at least 20% of either 0.26 AI or 0.19 AI 
are effective for the inhibition of increase in blood glucose level and 
for the inhibition of insulin secretion and can bring about duration in 
the feeling of satiety. 
EXAMPLE 5 
To 6.2 tons of a waste liquid of water washings of the dough discharged in 
the recovery of starch and gluten from wheat flour was added hydrochloric 
acid to adjust to pH 3.0. The mixture was allowed to stand for 30 minutes 
and adjusted to pH 6.5 with ammonia to precipitate insoluble matters. The 
precipitate was removed to recover 5.2 tons of a supernatant. To the 
supernatant was added 300 ppm of sodium alginate. The mixture was adjusted 
with hydrochloric acid to pH 4.2, stirred for 30 minutes and centrifuged 
to recover precipitates (65 kg). To the precipitates were added 650 liters 
of water to form a dispersion, to which was added 4.7 kg of calcium 
chloride and well stirred. The mixture was adjusted with ammonia to pH 8.5 
and allowed to stand for one hour. The precipitates were removed by 
centrifuge to recover 600 liters of supernatant. To the supernatant was 
added sodium phosphate so as to give a concentration of 0.1%, the mixture 
was adjusted with ammonia to pH 6.5 and heat treated at 80.degree. C. for 
15 minutes. The heat denatured precipitates were removed by centrifuge, 
desalted and concentrated using a ultrafiltration membrane (manufactured 
by Nitto Denko K.K., NTU-3250 CIR, 200 Dalton cut off) and lyophilized to 
give 1.4 kg of dry powders. 
The dry powders (500 mg) were dissolved in a 20 mM acetate buffer solution 
(pH 4.0) containing 5 ml of 100 mM of NaCl. Insoluble matters were removed 
by centrifuge. The resulting solution was subjected to gel filtration 
chromatography using Sephadex G-100. The active peak was separated and 
dialyzed at 4.degree. C. overnight against 20 mM acetate buffer (pH 4.0) 
using a dialyzing tube. The dialyzed solution was passed through 
CM-Toyopal which had been equilibrated with the same buffer to adsorb a 
target material thereon. The adsorbed material was passed through 20 mM 
acetate buffer (pH 4.0) containing 300 mM sodium chloride to elute 
impurities. Then, the target material was eluted with 0.05 N sodium 
hydroxide solution (pH 12.2). The eluate was immediately adjusted with HCl 
to pH 3.0, thoroughly desalted using a dialyzing tube and lyophilized to 
give white powders. The resultant white powders were determined for total 
protein content and 0.26 AI content, which were found 94% and 88%, 
respectively. The human pancreatic .alpha.-amylase inhibitory activity was 
determined as 22030 U/mg. 
EXAMPLE 6 
To 100 g of wheat flour was added one liter of 1% NaCl solution, the 
mixture was stirred at room temperature for 2 hours and centrifuged to 
separate a supernatant. To the supernatant was added ammonium sulfate 
until a 50% saturation was achieved. The mixture was stirred at room 
temperature for one hour and left to stand for one hour. 
Separation by centrifugation gave precipitates to which was added 200 ml of 
a distilled water to disperse it. The dispersion was dialyzed overnight 
against a deionized water. To the dialyzed solution was added hydrochloric 
acid to adjust to pH 4.0 and then the remaining precipitates were removed 
by centrifugation. The resultant solution was passed through a 
CM-Sepharose column which had been equilibrated with an acetate buffer (20 
mM, pH 4.0) to adsorb a target material on a resin. The resin was 
thoroughly washed with the same buffer and impurities were eluted with 20 
mM acetate buffer (pH 4.0) containing 400 mM NaCl. The target material was 
eluted with 0.05 N NaOH (pH 12.2), adjusted with HCl to pH 3.0, and 
thoroughly dialyzed against 50 mM acetate buffer solution (pH 4.0) 
containing 100 mM NaCl. The dialyzed solution was concentrated by passing 
through an ultrafiltration membrane (M.W. 20,000) and subjected to gel 
filtration chromatography (20 mM acetate buffer, pH 4.0, 100 mM sodium 
chloride) using Sephadex G-75 for further purification. The purified 
product was thoroughly dialyzed against a deionized water using a 
dialyzing tube and lyophilized to obtain white powders. The resultant 
white powders were determined for the total protein content and 0.26 AI 
content, which were found 96% and 93%, respectively. The human pancreatic 
.alpha.-amylase inhibitory activity was determined as 23500 .mu./mg. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 124 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
SerGlyProTrpMet CysTyrProGlyTyrAlaPheLysValProAla 
151015 
LeuProGlyCysArgProValLeuLysLeuGlnCysAsnGlySerGln 
20 2530 
ValProGluAlaValLeuArgAspCysCysGlnGlnLeuAlaAspIle 
354045 
SerGluTrpCysArgCys GlyAlaLeuTyrSerMetLeuAspSerMet 
505560 
TyrLysGluHisGlyValGlnGluGlyGlnAlaGlyThrGlyAlaPhe 
6570 7580 
ProSerCysArgArgGluValValLysLeuThrAlaAlaSerIleThr 
859095 
AlaValCysLysLeuPro IleValIleAspAlaSerGlyAspGlyAla 
100105110 
TyrValCysLysGlyValAlaAlaTyrProAspAla 
115120 
(2 ) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 124 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
SerGlyProTrpMetCysTyrProGlyGlnAlaPheGlnValProAla 
15 1015 
LeuProAlaCysArgProLeuLeuArgLeuGlnCysAsnGlySerGln 
202530 
ValProGluAlaValL euArgAspCysCysGlnGlnLeuAlaHisIle 
354045 
SerGluTrpCysArgCysGlyAlaLeuTyrSerMetLeuAspSerMet 
50 5560 
TyrLysGluHisGlyAlaGlnGluGlyGlnAlaGlyThrGlyAlaPhe 
65707580 
ProArgCysArgArgGluValV alLysLeuThrAlaAlaSerIleThr 
859095 
AlaValCysArgLeuProIleValValAspAlaSerGlyAspGlyAla 
100 105110 
TyrValCysLysAspValAlaAlaTyrProAspAla 
115120 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 124 amino acids 
(B) TYPE: amino acid 
( D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
SerGlyProTrpMetCysTyrProGlyGlnAlaPheGlnValProAla 
151015 
LeuProGlyCysArgPr oLeuLeuLysLeuGlnCysAsnGlySerGln 
202530 
ValProGluAlaValLeuArgAspCysCysGlnGlnLeuAlaAspIle 
35 4045 
SerGluTrpProArgCysGlyAlaLeuTyrSerMetLeuAspSerMet 
505560 
TyrLysGluHisGlyValSerGluGlyGl nAlaGlyThrGlyAlaPhe 
65707580 
ProSerCysArgArgGluValValLysLeuThrAlaAlaSerIleThr 
85 9095 
AlaValCysArgLeuProIleValValAspAlaSerGlyAspGlyAla 
100105110 
TyrValCysLysAspValAlaA laTyrProAspAla 
115120 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 123 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
SerGlyProTrpSerTrpCys AsnProAlaThrGlyTyrLysValSer 
151015 
AlaLeuThrGlyCysArgAlaMetValLysLeuGlnCysValGlySer 
20 2530 
GlnValProGluAlaValLeuArgAspCysCysGlnGlnLeuAlaAsp 
354045 
IleAsnAsnGluTrpCysArgCys GlyAspLeuSerSerMetLeuArg 
505560 
AlaValTyrGlnGluLeuGlyValArgGluGlyLysGluValLeuPro 
6570 7580 
GlyCysArgLysGluValMetLysLeuThrAlaAlaSerValProGlu 
859095 
ValCysLysValProIleProAsn ProSerGlyAspArgAlaGlyVal 
100105110 
CysTyrGlyAspTrpCysAlaTyrProAspVal 
115120