JP3480965B2 - Method for preparing amylase inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] BACKGROUND OF THE INVENTION The present invention relates to wheat, flour,
Acquire amylase inhibitor from rutin or starch wastewater
WayAbout. Amirror obtained by the method of the present invention
Inhibitors can be added to foods,
Useful as an elevation inhibitor and insulin secretion regulator
is there. [0002] 2. Description of the Related Art In recent years, the dietary life in Japan has been enriched,
Metabolic diseases such as urine are increasing rapidly. Excess
Nutrition is induced by inducing massive insulin secretion.
Indirectly causes metabolic balance breakdown and glucose tolerance function
Decrease in blood pressure (hyperglycemia), diabetes, hyperlipidemia, arteriosclerosis, etc.
It goes. Insufficient insulin action, especially in diabetics
Blood glucose levels after meals have risen markedly
Cause complications such as capillary damage and arteriosclerosis
Has become. [0003] For the prevention and treatment of the above-mentioned diseases
Had difficulty raising blood sugar even after taking necessary nutrition
Foods and substances that can suppress the massive secretion of insulin
Ingestion is said to be effective. Starch consumed for that
Substances that can inhibit or inhibit the degradation of
There is a need for a substance that can save insulin secretion. [0004] In view of the above, Amira which degrades starch into sugars
So-called amylase having the activity of inhibiting the activity of
Many studies on inhibitors have been conducted.
Amylase inhibitor in wheat
Has been reported on amylase inhibitors from wheat since
Research and development have been carried out in various ways. Like that
Conventional technologies include water, acid or hydrous alcohol from wheat.
Amylase inhibitor extracted with
(See, for example, JP-A-46-183).
No. 3, JP-A-61-171431). However, the above-mentioned conventional wheat-derived
Amylase inhibitors are effective when administered orally to humans.
Not as expected, especially cooked rice
The effect of inhibiting the decomposition of sugar into starch is low,
Moreover, it has disadvantages such as being expensive,
Effective for oral starch digestion
There has been a need for a method for economically obtaining a protease inhibitor. [0006] SUMMARY OF THE INVENTION The present applicant has obtained useful ingredients from wheat.
Has been conducting research for many years.
As a ring, the acquisition of amylase inhibitors from wheat
I have continued my research. As a result, wheat, flour or
Wheat gluten in water, dilute acid, dilute alkali or hydrated alcohol
Starch from the extract or wheat flour, etc.
Polysaccharides such as sodium alginate are added to the starch waste liquid obtained
Are added to form an insoluble complex, fractionated, and then
After separating and removing the polysaccharides in this complex,
Treatment with an ion exchanger, and the cation exchanger
When the amylase inhibitor was recovered from the excess fraction, it was obtained.
Amylase inhibitors have extremely high amylase inhibitory activity
Have no trypsin inhibitory activity while having
Amylase inhibitor obtained by leverage method is used for pancreatic juice
With high inhibitory activity against amylase contained in
And is extremely effective in reducing insulin secretion
And filed an earlier application (Japanese Patent Application No. 4-127970).
issue). The above method developed by the present inventors is described above.
After further study on the above method,
Is it an insoluble complex of amylase inhibitor and polysaccharide?
Contains amylase inhibitor after separation and removal of polysaccharides
Instead of treating the solution with a cation exchanger,
40-70 of the protein in the solution containing the millase inhibitor
% Protein precipitation, dissolving the precipitated protein in water
To obtain a solution containing the amylase inhibitor again
Add calcium and phosphate ions to the solution
An insoluble complex containing an amylase inhibitor is formed,
Separating the insoluble complex of
Amylase inhibitor is solubilized in water from the insoluble complex
To obtain a solution containing an amylase inhibitor
In spite of the large amount of processing, the loss
Of the desired amylase inhibitor with good operability
The amount that can be obtained, and the resulting final product
The product has a high amylase inhibitor content and
The present invention has been found to have
Done. That is, the present invention relates to an amylase inhibitor
A method for preparing (A) Wheat, flour or wheat gluten is added to water, diluted acid, diluted
Amines obtained by extraction with alkali or aqueous alcohol
Solution containing an enzyme inhibitor or from flour to starch and
And / or amilla emitted when collecting gluten
The amylase inhibitor is added to the washing waste liquid containing the enzyme inhibitor.
Polysaccharides that form an insoluble complex with
To form an insoluble complex between the enzyme inhibitor and the polysaccharide,
Separating the complex from the liquid; (B) separating the polysaccharide from the complex separated in step (a)
To separate the solution containing the amylase inhibitor
Process; (C) containing the amylase inhibitor obtained in the above step (b)
Precipitate 40-70% of the protein in the solution
After collecting the precipitated protein, it is dissolved in water and
A step of obtaining a solution containing an enzyme inhibitor; (D) Calcium ions and lye are added to the solution obtained in the above step (c).
Insoluble complex containing amylase inhibitor with addition of phosphate ion
Forming a coalescence and fractionating the insoluble complex;
And (E) The insoluble complex separated in step (d) is
Amylase inhibitor by solubilizing the enzyme in water
Obtaining a liquid containing: and the above step
At any stage up to the end of (c), the impurity protein and its
Characterized by having a processing step for removing other impurities.
This is a method for preparing an amylase inhibitor. [0009]The amino prepared by the method of the present invention described above.
Rase inhibitors can be added to foods or used on blood sugar levels.
Used as an elevation inhibitor and an insulin secretion regulator
Can be. The method for preparing an amylase inhibitor of the present invention
Will be described. First, (a) wheat, flour or wheat glue
Extract water with water, dilute acid, dilute alkali or hydrous alcohol.
Solution containing the amylase inhibitor obtained
(A) is discharged when collecting starch and gluten from flour.
Containing dough or batter amylase inhibitors
Using the washing waste liquid
A polysaccharide that forms an insoluble complex with zease inhibitors
To form an insoluble complex of an amylase inhibitor and a polysaccharide.
Then, the complex is separated from the liquid [step (a)]. In the above step (a), the extraction process
Using the solution (A) containing the obtained amylase inhibitor
Water is the most preferred liquid for extraction,
Use dilute acid, dilute alkali or hydrous alcohol instead of water
May be used. When using dilute acid, use hydrochloric acid or phosphoric acid
PH was adjusted to about 2 to 6 with an inorganic acid such as acetic acid and an organic acid such as acetic acid.
It is preferable to use an acidic aqueous solution. Use dilute alkali
In the case, the pH is 8 to 1 with ammonia, sodium hydroxide, etc.
It is preferable to use an alkaline aqueous solution adjusted to zero. Ma
When using hydroalcohol, the alcohol concentration
Alcohol aqueous solution of about 1-50% is good.
Calls include methanol, ethanol, isopropyl
Alcohol and the like. In the extraction process, wheat, flour or wheat glute
Sufficient amount of water, dilute acid, dilute alkali
Usually, about 10 to 4
After performing the extraction while stirring at a temperature of 0 ° C.,
Solids can be removed by any method such as centrifugation, filtration, standing, etc.
To obtain an extract containing an amylase inhibitor.
No. In the above step (a), washing with water
When using liquid (a), starch or gluten is used from flour.
Dough or batter wash effluent drained during collection
Should be used directly as a solution containing an amylase inhibitor.
Can be. Especially when this washing waste liquid (a) is used,
Starch and gluten by Martin method or batter method
Also effectively uses the washing waste liquid discharged during the production of
This is extremely effective. Add water to flour and mix
Knead it to make dough or batter,
Allow the gluten to fully hydrate, then add water
Repeat washing the ground, gluten and starch milk (gluten wash
Liquid) and separated from the starch milk by mechanical separation.
The powder is recovered, but the washing waste liquid
Since this contains washing inhibitors,
The washing waste liquid containing the amylase inhibitor in the invention (a)
To use. [0013] The above-mentioned amylase inhibitor is contained.
To extract (a) or waste water (a)
A polysaccharide that can form an insoluble complex with
To form an insoluble complex. Extract (a) or water
By adjusting the temperature and pH of the washing waste liquid (a),
The types of polysaccharides that can form soluble complexes are widely selected,
Although it is possible to change it, a specific example is
Sodium carboxymethylcellulose, κ-f
Laginan, ν-carrageenan, λ-carrageenan, etc.
, Pectin, and xanth having cation exchange function
Tangum, gellan gum and the like can be mentioned. That
Among them, sodium alginate is preferred in terms of yield.
No. Polysaccharides usually contain an amylase inhibitor
Add 50-600p to the extract (a) or the waste water (a)
pm. In addition,
The formation of a soluble complex will bring the pH of the solution to a range of about 1-6.
It is good to do it. Insolubility of amylase inhibitors and polysaccharides
The formation of the complex can be performed under heating, but at room temperature or
It is preferably carried out under cooling. Generally, amylase inhibitors
Of polysaccharides to the extract containing water (a) or waste water (a)
After that, adjust the pH of the solution to 2-5, and cool to room temperature or
Continue stirring for several tens to several hours under (usually about 1 to 30 ° C)
This forms an insoluble complex. And the generated
Filter the soluble complex by filtration, centrifugation, or any other appropriate method.
To separate from. Incidentally, sodium alginate is added to the washing waste liquid (a).
Anionic polymer electrolytes such as thorium and calcium chloride
Added to the wastewater by adding polycationic inorganic salts such as
It has been known to recover water-soluble polymer substances by precipitation.
However, in the method of the present invention, the form of the insoluble complex with the polysaccharide is
Add a polycationic inorganic salt such as calcium chloride during formation
Is different from such prior art in that
The purpose is achieved by not adding cationic inorganic salts.
Amylase inhibitor recovery rate
You. Next, the polyamide obtained in the above step (a) is separated.
Insoluble complex of enzyme inhibitor and polysaccharide into dissociation solution
When inserted, the amylase inhibitor that had formed the complex
Polysaccharides are water soluble or swollen dissociated from each other
The polysaccharide is separated and removed from this aqueous solution
Separate the solution containing the mylase inhibitor [Step
(b)]. In that case, the dissociation solution may simply be water,
Weak alkaline such as ammonia and ammonium bicarbonate
Aqueous solution or salt water without calcium and potassium
A solution may be used. Amylase inhibitors and polysaccharides
Dissociation can occur at room temperature, but is usually about 30 to 70
It is preferable to perform the heating at a temperature of ° C., because dissociation is further promoted. Amylase inhibitor and polysaccharide dissociated from each other
In the above solution containing
Metal ions such as magnesium and magnesium ions
The polysaccharide gels to a solid insoluble material, while
Rase inhibitors dissolve in the solution and remain
The gelled polysaccharide is separated and removed from the liquid by an appropriate method.
The solution containing the amylase inhibitor.
Can be collected. Among the above polysaccharides, especially algin
Sodium acid, κ-carrageenan, ν-carrageenan,
λ-carrageenan is potassium ion, calcium ion
ON, addition of metal ions such as magnesium ions
Easily forms an insoluble gel. For example, dissociation
Add calcium chloride to the solution containing sodium alginate
When added, a gel of calcium alginate easily forms
Is done. The amylase inhibitor obtained in the above step (b)
The harmful substance-containing liquid may be used as it is in the next step.
To make the process easier, concentrate the amylase
It is desirable to reduce the amount of processing of the solution containing the inhibitor.
No. In this case, the concentration method is a membrane concentration method, a vacuum concentration method.
Method, etc., especially using the membrane concentration method
In this case, it is preferable to use a membrane having a molecular weight cut-off of 20,000 or less.
New Next, the amylar obtained in the above step (b)
Contained in the solution containing zease inhibitor or its concentrated solution
Precipitate 40-70% of the protein, and
Amylase inhibition by dissolving white matter after dissolving it in water
A liquid containing the substance is obtained [Step (c)]. At that time,
Included in the solution containing the myrase inhibitor or its concentrated solution
As a method for precipitating a protein, (i) adding a salt
Method, (ii) method of adding an organic solvent, or (iii) water
Examples thereof include a method of adding a soluble polymer. Kinds of salt used in the above method (i)
Is any salt commonly used for salting out.
For example, ammonium sulfate, sodium sulfate, etc.
Can be mentioned. Also used in method (ii)
As the type of the organic solvent, for example, methanol, ethanol
And acetone, etc., and the method (ii)
If it is, settle at low temperature (usually below 8 ° C)
Is preferred. Further, the aqueous polymer used in the method (iii)
Types include, for example, polyethylene glycol, dex
Tolan, methylcellulose and the like can be mentioned.
In the above methods (i) to (iii), the protein is precipitated.
The amount of salt, organic solvent or water-soluble polymer required for
Depending on the protein concentration in the solution,
In the case of the method, 40-70% of the protein contained in the solution
Is desirably added in an amount that causes precipitation. To be precipitated
Amylase inhibitor of interest if white matter is less than 40%
If the quality yield is less than half, but more than 70%
Contamination of other irregular proteins, soluble polysaccharides, soluble dyes, etc.
Miscellaneous substances are increased and the purity is reduced. The protein precipitated as described above contains a large amount of
It contains a millase inhibitor and the precipitate is filtered,
After collecting and collecting by centrifugation and other methods, dissolve in water
Thus, a liquid containing an amylase inhibitor is obtained. The water temperature at this time
The temperature is preferably from 30 to 85 ° C, particularly preferably from 40 to 60 ° C. Ma
The amount of water used at this time was
Protein-containing solution) has a protein concentration of 1 to 10 mg / ml,
It is preferable to use at a ratio of 2 to 5 mg / ml.
New The amylase inhibitor obtained in the above step (c)
The harmful substance-containing liquid is then treated in the following steps (d) and after.
However, in the liquid obtained by the above-mentioned process,
Α-amylase, β
-Exist in an unstable state or enzyme protein such as amylase
Some soluble impurities such as albumin and globulin
Proteins, soluble sugars, soluble inorganic salts, soluble pigments, etc.
Which impurities are often contained,
Before performing the process of the next step (d),
Α-amylase or β-amylase
Impurities such as lase, soluble sugars, inorganic salts, and soluble
Provide a process for removing impurities such as dyes, especially impure proteins
To keep. Specifically, impurities such as impure proteins
The removal is carried out in step (a) by extraction with an amylase inhibitor.
Before adding polysaccharide to effluent (a) or washing waste liquid (a)
Amylase inhibitor-containing liquid obtained in step (b)
Obtained before the step (c) or in the step (c).
The obtained amylase inhibitor-containing liquid is subjected to step (d).
This can be done at any of the previous stages. The method for removing impurities is as described above.
In this step, the solution containing the amylase inhibitor is also used for 7 minutes.
A method of heating to 0 to 90 ° C, preferably 85 to 90 ° C,
Adjust the pH of the solution containing amylase inhibitor to 2 or more and less than 4
Method, or a combination of both.
Can be. By performing such processing,
Soluble impurities such as dissolved impure proteins are denatured.
The resulting insoluble material is removed by a suitable method.
And can be removed separately. Also, like this
The liquid from which impurities have been removed is further purified as necessary by a microfiltration membrane, etc.
To remove bacteria. This eradication treatment is, for example,
Porous polymer membrane with 0.2 μm pores, ceramic foil
Performed by passing through a microfiltration membrane such as an ilter
be able to. Next, the amylase obtained in the above step (c)
Add calcium ions and phosphate ions to the inhibitor-containing solution.
Calcium phosphate adsorbed amylase inhibitor
To form an insoluble complex consisting of
The body is separated [step (d)]. Calcium compound in that case
Calcium hydroxide such as calcium chloride
Water-soluble inorganic calcium salts such as calcium nitrate, potassium acetate
Water-soluble organic calcium salts such as calcium and calcium lactate
And the like. On the other hand, providing phosphate ions
Phosphoric acid such as sodium phosphate,
Various water-soluble resins such as potassium phosphate and ammonium phosphate
And the like. In this step (d), amylase inhibition
Calcium ion and phosphoric acid added to substance-containing liquid
The amount of ions is adjusted according to the protein concentration in the solution
It is necessary to perform
Preferably, 80 to 90% of the insoluble complex (phosphate
Calcium gel)
It is better to add on and phosphate ions. Do so
In general, the calcium ion concentration in the liquid
50-300%, preferably 80-300% of the protein concentration
200% and the phosphate ion is calcium ion
30 to 100%, preferably 50 to 100% of the
To provide calcium compounds and phosphate ions
It is advisable to add each compound to the solution. Then, this step (d) is carried out in an amylase inhibitor.
PH of the harmful substance-containing liquid is 5 to 8, preferably 6 to 7,
The operation is carried out at a temperature of 30 to 80 ° C, preferably 40 to 60 ° C.
Is preferred. As a result, adsorb amylase inhibitor
A filtered insoluble complex is formed.
Separate and collect by any method such as heart separation. Next, the insoluble fraction separated in the above step (d)
Of amylase inhibitor from water-soluble complex
A solution containing a mylase inhibitor is obtained [step (e)].
In this step (e), the amylase is removed from the insoluble complex.
As a method of solubilizing the inhibitor, (i) adding an acid
(Ii) a method of adding water-soluble salts, (iii)
A heating method and the like can be given. In the case of the method (i), the acid
For example, inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as acetic acid and lactic acid.
Can be used, at which time the pH is 4 or less, especially 1
It is desirable to adjust to ~ 3. In addition, the above (ii)
In the case of the method, calcium ions are used as water-soluble salts.
It is better to use one that has a strong binding force with
Sodium, potassium, ammonium and phosphoric acid
Sodium salts, potassium salts, ammonium salts and the like
Water-soluble salts can be used to separate insoluble complexes.
The content is preferably about 1 to 10% by weight of the dispersed liquid. Ma
In the case of the above method (iii), the insoluble complex is
Disperse in a sufficient amount of water at 50-90 ° C, preferably 7
The heating is preferably performed by heating to 0 to 85 ° C. Then, the solubilized product obtained in the step (e) is obtained.
Add an aqueous solution containing an amylase inhibitor as needed.
Water-insoluble impurities were removed by centrifugation, filtration, etc.
And, if necessary, sterilization treatment or sterilization treatment (eg, heating
Sterilization, alcohol sterilization, sterilization filtration, etc.), cation exchange tree
Fat treatment (further removal of trypsin inhibitor), desalting
After drying, after processing and concentration
Amylase inhibitors can be obtained in solid form such as powder.
Wear. The drying process at this time is, for example, freeze drying, vacuum drying.
Drying, spray drying, ball drying, etc.
Can be. The above method increases the processing amount.
With good operability while suppressing loss
No or very low syn-inhibitory activity
High yield of amylase inhibitor with extremely high inhibitory activity
Can be obtained in large quantities. This amylase inhibitor is
High inhibitory activity against amylase contained in
It is effective in suppressing insulin secretion and is like rice cooked.
Of starch cooked at high temperatures, ie, decomposition into sugar
It has a high effect on suppression. And the present inventors,
The amylase inhibitor obtained by the above method of the present invention
The method of Davis et al. (Annals New York Academy of
Science, 121, p404, 1985)
When subjected to electrophoresis, it has a molecular weight of about 0.19.
Approximately 24,000 proteins were found in the aforementioned Japanese Patent Application No. 4-12797.
It was found that it was contained in a larger amount than the method of No. 0
Was. In some cases, the above step (e) is performed
Insoluble complex recovered in step (d) above
Dry the body directly and use it directly as an amylase inhibitor-containing product.
May be commercialized. The aboveObtained by the method of the present inventionA
Millase inhibitors can be used as is, or as known carriers or
Use auxiliary agents to form liquids, granules, tablets, etc.
Sugar level elevation inhibitor and / or insulin secretion regulator
Can be used as In addition, food, especially starchy
Add to carbohydrate foods such as bread and cookies
Use, teas, soups, sprinkles and butters,
Can be used by adding to spreads such as jam
You. The dosage of the amylase inhibitor and the amount added to food
Depends on the condition, symptoms, type of food and
It can be adjusted appropriately according to the amount taken, for example, for food
If ingested, add the amilla per serving
The dose of the protease inhibitor is about 0.1-20 g, preferably
It may be in the range of about 0.4 to 8 g. [0034] The present invention will be specifically described below with reference to examples.
As will be appreciated, the invention is not limited by those examples. Less than
In the example below, the above product contained in the resulting product
For polyacrylamide electrophoresis by the method of Davis et al.
A protein having an electrophoretic mobility of about 0.19 when weighed (hereinafter referred to as "0.1
9AI ") and human pancreatic amylase
The inhibitory activity was determined by the following method. [0035]Determination of 0.19AI content: Sample (product)
Was dissolved in a 0.1% aqueous trifluoroacetic acid solution.
Perform high-performance liquid chromatography under the conditions shown in
Measure the peak area of 0.19 AI in the chromatogram.
Was. On the other hand, the 0.19 AI standard (purity 100%)
Subject to high-performance liquid chromatography
The peak area of 0.19 AI in the ram was measured and
Calculate the 0.19 AI content (%) in the sample (product) according to Equation 1.
Issued. [0036] (Equation 1) 0.19AI content (%) in sample = (Sa / St) × 10
0 Where Sa = peak area of 0.19 AI for sample St = 0.19 AI peak area of the sample [0037] [Table 1]                       High-speed chromatography conditions Sample: 1% solution   Column injection volume： 10μl   column:     Filler: CAPCELL PAK C18 SG120A (particle size 5 μm) (manufactured by Shiseido)     Size: 4.6mmφ × 250mm     Temperature: 50 ° C   Speed: 0.5 ml / min   Detection: Absorbance at 280 nm   Mobile phase (eluent):     Solution A: 0.1% trifluoroacetic acid aqueous solution     Solution B: 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution     High pressure gradient end elution with the following time / concentration gradient consisting of        Time (min) Solution A (%) Solution B (%)             0 100 0             5 62 38           25 62 38           35 57 43           40 0 100           45 0 100           46 100 0 [0038]Measurement of amylase inhibitory activity: Human pancreas Amira
Of the sample and 50 mM NaCl, 5 mM CaCl_TwoYou
PIP containing 0.04% ovalbumin and 0.04% ovalbumin
Leave for 30 minutes at 37 ° C in ES buffer (pH 6.9)
After that, a 1.5% soluble starch solution (pH 6.9) 0.5
ml were mixed. This solution was reacted at 37 ° C. for 10 minutes.
Then, 2.5 ml of stop solution (0.08 M HCl, 0.4 M
Acetic acid). 1. 0.2 ml was collected from the reaction solution.
5 ml iodine solution (0.05% KI, 0.005% iodine
And the absorbance at 660 nm was measured. What
Note that the absorbance is reduced by 80% when no sample solution is included in the measurement.
50% inhibition of enzyme activity at this time by using less amount of enzyme
The inhibitor amount was expressed as 1 unit. << Embodiment 1 >> (1) Add 410 liters of water to 800 kg of flour,
The mixture was kneaded to form a dough. 7600 liters of this dough
Wash with water and add 410 kg of gluten and small
505 kg of wheat starch was recovered. At that time,
Water was generated, and hydrochloric acid was added to the water.
In addition, adjust to pH 3 and leave for 30 minutes.
When the pH was adjusted to 6.5 with, insolubles precipitated. Precipitation
After removal, 5,200 liters of the supernatant was recovered. (2) To the supernatant recovered in (1) above
After adding 300 ppm of sodium alginate, the pH was increased.
It adjusted to 4.2 and stirred for 30 minutes. As a result, water insoluble
The water-insoluble matter was separated by a Laval type centrifuge.
Collected using a machine. This collected product is 10 times as much water
And then add 4.7 kg of calcium chloride.
Stir well and adjust pH to 8.5 with ammonia for 1 hour
It was left still. The solids are then solidified using a Laval type centrifuge.
The material was separated and removed, and 600 liters of the supernatant was recovered. (3) The supernatant recovered in the above (2) is
Neutralize with hydrochloric acid, heat the neutralized solution to 80 ° C for 30 minutes,
Separating the generated insoluble matter with a Laval type centrifuge
The supernatant is collected, and the supernatant is subjected to an ultrafiltration membrane [Nittoden
NTU-3250CIR (MW: 20,000)]
And concentrated to give 55 liters of concentrated liquid. This concentration
The protein concentration in the solution was 25 mg / ml. (4) In the concentration solution obtained in the above (3),
The protein concentration in the supernatant of the concentrate will be 10mg / ml
Ammonium sulfate (sulfuric acid per 1 ml of concentrated solution)
100 mg of ammonium acid) and stirred for about 1 hour
After that, the resulting precipitate is centrifuged using a DeLaval centrifuge.
Separate and collect, then add water to the precipitate and bring to 50 ° C.
After heating to completely dissolve the precipitate, the protein concentration
Add about 250 liters of water to 3mg / ml
It was adjusted. (5) Water of the aqueous solution obtained in (4) above
While maintaining the temperature at 50 ° C., the calcium ion concentration becomes 30
00 ppm (about 100% of the protein concentration in the solution)
About 2 kg of calcium chloride,
Sodium phosphate so that the concentration of
750 g was added and the pH was adjusted to 6.5. Stir enough
Contains an amylase inhibitor if left to stir
A precipitate of insoluble complex formed. (6) Insoluble complex produced in (5) above
After the body was collected using a Lavalval centrifuge, 250
Heated to 80 ° C. in 1 liter of water, solubilized
Processing of a solution containing a millase inhibitor using a DeLaval centrifuge
To remove insoluble impurities,
A liquid was obtained. (7) Amylase obtained in (6) above
After concentrating and desalting the inhibitor-containing liquid using an ultrafiltration membrane
Lyophilized, dry powder containing amylase inhibitor
1.4 kg were obtained. 1 kg of raw wheat flour of the obtained product
The amount recovered per unit is 1.75 g as shown in Table 2 below.
there were. Also, 0.19AI content in the product and
Amylase inhibitory activity of the product is measured by the method described above
The result was as shown in Table 2 below. << Reference Example 1 >> [Amylase inhibition by the method of Japanese Patent Application No. 4-127970]
Preparation of harmful substances] (1) The same steps as (1) to (3) of Example 1 are sequentially performed.
And simultaneously remove excess calcium salt from the resulting solution.
Salting gave a concentrate. (2) 140 l of the concentrated solution obtained in the above (1)
The pH was adjusted to 7.5 with Monia, and the cation exchange resin [Die
Yaion HPK-55; manufactured by Mitsubishi Kasei Corporation] 28 liters
Column (length 900mm, inner diameter 200m)
m) at a flow rate of 1 liter / min.
The fraction eluted without adsorbing to the fat was collected. Eradication of the above eluted fraction using a ceramic filter
After filtration, freeze-dry to contain amylase inhibitor.
1.4 kg of dry powder was obtained. Small amount of raw material of obtained product
The amount recovered per kg of wheat flour is as shown in Table 2 below.
The weight was 1.75 g. 0.19A in the product
I content and amylase inhibitory activity of the product as described above
As shown in Table 2 below,
Was. [0047] [Table 2]                   Recovered amount 0.19AI content Amylase inhibitory activity              (g / kg flour) (%) (unit / mg)   Example 1 1.75 55 12000   Reference Example 1 1.75 35 8000 From the results in Table 2 above, it can be seen that Example 1 (the present invention)
Method), the final generation compared to the case of Reference Example 1
The content of 0.19AI in the product is about 1.5 times
As a result, the amylase inhibitory activity of the final product is also about
It turns out that it has become 1.5 times. [0049] According to the method of the present invention, the conventional mass processing
The point of difficulty was solved, and the processing amount increased
Operability with low loss
Inexpensive large products containing amylase inhibitors at high rates
Volume. Then, according to the method of the present invention,
The resulting amylase inhibitor has high amylase inhibition
It has no harmful activity but no trypsin inhibitory activity.
Has high inhibitory activity against amylase in pancreatic juice
Effectively suppresses insulin secretion
Hyperglycemia, diabetes, hyperlipidemia, arterial stiffness
It is effective for prevention and treatment of hyperplasia and obesity. Furthermore, the book
The amylase inhibitor obtained by the method of the invention was ingested
No side effects such as diarrhea or nausea in case
Easy to take in.

Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12N 9/99 C12N 9/99 (72) Inventor Toshihisa Morimoto 5-3-1 Tsurugaoka, Oimachi, Iruma-gun, Saitama Nisshin Seifun KK Research Ryuji Murayama, Inventor Ryuji Murayama 40-1 Mitani, Yamazaki-cho, Shiso-gun, Hyogo Prefecture (56) References JP-A-46-1833 (JP, A) JP-A-4-182500 (JP, A) Biochim . Biophysics a. Acta, 1985, Vol. 828, no. 3, pp. 213-221 J.C. Food Biochem. , 1977, Vol. 1, No. 4, pp. 385-401 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 A23L 1/30

Claims (1)

(57) [Claim 1] A method for preparing an amylase inhibitor, comprising: (a) extracting wheat, flour or wheat gluten with water, a dilute acid, a dilute alkali or a hydroalcohol; A polysaccharide that forms an insoluble complex with the amylase inhibitor is added to the solution containing the amylase inhibitor or the washing waste liquid containing the amylase inhibitor discharged when collecting starch and / or gluten from wheat flour. To form an insoluble complex of the amylase inhibitor and the polysaccharide,
A step of separating the complex from the liquid; (b) a step of separating and removing a polysaccharide from the complex separated in the step (a) to separate a liquid containing an amylase inhibitor; (c) the step of (c) Precipitating 40-70% of the protein in the solution containing the amylase inhibitor obtained in b),
Collecting the precipitated protein and dissolving it in water to obtain a solution containing an amylase inhibitor; (d) adding calcium ions and phosphate ions to the solution obtained in the above step (c) to inhibit amylase inhibition. Generating an insoluble complex containing the substance and separating the insoluble complex; and (e) solubilizing the amylase inhibitor in water from the insoluble complex separated in the step (d) to contain the amylase inhibitor. A method for preparing an amylase-inhibiting substance, which comprises a step of removing an impurity protein and other impurities at an arbitrary stage until the end of the step (c).