Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 

 

 


Title:  Process for the preparation of amylase inhibitor

United States Patent:  6,858,234

Issued:  February 22, 2005

Inventors:  Murayama; Ryuji (Hyogo, JP); Kanafuji; Takeo (Hyogo, JP); Muranaka; Yasuhito (Hyogo, JP); Muranaka; Rumiko (Hyogo, JP); Sato; Kazuo (Ueda, JP); Sekigawa; Akira (Ueda, JP); Yamada; Kazuhiko (Saitama, JP); Suzuki; Yoshio (Saitama, JP); Ikemoto; Hiroyuki (Saitama, JP)

Assignee:  Nisshin Pharma Inc. (JP); Nagata Sangyo Co., Ltd. (JP)

Appl. No.:  349714

Filed:  January 23, 2003

Abstract

The process for the preparation of amylase inhibitor including the steps of: (A) obtaining an extract solution containing the amylase inhibitor; (B) insolubilizing the amylase inhibitor by salting out by addition of a salt or salts to the solution obtained in the step (A) and recovering the insolubilized substance resulting from the salting out; and (C) directly drying the insolubilized substance recovered in the step (B) or dissolving the insolubilized substance in water to prepare an aqueous solution, and desalting and drying the aqueous solution to recover the amylase inhibitor, whereby, an amylase inhibitor in high concentrations 0.19 AI is prepared that shows highly inhibitive activity against the amylase in high yield and with good productivity.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of amylase inhibitor from wheat flour or wheat gluten, which is simple but productive to realize high yields.

BACKGROUND OF THE INVENTION

Patients suffering metabolic diseases, such as diabetes, are rapidly increasing due to the recent rich and varied diet. Intake of excessive nutrients induces oversecretion of insulin to cause indirectly a metabolic unbalance, thus leading to glucose intolerance (hyperglycemia), diabetes, hyperlipemia, arteriosclerosis, etc. Diabetic patients in particular suffer insufficient insulin function and glucose intolerance so that their blood glucose level drastically increases after meals to invite complications such as damages in blood capillaries and arteriosclerosis.

It is thought effective for prevention and treatment of such diseases to take in medications or foods that will depress the rise of blood glucose level after intake of essential nutrients or that will inhibit excessive secretion of insulin. Therefore a substance capable of controlling or inhibiting the hydrolysis of ingested starch into glucose and a substance capable of controlling the insulin secretion are demanded.

From the above aspects, various studies have been made on amylase inhibitors that inhibit an activity of an amylase from hydrolyzing starch into glucose. Since the report that wheat contains the amylase inhibitor, the research and development of the amylase inhibitors of wheat origin have been carried out.

The wheat-origin amylase inhibitors include 0.19 AI (AI: amylase inhibitor), a protein constructed of two subunits each consisting of 124 amino acid residues and having the molecular weight 13,337, in which a single band is observed at the mobility 0.19 by polyacrylamide gel electrophoresis (Swiss Port: ID=IAA1_WHEAT); 0.28 AI, a protein constructed of two subunits each consisting of 123 amino acid residues and having the molecular weight 13,326, in which a single band is observed at the mobility 0.28 by polyacrylamide gel electrophoresis (U.S. Pat. No. 5,444,046, Swiss Port: ID=IAA2_WHEAT); and a protein constructed of two subunits each consisting of 124 amino acid residues and having the molecular weight 13,185, in which a single band is observed at the mobility 0.53 by polyacrylamide gel electrophoresis (U.S. Pat. No. 5,726,291, Swiss Port: ID=IAA5_WHEAT). These amylase inhibitors are known to be effective in inhibiting the rise in the blood glucose level and controlling the insulin secretion. The mobility according to the polyacrylamide gel electrophoresis is based on the mobility 1 of bromphenol phenol according to the polyacrylamide gel electrophoresis (7.5%, pH 9.5), as described in J Sci. Food Agric., 20, pp. 260-261 (1969).

JP-A-46(1971)/1833 and JP-A-61(1986)/171431 disclose that the amylase inhibitors extracted from wheat with water, an acid or an aqueous alcohol may be used in treatments of diabetes and obesity. However, these conventional amylase inhibitors of wheat origin do not produce as much results as expected when orally administered to humans; they can achieve only a limited effect of inhibiting the digestion (hydrolysis) of heat cooked starch, such as of cooked rice, into glucose and are expensive.

U.S. Pat. No. 5,332,803 discloses a process for the preparation of amylase inhibitor, which comprises the steps of extracting wheat, wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol to produce an extract solution (the solution may be otherwise a starch wastewater discharged in the process of recovery of starch from wheat flour, etc.); adding a polysaccharide, such as sodium alginate, to the extract solution to form an insoluble complex; recovering the complex from the solution and dissolving or dispersing it in a solvent; dissociating the polysaccharide from the complex and removing it from the solution; treating the resultant solution with a cation exchange resin; and recovering the amylase inhibitor from the fractions passed through the cation exchange resin. The amylase inhibitor produced by the above process shows a very high inhibitory activity against the amylase but hardly against trypsin. The amylase inhibitor also has a high inhibitory activity against the amylase contained in pancreatic juice so that it is very effective in controlling the insulin secretion.

The method of JP-A-7 (1995)/48268 is capable of treating a large amount of materials with good operability while reducing wastes so that the mass production of the objective amylase inhibitor can be obtained. The method, which also uses the above amylase inhibitor-containing solution (of the U.S. Pat. No. 5,332,803) obtained by removing from the liquid the polysaccharide dissociated from the insoluble complex of the amylase inhibitor and the polysaccharide, comprises the steps of precipitating 40-70% of the protein contained in the solution; dissolving the precipitated protein in water to prepare another solution containing the amylase inhibitor; adding calcium and phosphoric ions to the newly obtained solution to insolubilize a complex containing the amylase inhibitor and recovering it from the solution; and solubilizing the amylase inhibitor of the insolubilized complex in water to obtain a solution containing the amylase inhibitor. The resulting final product contains the amylase inhibitor in high concentration and has a stronger amylase inhibitory activity.

According to the methods disclosed in U.S. Pat. No. 5,332,803 and JP-A-7(1995)/48268, the obtainable amylase inhibitors contain in high concentrations 0.19 AI that has a very high inhibitory activity against the amylase but not against or hardly against trypsin. The amylase inhibitors obtained by these methods are highly inhibitive against the amylase contained in pancreatic juice and are therefore effective in controlling the insulin secretion. Accordingly those amylase inhibitors can be an effective suppressant for the hydrolysis of heat cooked starch, such as of cooked rice, into glucose.

However, the method of U.S. Pat. No. 5,332,803 involves the use of cation exchange resin, which necessitates washing of the cation exchange resin after preparation of the amylase inhibitor so that it can be reused. The process of JP-A-7(1995)/48268 comprises so many steps that it is complicated. Thus, the advent of a simpler and more efficient process capable of quick production of the amylase inhibitor with good productivity is demanded.

It is accordingly an object of the present invention to provide a process for the preparation of amylase inhibitor, the process being simple and productive so that the amylase inhibitor containing in high concentrations 0.19 AI that is highly inhibitive against the amylase can be obtained quickly and in high yields.

SUMMARY OF THE INVENTION

The process for the preparation of amylase inhibitor according to the present invention comprises:

(A) a step of obtaining an extract solution containing the amylase inhibitor by:

(A1) extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol,

(A2) extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol; acid-treating and/or heat-treating the resulting solution to denature the contaminants; and removing the denatured contaminants from the solution, or

(A3) adding a polysaccharide to the amylase inhibitor-containing solution obtained in (A1) or (A2) to form an insoluble complex of the amylase inhibitor and the polysaccharide; and dissociating the polysaccharide from the insoluble complex to remove the polysaccharide in an insolubilized form from the solution;

(B) a step of insolubilizing the amylase inhibitor by salting out by addition of a salt or salts to the amylase inhibitor-containing solution obtained in the step (A) and recovering the insolubilized substance resulting from the salting out; and

(C) a step of directly drying the insolubilized substance recovered in the step (B) or dissolving the insolubilized substance in water to prepare an aqueous solution, and desalting and drying the aqueous solution to recover the amylase inhibitor.

In the above process, the salt in the step (B) is preferably sodium chloride. The salting out is preferably carried out in the presence of calcium ions in the extract solution. Also preferably, the salting out is carried out in the extract solution adjusted the pH within the range of 3 to 4.

In the above process, at least one of the steps (B) and (C) preferably includes addition of ascorbic acid and/or cysteine.

Preferably, the solution containing the amylase inhibitor for use in the salting out in the step (B) is a concentrate of the solution obtained in the step (A). The concentrate preferably has a protein concentration of 1 to 100 mg/cm3.

The process for the concentration of extract solution containing the amylase inhibitor according to the present invention comprises the steps of:

(I) adjusting the pH of an extract solution containing the amylase inhibitor within the range of 4.5 to 5.5, adding a polysaccharide to the solution to form an association product of the amylase inhibitor and the polysaccharide in the solution and then adjusting the pH within the range of 3.0 to 4.0 to form an insoluble complex of the amylase inhibitor and the polysaccharide; and

(II) separating the insoluble complex from the solution, dissociating the polysaccharide from the insoluble complex, and removing the polysaccharide in an insolubilized form from the solution to recover the amylase inhibitor in the form of solution.

In the above concentration process, the polysaccharide is preferably dissociated from the insoluble complex of the amylase inhibitor and the polysaccharide under the influence of glucanase, and the dissociated polysaccharide is preferably removed in an insolubilized form by filtration. The filtration is preferably carried out with addition of a filter aid. Preferably, the solution containing the amylase inhibitor is heated at 50oC. or above during or after the dissociation of the polysaccharide.

The present patent application is claiming priority based on Japanese Patent Application Nos. 2002-16573 and 2002-16574, which will be incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

The process for the preparation of amylase inhibitor according to the invention is described below.

Step (A)

In the step (A), the extract solution containing the amylase inhibitor is prepared from wheat flour or wheat gluten. The extract solution containing the amylase inhibitor is preferably obtained by either the step (A1), (A2) or the step (A3):

(A1) the step of extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol;

(A2) the step of extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol; acid-treating and/or heat-treating the resulting solution to denature the contaminants; and removing the denatured contaminants from the solution;

(A3) adding a polysaccharide to the amylase inhibitor-containing extract solution obtained in (A1) or (A2) to form an insoluble complex of the amylase inhibitor and the polysaccharide; and dissociating the polysaccharide from the insoluble complex to remove the polysaccharide in an insolubilized form from the solution.

The extraction in the step (A1) or (A2) may be conducted with the use of any of water, an acidic aqueous solution, an alkali aqueous solution and an aqueous alcohol.

When the extraction in the step (A1) or (A2) is conducted using water, the extraction conditions are not particularly limited and the extract solution may be obtained from wheat flour or wheat gluten by any appropriate method.

For example, starch or gluten is generally obtained from wheat flour as follows: wheat flour and water are kneaded together to form a dough or batter, which is then aged to thoroughly hydrate the gluten, and the dough is repeatedly washed with water to give gluten and starch milk (gluten wash liquid), and the starch milk is subjected to a separation such as mechanical separation, thereby recovering the starch. The solution discharged as above contains the amylase inhibitor so that it can be used as the extract solution in the present invention. This is extremely advantageous since the liquid usable as the extract solution is a waste liquid discharged in production of starch and gluten by Martin's or Batter's method.

When the extraction in the step (A1) or (A2) is conducted using an acidic aqueous solution, the acidic aqueous solution is desirably prepared by adding water to an inorganic acid, such as hydrochloric acid, phosphoric acid, etc., or an organic acid, such as acetic acid, etc., such that the resulting acidic aqueous solution is adjusted to the pH within the range of about 2 to 6, preferably 2 to 4.

When the extraction in the step (A1) or (A2) is conducted using an alkali aqueous solution, the alkali aqueous solution is desirably prepared by adding ammonia or sodium hydroxide or the like to water such that the resulting alkaline aqueous solution is adjusted to the pH about 8 to 10.

When the extraction in the step (A1) or (A2) is conducted using an aqueous alcohol, it is ideal to use an alcoholic aqueous solution of about 1 to 50% alcohol concentration. Examples of the alcohol used herein include methanol, ethanol and isopropyl alcohol.

The extraction in the step (A1) or (A2) is ordinary carried out with stirring at temperatures of about 10 to 40oC., for example at room temperature.

In the step (A1), the solid matters in the liquid are removed by such an appropriate means as centrifugal separation, filtration or standing, thereby obtaining the extract solution containing the amylase inhibitor.

In the step (A2), the extract solution is subjected to acid treatment and/or heat treatment to denature the contaminating proteins and those proteins are removed from the solution so that the extract solution containing the amylase inhibitor may be obtained. The acid treatment is preferably carried out by allowing the extract solution containing the amylase inhibitor to stand at the pH 2 to 4, and the heat treatment is preferably carried out by heating the solution at temperatures from 70 to 90oC., more preferably from 85 to 90oC. Those treatments are capable of denaturing the contaminating proteins, contained in the amylase inhibitor-containing extract solution, to render them insoluble in water so that the water-insolubilized contaminating proteins can be removed from the solution by such an appropriate means as centrifugal separation, filtration or standing. The thus-obtained extract solution containing the amylase inhibitor is then subjected to the step (B).

In the step (A3), a polysaccharide capable of forming an insoluble complex together with the amylase inhibitor is added to the amylase inhibitor-containing extract solution obtained in the step (A1) or (A2) to form such an insoluble complex. The insoluble complex thus formed is separated from the solution thus a dissociation liquid is added to the separated insoluble complex. The polysaccharide is then dissociated from the insoluble complex and removed in an insolubilized form from the dissociation liquid. Thus, the amylase inhibitor can be obtained in the form of solution. The extract solution obtained in the step (A3) has been concentrated so that it contains the amylase inhibitor in higher concentrations.

The polysaccharide for use in the step (A3) may be any one capable of forming the insoluble complex together with the amylase inhibitor. Examples include polysaccharides capable of cation exchange, such as sodium alginate, carboxymethyl cellulose, K-carrageenan, .nu.-carrageenan and .lambda.-carrageenan; pectin, xanthan gum and gellan gum. Of these, sodium alginate is preferable from the viewpoint of yield of the insoluble complex.

The polysaccharide is added usually in an amount of 50 to 600 ppm to the extract solution containing the amylase inhibitor obtained in the step (A1) or (A2).

In the step (A3), the association product of the amylase inhibitor and the polysaccharide is formed by adjusting the pH of the extract solution within the range of 2 to 5.5, preferably 4.5 to 5.5, and adding a polysaccharide to the solution. The pH of the solution is then adjusted to 3.0 to 4.0 to induce formation of the insoluble complex of the amylase inhibitor and the polysaccharide. The insoluble complex thus formed is so a large agglomerate that it can be readily recovered by an appropriate means such as filtration.

In the step (A3), the formation of the insoluble complex of the amylase inhibitor and the polysaccharide may take place on heating, but preferably at room temperature or with cooling. The resulting insoluble complex may be separated from the solution by such an appropriate means as gravity setting, filtration, centrifugal separation, etc.

The polysaccharide is dissociated from the insoluble complex of the amylase inhibitor and the polysaccharide in a dissociation liquid and then removed in an insolubilized form from the liquid. Thus, the amylase inhibitor is recovered in the form of solution.

Step (B)

In the step (B), the amylase inhibitor is insolubilized by salting it out by addition of a salt or salts to the solution obtained in the step (A) and the insolubilized substance resulting from the salting out is recovered.

The extract solution obtained in the step (A1) or (A2) may be directly subjected to the step (B) or maybe concentrated by the method of the step (A3) or other conventional concentration method.

The use of the concentrated solution is preferable since the amylase inhibitor has higher concentration in the solution so that it can be salted out in high yield by addition of a salt or salts, thereby realizing higher yield of the amylase inhibitor. The degree of concentration, which is appropriately adjusted depending on the amylase inhibitor content in the extract solution to be concentrated, is usually such that the protein concentration in the concentrated solution falls within 1 to 100 mg/cm3. This concentration is preferable from the viewpoints of yield of the amylase inhibitor, control of the contamination of the solution by impurities, operability, etc.

The extract solution containing the amylase inhibitor may be concentrated by the method of the step (A3), vacuum concentration, ultrafiltration or the like.

When the extract solution contains any proteins other than the amylase inhibitor that are dispersed or precipitated therein as insoluble impurities, those impurities are preferably removed from the solution by filtration, centrifugal separation, decantation, etc., before the extract solution is subjected to the salting out in the step (B).

Since the concentrated extract solution often contains dispersed impurities such as proteins besides the amylase inhibitor, the salting out is ideally carried out after those impurities are removed by acid treatment or alginic acid treatment or the like.

Examples of the salt for use in the salting out of the amylase inhibitor in the step (B) include sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, potassium sulfate and calcium phosphate, with sodium chloride and ammonium sulfate being preferred. Above all, sodium chloride is particularly preferable since it is capable of satisfactorily separating the amylase inhibitor from the solution obtained in the step (A) and it can be readily removed in the following step (C). Also, sodium chloride causes little environmental pollution.

The amount of salt used for the salting out may be adjusted depending on the type of the salt, the concentration of the amylase inhibitor in the extract solution, whether the extract solution has been concentrated, and the degree of concentration. The salt is generally used in an amount such that its concentration in the solution falls within 1 to 20 wt %, especially about 3 to 15 wt %, this concentration being preferable since it allows favorable separation of the amylase inhibitor while preventing other proteins from being salted out.

The salt, such as sodium chloride or ammonium sulfate, may be used for the salting out individually or in combination with a compound that generates calcium ions (calcium chloride, calcium bromide, calcium carbonate, etc.). The salting out in the presence of calcium ions allows for separation of the amylase inhibitor in higher yields. The concentration of calcium ions in the solution is preferably about 100 to 10,000 ppm in view of accelerated effect for the salting out.

The temperature of the solution during the salting out in the step (B) is preferably 50oC. or below, particularly 30oC. or below since the amylase inhibitor can be smoothly salted out at the temperatures. The salting out at liquid temperatures over 50oC. may result in denaturation or deactivation of the amylase inhibitor.

The pH of the solution at the salting out is preferably within 2 to 8, more preferably 3 to 4 from the viewpoint of efficient salting out of the amylase inhibitor. The salting out at the pH of the liquid less than 2 or exceeding 8 results in decreased efficiency of the salting out, lowering the yield of the amylase inhibitor.

The insolubilized substance (salted out product) resulting from the salting out that contains the amylase inhibitor is separated and recovered from the solution. The method for separating and recovering the salted out product is not particularly limited and can be decantation, filtration, centrifugal separation or gravity setting. The salted out product obtained in the step (B) can be readily separated from the liquid phase so that the separation and recovery thereof can be smoothly achieved by a general solid-liquid separation method.

The salted out product obtained in the step (B) is then subjected to the step (C).

Step (C)

The step (C) is either (i) a step of directly drying the insolubilized substance recovered in the step (B.) or (ii) a step of dissolving the insolubilized substance in water to prepare an aqueous solution, and desalting and drying the solution to recover the amylase inhibitor.

In the case of the step (ii), the insolubilized substance (salted out product) containing the amylase inhibitor is preferably dissolved in water which temperature of 10 to 85oC., particularly 20 to 40oC. since the water temperature accelerates dissolution of the salted out product in water and causes no denaturation of the amylase inhibitor.

The water is preferably used in a mass 3 to 30 times that of the salted out product.

In the step (ii), the salt may be removed from the aqueous solution of the salted out product by means of ultrafiltration, dialysis, ion exchange, etc. Of these, the ultrafiltration is preferable because of easy maintenance of the equipment.

In the step (ii), a treatment (such as filtration) to remove impurities and bacteria may be optionally carried out before, during or after the removal of the salt from the aqueous solution of the salted out product. The removal of impurities and bacteria may be achieved by means of a microfiltration membrane, a porous polymer membrane, a ceramic filter, etc.

The drying treatment in the step (C) to recover the amylase inhibitor may be freeze drying, vacuum drying, spray drying or ball drying. Of these, freeze drying or vacuum drying is preferable from the viewpoint of prevention of denaturation of the amylase inhibitor. The aqueous solution containing the amylase inhibitor may be subjected to the drying treatment directly or after it is concentrated. The latter case is preferable in terms of drying efficiency and bulk specific gravity of the final product.

At least one of the steps (B) and (C) preferably contains addition of ascorbic acid and/or cysteine. The addition leads to inhibition of coloring of the solution so that the resulting amylase inhibitor will be excellent in color, taking on a white or like tone.

The amount of ascorbic acid and/or cysteine used is preferably 1 to 1,000 g/m3 based on the liquid amount. The reason as to why the ascorbic acid and cysteine have capability of inhibiting the coloring of the amylase inhibitor-containing solution is presumably that they are able to neutralize or decrease the activity of enzyme contained in the solution that is partially responsible for the coloring of the liquid.

By the above process for the preparation of amylase inhibitor comprising the steps (A) to (C), the amylase inhibitor containing in high concentrations 0.19 AI, which is highly inhibitive against the amylase, can be obtained quite readily, quickly, in large amounts and with good productivity.

Next, the process for the concentration of extract solution containing the amylase inhibitor will be described.

The concentration process comprises the steps of:

(I) adjusting the pH of an extract solution containing the amylase inhibitor within the range of 4.5 to 5.5, adding a polysaccharide to the solution to form an association product of the amylase inhibitor and the polysaccharide in the solution and then adjusting the pH within the range of 3.0 to 4.0 to form an insoluble complex of the amylase inhibitor and the polysaccharide; and

(II) separating the insoluble complex from the solution, dissociating the polysaccharide from the insoluble complex, and removing the polysaccharide in an insolubilized form from the liquid to recover the amylase inhibitor in the form of solution.

Step (I)

Although it is not particularly limited thereto, the extract solution containing the amylase inhibitor for use in the concentration process is preferably obtained by either the step (A1) or the step (A2) of the present process for the preparation of amylase inhibitor.

The polysaccharide for use in the step (I) may be any one capable of forming the insoluble complex together with the amylase inhibitor. Examples include polysaccharides capable of cation exchange, such as sodium alginate, carboxymethyl cellulose, K-carrageenan, .nu.-carrageenan and .lambda.-carrageenan; pectin, xanthan gum, gellan gum and the like. Sodium alginate is preferable from the viewpoint of yield of the insoluble complex.

The polysaccharide is added usually in an amount of 50 to 600 ppm to the extract solution containing the amylase inhibitor.

In the step (I), the extract solution containing the amylase inhibitor is first adjusted to the pH 4.5 to 5.5, then the polysaccharide is added to the solution to form the association product, and the pH of the mixture solution is adjusted within the range of 3.0 to 4.0.

By previously adjusting the pH of the extract solution within the range of 4.5 to 5.5, the association product of the amylase inhibitor and the polysaccharide can be formed at a high efficiency once the polysaccharide is added to the solution. By the subsequent adjustment of the pH within 3.0 to 4.0, the insoluble complex will be formed so a large agglomerate that it can be readily recovered from the solution by gravity setting or centrifugal separation. As a result, the amylase inhibitor can be recovered from the extract solution in high yields. The recovery percentage for the amylase inhibitor from the extract solution is generally not less than 80 wt %.

When the polysaccharide is added to the extract solution whose pH is less than 4.5 or exceeds 5.5, the association of the amylase inhibitor and the polysaccharide cannot be thoroughly achieved so that the recovery of the amylase inhibitor will be lowered.

The pH adjustment of the extract solution may be carried out by use of an acid, such as hydrochloric acid or phosphoric acid, or an alkali, such as sodium hydroxide or potassium hydroxide, which can be appropriately selected depending on the pH of the extract solution.

In the step (I), the formation of the insoluble complex of the amylase inhibitor and the polysaccharide may take place on heating, but preferably at room temperature or with cooling within the range of 1 to 30oC.

In general, the insoluble complex is formed at temperatures about 1 to 30oC. in the following manner: the polysaccharide is added to the extract solution previously adjusted to the pH 4.5 to 5.5, the mixture solution is continuously stirred for several tens of minutes to several hours, the pH is then adjusted to 3.0 to 4.0 and the mixture solution is continuously stirred for several tens of minutes to several hours. The insoluble complex thus formed is so a large agglomerate that it can be readily recovered from the solution in the step (II) by an appropriate means such as gravity setting, filtration, centrifugal separation, etc. According to the step (I), the insoluble complex of the amylase inhibitor and the polysaccharide forms a large agglomerate to allow for easy separation.

Step (II)

In this step, the insoluble complex of the amylase inhibitor and the polysaccharide obtained in the step (I) is separated from the solution, a dissociation liquid is added to the separated insoluble complex, and the polysaccharide in an insolubilized form (insolubilized polysaccharide) is removed from the liquid to obtain the amylase inhibitor in the form of solution.

In the step (II), the insoluble complex can be recovered by an appropriate means such as gravity setting, filtration, centrifugal separation, etc.

In the step (II), the separation of the polysaccharide from the insoluble complex is performed in a dissociation liquid by adding a dissociation liquid to the recovered insoluble complex. In the dissociation liquid, the amylase inhibitor and the polysaccharide that have formed the insoluble complex are dissociated from one another and are dissolved or swollen in the dissociation liquid. The dissociation liquid used herein may be water, a weak-alkaline aqueous solution containing ammonia, ammonium hydrogen carbonate carbonate, etc., or an aqueous solution of a salt containing neither calcium nor potassium.

Although the dissociation of the insoluble complex into the amylase inhibitor and the polysaccharide can take place at room temperature, the dissociation liquid is preferably heated at 30oC. or above to accelerate the dissociation.

In particular, the solution containing the amylase inhibitor is preferably heated at 50oC. or above, more preferably 60oC. or above, particularly 80oC. or above at least once during or after the dissociation of the insoluble complex, since the resulting liquid will be colorless or a like color (light yellow) instead of the usual brown so that the final product of the amylase inhibitor in dry powder will take on a white or like tone.

When metal ions, such as of calcium or magnesium, are added to the solution in which the amylase inhibitor and the polysaccharide are dissociated from each other, the polysaccharide is insolubilized to form a solid gel while the amylase inhibitor stays dissolved in the liquid. The resulting solution is then subjected to an appropriate separation method to remove the gel of insolubilized polysaccharide. Thus, the solution containing the amylase inhibitor can be recovered.

Sodium alginate, K-carrageenan, .nu.-carrageenan and .lambda.-carrageenan are readily insolubilized to form a solid gel on addition of the metal ions, such as of calcium or magnesium. For example, when calcium chloride is added to the solution that contains dissociated sodium alginate, the gel of calcium alginate is readily formed.

The insolubilized polysaccharide may be separated or removed by means of filtration, centrifugal separation or other separation methods, with the filtration being preferable in terms of yield and purity of the amylase inhibitor.

When the insolubilized polysaccharide, resulting from the dissociation of the insoluble complex, is removed by filtration, the filtration will suffer clogging due to the poor filterability of insolubilized polysaccharide, taking long time for completion.

Therefore, the solid polysaccharide (insolubilized polysaccharide) can be removed under the influence of glucanase, which is added to the solution when the insoluble complex is dissociated into the amylase inhibitor and the polysaccharide and/or when the dissociated polysaccharide is insolubilized (gelled). The addition of glucanase enhances the filterability of insolubilized polysaccharide so that the insolubilized polysaccharide can be efficiently removed by filtration.

The term glucanase means the all enzymes capable of hydrolyzing glucose polysaccharide (glucan) into oligosaccharide or glucose. The glucanase for use in the invention may be any enzyme, for example Cellulosin (available from HANKYU KYOEI BUSSAN).

The glucanase may be added to the solution prior to the dissociation of polysaccharide from the insoluble complex, during the dissociation of the insoluble complex into the amylase inhibitor and the polysaccharide, or at the insolubilization (gelatinization) of the dissociated polysaccharide. The glucanase is preferably added during the dissociation of the insoluble complex in view of enhanced filterability of the resulting polysaccharide gel (insolubilized polysaccharide).

The glucanase is added preferably in an amount of at least 0.1 mg, more preferably from 1 to 100 mg based on 1 liter of the solution or suspension containing either the insoluble complex or the dissociated amylase inhibitor and polysaccharide.

The filterability of the insolubilized polysaccharide may be further enhanced by addition of a filter aid together with the glucanase so that the insolubilized polysaccharide dissociated from the insoluble complex may be removed by filtration even more smoothly. Examples of the filter aid include the Radiolite series produced by Showa Chemical Industry Co., Ltd., the Celite series produced by Celite Corporation, talk and the like, which may be used either individually or in combination.

The filter aid is used preferably in a fortieth mass or more, still preferably a thirtieth mass or more of the solid matters to be filtered out (mainly the insolubilized polysaccharide).

The filter aid may be added to the solution prior to the dissociation of polysaccharide from the insoluble complex, during the dissociation of the insoluble complex into the amylase inhibitor and the polysaccharide, or at the insolubilization (gelation) of the dissociated polysaccharide. The filter aid is preferably added during the dissociation of the insoluble complex since the filterability of the insolubilized polysaccharide is further enhanced.

The separation of insolubilized polysaccharide may be achieved by means of such a separation apparatus as a pressure filtration device or a centrifugal separator, with the pressure filtration device being preferable in terms of microfiltration. The type of pressure filtration device is not particularly limited and may be a conventional one, such as YABUTA automatic filtration presser or YAEGAKI automatic filtration presser.

The amylase inhibitor-containing solution recovered in the step (II) may be optionally subjected to sterilization, sanitization, treatment with cation exchange resin (to remove a trypsin inhibitor), etc. Impurities and bacteria may be removed by means of a microfiltration membrane, a porous polymer membrane, a ceramic filter, etc.

The recovered solution containing the amylase inhibitor is then dried by an appropriate method to obtain the amylase inhibitor in the form of solid, such as powder. The amylase inhibitor in dry powder may be obtained by directly drying the concentrated solution of the amylase inhibitor obtained as above or by concentrating the same by vacuum concentration or ultrafiltration and drying the resulting concentrate. The drying may be performed by freeze drying, vacuum drying, spray drying or ball drying.

Otherwise, the concentrate of the amylase inhibitor may be subjected to the steps (B) and (C) of the present process for the preparation of amylase inhibitor, in which the salt is added to the concentrate to insolubilize and separate (salt out) the amylase inhibitor, optionally followed by desalting of the solution, and the solution is dried to obtain the amylase inhibitor in dry powder.

The amylase inhibitor prepared by the present process may be used alone or in combination with conventional carriers or adjuvants in the form of liquid drug, granule or tablet as a hypoglycemic agent and/or an insulin secretion controller. The amylase inhibitor may be also used as food additives, particularly for starch-rich carbohydrate foods, such as bread and cookies; tea; soup; fish or vegetable flakes; and spread, such as butter and jam.

According to the invention, the amylase inhibitor containing in high concentrations 0.19 AI, which shows highly inhibitive activity against the amylase, can be prepared more readily, quickly and with better productivity than conventional methods.

The amylase inhibitor prepared by the present process shows a very high inhibitory activity against the amylase but hardly against trypsin. The amylase inhibitor has a particularly high inhibitory activity against the amylase contained in pancreatic juice so that the insulin secretion can be effectively controlled. Therefore the amylase inhibitor is useful for the prevention and/or treatment of such diseases as hyperglycemia, diabetes, hyperlipidemia, arteriosclerosis and adiposis. Further, the amylase inhibitor obtained by the present process is free from side effects, such as diarrhea and nausea after intake, and is easy to take orally because of its mild taste.

Claim 1 of 13 Claims

What is claimed is:

1. A process for a preparation of amylase inhibitor, comprising:

(A) a step of obtaining an extract solution containing the amylase inhibitor by:

(A1) extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol,

(A2) extracting wheat flour or wheat gluten with water, an acidic aqueous solution, an alkali aqueous solution or an aqueous alcohol; acid-treating and/or heat-treating the resulting solution to denature the contaminants; and removing the denatured contaminants from the solution, or

(A3) adjusting the pH of an extract solution containing the amytase inhibitor obtained in (A1) or (A2) within the range of 4.5 to 5.5, adding a polysaccharide to the solution to form an association product of the amylase inhibitor and the polysaccharide in the solution and then adjusting the pH within the range of 3.0 to 4.0 to form an insoluble complex of the amylase inhibitor and the polysaccharide;

recovering the insoluble complex;

and dissociating the polysaccharide from the insoluble complex in a liquid to remove the polysaccharide in an insolubilized form from the liquid;

(B) a step of insolubilizing the amylase inhibitor by salting out by addition of a salt or salts to the amylase inhibitor-containing solution obtained in the step (A) and recovering the insolubilized substance resulting from the salting out; and

(C) a step of directly drying the insolubilized substance recovered in the step (B) or dissolving the insolubilized substance in water to prepare an aqueous solution, and desalting and drying the aqueous solution to recover the amylase inhibitor.


____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]