WO2010107414A1

WO2010107414A1 - Enzyme compositions and use thereof

Info

Publication number: WO2010107414A1
Application number: PCT/US2009/001719
Authority: WO
Inventors: Yuzo Kojima; Satoshi Koikeda; Alan E. Kligerman; Sarah R. Finnegan
Original assignee: Amano Enzyme Inc
Current assignee: Amano Enzyme Inc
Priority date: 2009-03-19
Filing date: 2009-03-19
Publication date: 2010-09-23
Anticipated expiration: 2011-09-19

Abstract

A method for preventing or treating (ameliorating) a discomfort accompanied by fructose absorption deficiency (fructose intolerance) is provided. An enzyme composition for mammals contains an enzyme capable of converting fructose in a food to be ingested to another substance. The enzyme composition can be administered before the ingestion of a fructose-containing food, simultaneously with the ingestion of the fructose-containing food or after the ingestion of the fructose-containing food.

Description

DESCRIPTION

ENZYME COMPOSITIONS AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to an enzyme composition. More specifically, it relates to an enzyme composition used in prevention or treatment of symptoms accompanied by fructose absorption deficiency in mammals, a method for using the same, and a food containing an enzyme.

BACKGROUND ART

As symptoms of saccharide absorption deficiency, lactose intolerance has been so far well known. It has been generally known that in humans who are lactose-intolerant, insufficient production of a digestive enzyme, lactase, in the small intestine leads to insufficient decomposition and absorption of lactose contained in milk with the result that the unabsorbed lactose reaches the large intestine where osmotic irritation and catabolism of lactose by intestinal flora occur to induce discomfort symptoms such as excess intestinal gas retention, nausea, diarrhea and abdominal spasm. Meanwhile, as similar intolerance, fructose intolerance has been reported. The absorption of fructose in the small intestine is limited in comparison to the absorption of glucose, and fructose that reaches the large intestine as a result of excess ingestion of fructose is known to induce discomfort symptoms similarly to lactose intolerance. Fructose is present at high levels in fruits and vegetables, and is contained in edible sugar as a constituent monosaccharide of sucrose. In addition, it has been recently added as a sweetener of various foods.

The small intestinal absorption of fructose widely used as saccharide of many refreshing drinks is at present by far lower than that of glucose or sucrose, and the acceptable amount of fructose in a normal adult is said to be 25 to 50 g (10% solution) in a single oral intake. This acceptable amount is the level of absorption in the resting state, and the absorption of fructose is reported to be further reduced in a state of activity due to an increased intestinal tract peristaltic motion (E. R. Jacques et al., Am. J. Clin. Nutr., 58, 748-753(1993)).

The in-vivo absorption of fructose is limited in comparison to that of glucose. Meanwhile, it has been reported that the absorption of fructose is prompted in the presence of glucose and the absorption rate is increased in proportion to the glucose level (A. S. Truswell et al., Am. J. Clin. Nutr., 48, 1424- 1430 (1988)).

DISCLOSURE OF INVENTION

An effective method for preventing or treating fructose intolerance has not been established so far owing to poor recognition and insufficient studies of fructose intolerance. For a person who has a discomfort due to insufficient absorption of fructose, there has been no choice but to take a countermeasurement of refraining from ingestion of a fructose-containing food. However, since this countermeasure disturbs an enjoyable eating habit, it has not been accepted by the moderns. Under these circumstances, it is an object of the invention to provide a method for preventing or treating (ameliorating) discomfort symptoms accompanied by fructose absorption deficiency (fructose intolerance).

The present inventors have assiduously conducted investigations to achieve the object, and have consequently found that when an enzyme capable of converting fructose into another substance is present in vivo at the time of ingesting fructose, the discomfort accompanied by fructose absorption deficiency (fructose intolerance) can effectively be prevented or treated. This finding has led to the completion of the invention.

SUMMARY OF THE INVENTION

The invention includes the following.

[l] An enzyme composition for mammals containing an enzyme capable of converting fructose present in a food to be ingested to another substance. [2] The enzyme composition according to [I]₁ wherein the enzyme is at least one enzyme selected from the group comprising of glucose isomerase, fructose dehydrogenase and mannose isomerase.

[3] The enzyme composition according to [l] , wherein the enzyme comprises of glucose isomerase having the characteristics that: it has optimum pH of from 6 to 8, and, it is relatively stable under acidic conditions and maintains activity of 50% or more when allowed to stand in a buffer solution of pH 6.5 at 37°C for 1 hour.

[4] A method for preventing or treating fructose intolerance, comprising a step of administering the enzyme composition according to any one of [l] to [3] to a subject.

[5] The method for preventing or treating fructose intolerance according to [4], wherein the administration is conducted i) before the ingestion of a fructose-containing food, ii) simultaneously with the ingestion of a fructose-containing food or iii) after the ingestion of a fructose-containing food.

[6] A method for decreasing a fructose level in a food, comprising a step of adding the enzyme composition according to any one of [l] to [3] to a fructose-containing food. [7] A food prepared by adding an enzyme capable of converting fructose to another substance.

[8] The food according to [7], wherein the enzyme is at least one enzyme selected from the group comprising of fructose isomerase, fructose dehydrogenase and mannose isomerase. When the enzyme composition of the invention is used, the enzyme contained therein serves to convert fructose contained in a food into another substance. Accordingly, the level of fructose which is present in vivo as a result of the ingestion of a fructose-containing food is decreased to bring forth an effect of preventing or treating symptoms caused by fructose absorption deficiency. Meanwhile, the enzyme-containing food of the invention can exhibit the prevention or treatment effect similar to the foregoing effect without separately administering the enzyme composition because the food itself contains the enzyme effective for decreasing the fructose level. Even when the food is free of fructose or contains a small amount of fructose, the prevention or treatment effect similar to the foregoing effect can be exerted on a fructose-containing food ingested simultaneously.

BRIEF DESCRIPTION OF THE DRAWGINS

FIG. 1 is a graph showing the results of measuring reactivity of various glucose isomerases,

FIG. 2 is a graph showing the results of measuring stability of various glucose isomerases,

FIG. 3 is a graph showing the effect of glucose isomerase (enzyme preparation 1) on fructose absorption deficiency, and FIG. 4 is a graph showing the effect of glucose isomerase (enzyme preparation 2) on fructose absorption deficiency.

DETAILED DESCRIPTION OF THE INVENTION

An enzyme composition of the invention contains an enzyme capable of acting on fructose to convert it into another substance. As an enzyme capable of exhibiting such an activity, glucose isomerase, fructose dehydrogenase and mannose isomerase are known, and any of these enzymes can advantageously be used in the invention. Of these, glucose isomerase is preferably used. Since the use of an enzyme composition containing glucose isomerase decreases the fructose level and it also produces glucose, the presence of glucose can be expected to increase the absorption of fructose.

Thus, the use of glucose isomerase provides both the decrease in fructose level and the increase in fructose absorption, and the enzyme composition which is all the more effective is provided. Glucose isomerase (EC. 5.3.1.5) here is a general term of an enzyme capable of converting fructose into glucose. Glucose isomerase which can be used in the invention is not particularly limited. For example, glucose isomerase derived from a microorganism belonging to the genus Streptomyces, Bacillus, Flavobacterium, Actinoplanes or the like can advantageously be used. Glucose isomerases derived from animals and vegetables are also available.

For example, glucose isomerase having optimum pH of from 6 to 8 (preferably from 7 to 8) may be used. It is preferable to use glucose isomerase which is relatively stable under acidic conditions and which can maintain activity of 50% or more when allowed to stand in a buffer solution of pH 6.5 at 37°C for 1 hour.

Meanwhile, fructose dehydrogenase (EC.1.1.99) is a general term of an enzyme capable of converting fructose into 5-keto-D-gluconic acid. Fructose dehydrogenase which can be used in the invention is not particularly limited. For example, fructose dehydrogenase derived from a microorganism belonging to the genus Gluconobacter or the like can advantageously be used. Fructose dehydrogenases derived from animals and vegetables are also available.

Mannose isomerase (EC.5.3.1.7) is a general term of an enzyme capable of converting fructose into mannose. Mannose isomerase which can be used in the invention is not particularly limited. For example, mannose isomerase derived from a microorganism belonging to the genus Pseudomonas, Agrobacterium or the like can advantageously be used. Mannose isomerases derived from animals and vegetables are also available. The enzymes derived from natural sources may be prepared by usual methods. The enzyme composition of the invention may be prepared using commercial enzymes. By the way, not only enzymes from natural sources but also genetically modified enzymes may be used in the invention.

The enzyme composition of the invention may contain two or more enzymes. In this case, a combination of enzymes of the same types (for example, glucose isomerases of two types derived from different sources) or a combination of enzymes of different types (for example, glucose isomerase and fructose dehydrogenase) may be used.

A food ingested by mammals is usually first decomposed by an cramylase secreted from the salivary gland, then decomposed by a pepsin under an acidic condition of hydrochloric acid in the stomach, further decomposed in the intestines by various enzymes secreted from the pancreas, and finally absorbed as nutrients via the intestinal tract wall. An abnormality or discomfort due to the ingestion of fructose may be considered to be attributable mainly to fructose which reaches the large intestine without being absorbed via the intestinal tract wall. Accordingly, the decrease in level of fructose which reaches the intestines is effective for preventing an abnormality or the like due to the ingestion of fructose. In view of the foregoing, it is more effective to use an enzyme which can act on fructose at a stage before reaching the intestines, namely in the stomach. In other words, it is especially preferable to use an enzyme capable of satisfactorily acting even in the acidic environment of the stomach.

The enzyme composition of the invention is typically prepared for oral administration. It is orally administered such that a fructose-containing food and the enzyme composition coexist within the body. In this case, the enzyme composition of the invention may be prepared for previously decreasing the fructose level of a food. Such an enzyme composition is not orally administered, but added to a fructose-containing food to decrease the fructose level in the food. This enzyme composition can be used, for example, for decreasing the fructose level of beverages containing fruit juices. It is also possible that the enzyme composition added to a food may be removed before the ingestion of the food.

The formulation may be conducted in a usual manner. In the formulation, other pharmaceutically acceptable components (for example, a carrier, an excipient, a disintegrant, a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an antiseptic and a physiological saline) may be incorporated. As the excipient, a lactose, a starch, sorbitol, D-mannitol, sugar and the like are available . As the disintegrant, a starch, carboxymethyl cellulose, calcium carbonate and the like are available. As the buffer, a phosphate, a citrate, an acetate and the like are available. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like are available. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like are available. As the stabilizer, propylene glycol, diethyl sulfite, ascorbic acid and the like are available. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like are available. As the antiseptic, benzalkonium chloride, p-oxybenzoic acid, chlorobutanol and the like are available. It is also possible to use an antacid and various medicaments acting on the digestive tract, such as an H2 blocker and a proton pump inhibitor in combination.

In the formulation, the dosage form is not particularly limited, and the enzyme composition can be formulated as tablets, a powder, fine particles, granules, capsules, a syrup and the like. The enzyme composition of the invention contains an enzyme (enzyme acting on fructose to convert it into another substance) in an amount required to obtain an expected prevention or treatment effect (namely a therapeutically effective amount). The amount of the enzyme in the enzyme composition varies with the nature or the purity of the enzyme used, the dosage form, the use condition (fructose level of a food) and the like. It is, for example, from approximately 100 U (enzyme unit)/g to approximately 100,000 U/g in order to achieve a required dose, for example, in case of using glucose isomerase. An enzyme unit (U) here means a value determined by the following method. First, a solution obtained by adding 0.3 ml of an enzyme solution to

0.3 ml of a 1 M fructose solution dissolved in a 0.2 M PIPES-NaOH buffer solution (pH 7.0) containing 10 mM MgSCh is provided (reaction solution). The reaction solution is warmed at 37°C for 10 minutes for an enzyme reaction, and 0.6 ml of a 0.3 N HCl solution is then added to stop the reaction. The glucose level of the solution with the reaction stopped is measured, and the rate of conversion from fructose to glucose in the reaction solution is calculated. The amount of the enzyme which produces 1 μmol of glucose for 1 minute under the foregoing conditions is defined as one unit (U).

The subject to which the enzyme composition of the invention is applied (administered) is not particularly limited. The enzyme composition of the invention can be used for various mammals such as humans, livestock (cattle, hog and the like), pet animals (dog, cat and the like) and experimental animals (rat, mouse, monkey and the like) . The function and the effect of the enzyme composition of the invention are considered to be provided in mammals including humans in view of the biological common qualities regarding the structure of the digestive tract, the liver or the like and the physiological performance.

The enzyme composition of the invention is administered to a subject for prevention or treatment of fructose intolerance. The term "fructose intolerance" here means a condition in which a certain abnormality (including a discomfort) is occurring in the body due to the ingestion of fructose . Such a condition may be long-lasting or transient. The term "prevention" here includes not only previous prevention of the development of fructose intolerance symptoms but also the consequential decrease in severity of the symptoms. The term "treatment" here means amelioration (including partial or complete cure) of fructose intolerance symptoms.

It is preferable to determine the dose of the enzyme composition such that the expected effect can satisfactorily be obtained. The dose is generally determined upon taking into consideration various factors such as the condition, the age, the sex, the body weight and the like of the subject. An appropriate dose can be determined by a skilled person upon taking these matters into consideration. For example, when using an enzyme composition containing glucose isomerase as an active ingredient, the dose can be determined such that a single enzyme dose in an adult (body weight approximately 60 kg) is from approximately 100 U (enzyme unit) to approximately 100,000 U, preferably from approximately 300 U to approximately 30,000 U. The "U (enzyme unit)" here is determined by the foregoing method.

The enzyme composition of the invention is administered such that it is present simultaneously with fructose in vivo. When such a condition is realized, the enzyme composition of the invention can exhibit its peculiar activity. Specifically, the enzyme composition of the invention is administered preferably i) before the ingestion of a fructose-containing food, ii) simultaneously with the ingestion of a fructose-containing food or iii) after the ingestion of a fructose-containing food.

When the enzyme composition is administered before the ingestion of the fructose-containing food, the administration of the enzyme composition and the ingestion of the fructose-containing food are conducted preferably at an interval which is as short as possible for exhibiting the activity of the enzyme composition effectively. From this standpoint, it is preferable that the enzyme composition is administered just before the ingestion of the fructose-containing food.

Similarly, when the enzyme composition is administered after the ingestion of the fructose-containing food, the ingestion of the fructose-containing food and the administration of the enzyme composition are conducted preferably at an interval which is as short as possible for exhibiting the activity of the enzyme composition effectively. From this standpoint, it is preferable that the enzyme composition is administered immediately after the ingestion of the fructose-containing food. It is also possible to administer the enzyme composition of the invention simultaneously with the fructose-containing food. In this case, the enzyme composition is provided separately from the fructose-containing food, and the administration (ingestion) of both is conducted without a substantial interval, or the enzyme composition is added in advance to the fructose-containing food, and this fructose-containing food is then ingested. In the latter case, the fructose-containing food to which the enzyme composition has been added is prepared. Thus, the enzyme composition of the invention can be used also as an enzyme which is a food additive. Instead of adding the enzyme composition of the invention, it is also possible to add an enzyme capable of acting on fructose to convert it to another substance either singly or along with other substances to a fructose-containing food.

As described above, one embodiment of the invention provides a fructose-containing food to which either an enzyme capable of acting on fructose to convert it to another substance or an enzyme composition containing the enzyme has been added. Such a fructose-containing food can include a fruit such as pear or apple, soft drinks, confectionery, bread and the like.

EXAMPLES EXAMPLE 1

Investigation of reactivity and stability of glucose isomerases in an acidic pH region

The decrease in level of fructose which reaches the small intestine is effective for preventing the discomfort accompanied by absorption deficiency of ingested fructose. It is therefore required to use an enzyme which satisfactorily acts even in the stomach, the first digestive organ. Accordingly, three commercially available glucose isomerases were compared with respect to reactivity and stability in an acidic pH region. 1 - 1. Method for measuring enzyme activity of glucose isomerases The method for measuring enzyme activity of glucose isomerases used in the following experiments is described below.

First, a solution obtained by adding 0.3 ml of an enzyme solution to 0.3 ml of a 1 M fructose solution dissolved in a 0.2 M PIPES-NaOH buffer solution (pH 7.0) containing 10 mM MgSO4 was provided as a reaction solution. The reaction solution was warmed at 37°C for 10 minutes for an enzyme reaction, and 0.6 ml of a 0.3 N HCl solution was added to stop the reaction. The glucose level of the solution with the reaction stopped was measured by a commercially available glucose measurement kit, and the rate of conversion from fructose to glucose in the reaction solution was calculated. The amount of the enzyme which produces 1 μmol of glucose for 1 minute under the foregoing conditions was defined as one unit (U) . 1-2. Test for reactivity of glucose isomerases

Three commercially available glucose isomerases were measured for reactivity at various pHs. As glucose isomerase preparations, enzyme preparation 1 (derived from Streptomyces rubiginosus, trade name : GI powder, Nagase ChemteX Corporation), enzyme preparation 2 (derived from Streptomyces griseofuscus, trade name : GODO AGI, GODO SHUSE Co., Ltd.) and enzyme preparation 3 (derived from Streptomyces ollvochromogenes, trade name : G-zyme G993, Enzyme Biosystems) were used. The activity of the glucose isomerases was measured with a Mcllvaine buffer solution of each pH, and the results are shown in FIG. 1. As is apparent from this figure, all of the enzyme preparations are satisfactorily reacted in a neutral pH region. Meanwhile, at lower pH, the activity of all enzyme preparations is decreased, but enzyme preparations 1 and 2 still maintain the activity relatively even in an acidic pH region. The optimum pH of enzyme preparations 1 and 2 is within the range of from 7.0 to 8.0 (near 7.5), and the reactivities of these enzyme preparations exhibit similar profiles in response to the change in pH. It is found that enzyme preparation 1 maintains activity of approximately 70% at pH 6.5, while enzyme preparation 2 maintains activity of approximately 50% under the same condition. 1 " 3. Test for stability of glucose isomerases

The pH stability of the foregoing enzyme preparations was verified. The results of measuring activity of glucose isomerases remaining after allowed to stand at 37°C for 1 hour in a Mcllvaine buffer solution of each pH are shown in FIG. 2. As is apparent from FIG. 2, the increase in acidity leads to the decrease in reactivity of all enzyme preparations, but enzyme preparation 1 exhibits higher reactivity than the other enzyme preparations in the acidic pH region.

The foregoing results have revealed that there is also a great difference in reactivity and stability in the acidic pH region even among the enzymes used in this experiment. The use of enzyme preparation 1 excellent in reactivity and stability in the acidic pH region can be expected to give a greater effect on fructose in the stomach than that of the other two enzymes. EXAMPLE 2 Validation of an effect of glucose isomerases on fructose absorption deficiency

As a method for diagnosing carbohydrate digestive absorption deficiency, a method for measuring an expiratory hydrogen gas is well known.

- When carbohydrate is not absorbed efficiently, carbohydrate which has not been absorbed in the small intestine reaches the large intestine, and the large intestinal flora (hydrogen-producing microorganism Clostridium) causes fermentation using the same to produce a hydrogen gas. The hydrogen gas thus generated is excreted in an expiratory gas via blood circulation. When absorption deficiency is induced by the administration of fructose, an increase in level of hydrogen in an expiratory gas due to the abnormal fermentation of the fructose in the large intestine can be observed. When the symptoms of the fructose absorption deficiency are eliminated or ameliorated, the production of a hydrogen gas in the expiratory gas is suppressed.

Accordingly, the following animal experiment was conducted using the hydrogen gas production level as an index to validate the effect of glucose isomerase on fructose absorption deficiency.

As a test enzyme, enzyme preparation 1 used in EXAMPLE 1 (activity of enzyme preparation: 631 U/g) was used in the following experiment. The experimental animal was a 6-year-old healthy beagle dog (body weight 10 kg) . After fasting for 12 hours, a catheter was inserted via the nose, and enzyme preparation 1 was administered such that an amount of a 20% fructose solution was 3 g per kg body weight. To the enzyme preparation administration group, 30 ml of a 5% enzyme preparation 1 solution was then administered after the fructose administration. After the administration, the expiratory gas was sampled intermittently, and measured for the hydrogen gas level using an expiratory gas analyzer (TGA-2000, TERRAMEX) .

The results of the measurement are shown in FIG. 3. It can be confirmed that in the enzyme preparation non-administration group with only fructose administered, the hydrogen gas level in the expiratory gas is markedly increased, while in the enzyme preparation administration group, the hydrogen gas level in the expiratory gas is decreased significantly. It is thus considered that in the enzyme preparation administration group, the ingested fructose is converted to readily absorbable glucose in the small intestine, whereby the level of saccharide that reaches the large intestine is decreased to significantly decrease the hydrogen gas level in the expiratory gas, and the fructose absorption deficiency symptoms might be eliminated or reduced. In addition, it is also assumed that the fructose absorption is increased in response to the production of glucose which would contribute to elimination of fructose absorption deficiency symptoms.

Enzyme preparation 2 was then used to conduct a similar experiment, and the effect was compared to that of enzyme preparation 1. Since enzyme preparation 2 was liquid, the excipient contained therein was removed by ultrafiltration concentration, and lyophilization was conducted to obtain a powder which was used in the experiment (activity of a powder: 1,586 U/g) . To the enzyme preparation administration group, after fructose was administered 99 ml of a 5% enzyme preparation 2 solution was then administered.

The results are shown in FIG. 4. Similarly to the experiment using enzyme preparation 1 , the enzyme preparation non-administration group with only fructose administered is markedly increased in hydrogen gas level in the expiratory gas, while in the enzyme preparation administration group, the hydrogen level in the expiratory gas is significantly decreased. Thus, an effect of ameliorating the fructose absorption deficiency is observed even when enzyme preparation 2 is used. Nevertheless, upon focussing on the dose of the enzyme preparation, the equal effect is obtained with a smaller amount of enzyme preparation 1.

The invention is not limited to the foregoing embodiments and Examples of the invention. Various modifications, obvious to a skilled person, which can be made without departing from the scope of the appended claims, are also encompassed by the invention.

INDUSTRIAL APPLICABILITY

The enzyme composition or the enzyme-containing food according to the invention is effective for preventing or treating the discomfort symptoms accompanied by fructose absorption deficiency. The invention can be applied not only to humans but also to other ordinary mammals such as livestock, pet animals and experimental animals.

Claims

CLAIMS What is claimed is:

1. An enzyme composition for mammals containing an enzyme capable of converting fructose present in a food to be ingested to another substance.

2. The enzyme composition according to Claim 1 , wherein the enzyme is at least one enzyme selected from the group comprising of glucose isomerase, fructose dehydrogenase and mannose isomerase.

3. The enzyme composition according to Claim 1, wherein the enzyme comprises of glucose isomerase having the characteristics that: it has optimum pH of from 6 to 8, and, it is relatively stable under acidic conditions and maintains activity of 50% or more when allowed to stand in a buffer solution of pH 6.5 at 37°C for 1 hour.

4. A method for preventing or treating fructose intolerance, comprising a step of administering the enzyme composition according to Claim

1 to a subject.

5. The method for preventing or treating fructose intolerance according to Claim 4, wherein the administration is conducted i) before the ingestion of a fructose-containing food, ii) simultaneously with the ingestion of a fructose-containing food or iii) after the ingestion of a fructose-containing food.

6. A method for decreasing a fructose level in a food, comprising a step of adding the enzyme composition according to Claim 1 to a fructose-containing food.

7. A food prepared by adding an enzyme capable of converting fructose to another substance.

8. The food according to Claim 7, wherein the enzyme is at least one enzyme selected from the group comprising of fructose isomerase, fructose dehydrogenase and mannose isomerase.