Agents and methods for reducing blood ammonia concentration

Abstract

To provide techniques that safely and effectively lower blood ammonia concentration.SOLUTION: Provided is an agent containing as an active ingredient clostridium butyricum, 1-kestose, or clostridium butyricum and 1-kestose, which lowers blood ammonia concentration. According to the present invention, blood ammonia concentration can be safely and effectively lowered. Therefore, hyperammonemia and hepatic encephalopathy can be prevented or ameliorated.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an agent for reducing blood ammonia concentration, which contains Clostridium butyricum and / or 1-kestose as an active ingredient.

Background Art

[0002] In vivo, ammonia is constantly produced during the metabolism of dietary proteins, body-constituting proteins, and nucleic acids. Since ammonia is a toxic substance, the living body has multiple systems for removing ammonia as shown in the following (i) to (iv). (i) Urea cycle (ornithine cycle) in the liver. (ii) Acid amide formation in all tissues. (iii) Reaction with keto acids mainly in the liver. (iv) Direct excretion into urine in the kidney.

[0003] The urea cycle in the above (i) is the center of the ammonia removal system. The ammonia taken into the urea cycle is converted into non-toxic urea and excreted outside the body as urine. In the acid amide formation of (ii), glutamine is produced from ammonia and glutamic acid by the action of glutaminase. Also, asparagine is produced from ammonia and aspartic acid by the action of asparaginase. In the reaction with keto acids of (iii), glutamic acid is produced from ammonia and α-ketoglutaric acid by the action of glutamate dehydrogenase. In the direct excretion of (iv), ammonia is produced from glutamic acid in the kidney and directly excreted into urine (Non-Patent Document 1).

[0004] When these multiple systems function, even if a slight disorder occurs in a part of the ammonia removal system, the ammonia in the blood is maintained at a low concentration (about 10 to 70 μg / dL). On the other hand, in cases of severe liver diseases such as cirrhosis, liver cancer, and fulminant hepatitis, or congenital urea cycle enzyme deficiency, when a severe disorder occurs in this system, the removal of ammonia becomes insufficient, and the ammonia in the blood becomes highly concentrated (hyperammonemia). If hyperammonemia progresses, it inhibits the function of the central nervous system and presents neuropsychiatric symptoms mainly characterized by disturbance of consciousness. In severe cases, it can lead to coma and death.

[0005] Therefore, in order to avoid a situation where the ammonia in the blood becomes highly concentrated, technologies for reducing the blood ammonia concentration have been researched and developed. For example, Patent Document 1 discloses a food or drink or an oral pharmaceutical composition containing collagen peptide for reducing the blood ammonia concentration.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, even though there is still no sufficient supply of technologies that can safely and effectively reduce blood ammonia concentration, the present invention has been made to solve such problems, and an object thereof is to provide a technology that can safely and effectively reduce blood ammonia concentration.

Means for Solving the Problems

[0009] As a result of intensive research, the present inventors have found that Clostridium butyricum and 1-kestose can reduce blood ammonia concentration. Based on such findings, the following inventions have been completed.

[0010] (1) A first aspect of an agent for reducing blood ammonia concentration according to the present invention (hereinafter sometimes referred to as "agent for reducing blood ammonia concentration" or "this agent") contains Clostridium butyricum as an active ingredient.

[0011] (2) A second aspect of the agent for reducing blood ammonia concentration according to the present invention contains 1-kestose as an active ingredient.

[0012] (3) A third aspect of the agent for reducing blood ammonia concentration according to the present invention contains Clostridium butyricum and 1-kestose as active ingredients.

[0013] (4) The agent for reducing blood ammonia concentration according to the present invention can be used for the prevention or improvement of hyperammonemia.

[0014] (5) The agent for reducing blood ammonia concentration according to the present invention can be used for the prevention or improvement of hepatic encephalopathy.

Effects of the Invention

[0015] According to the present invention, blood ammonia concentration can be reduced.

[0016] Clostridium butyricum is an anaerobic Gram-positive bacillus that forms spores. It is widely commercially available as a gastrointestinal agent and a feed additive, and has been ingested by mammals such as humans and livestock over a long period of time, so it has high safety. Therefore, according to the present invention, it is possible to safely reduce the blood ammonia concentration.

[0017] In addition, Clostridium butyricum has high resistance to various external environments such as heat and acid in the spore state. It can also be dried and processed into a powder form, and does not exhibit a strong odor or taste. From these facts, Clostridium butyricum can be easily incorporated as it is or into various foods, pharmaceuticals, feeds, etc., and can be ingested easily and conveniently on a daily basis. Therefore, according to the present invention, it is possible to obtain a blood ammonia concentration reducing agent that is highly safe and can be ingested easily and conveniently on a daily basis as it is or by being easily incorporated into various foods, pharmaceuticals, feeds, etc.

[0018] 1-Kestose is an oligosaccharide trisaccharide composed of one molecule of glucose and two molecules of fructose. 1-Kestose is also contained in vegetables and grains such as onions, garlic, barley, and rye, and has a long history of dietary experience. Since no toxicity has been observed in any of the mutagenicity test, acute toxicity test, subchronic toxicity test, and chronic toxicity test, it has high safety. Therefore, according to the present invention, it is possible to safely reduce the blood ammonia concentration.

[0019] In addition, 1-kestose has high solubility and heat resistance equivalent to that of sugar, and its sweetness is about one-third that of sugar, presenting a good sweetness similar to that of sugar. From this fact, it can be ingested easily and conveniently on a daily basis as it is or as a seasoning such as a sweetener, and can also be easily incorporated into various foods, pharmaceuticals, feeds, etc. Therefore, according to the present invention, it is possible to obtain a blood ammonia concentration reducing agent that is highly safe and can be ingested easily and conveniently on a daily basis as it is or by being easily incorporated into various foods, pharmaceuticals, feeds, etc.

[0020] Hyperammonemia refers to a condition in which the blood ammonia concentration deviates from the reference value (15 - 70 μg / dL) and becomes elevated. Hyperammonemia occurs when there is a severe impairment in the ammonia removal system due to liver damage, drugs, infections, etc. If it progresses, it can present various disturbances of consciousness and, in the worst cases, lead to death. According to the present invention, by reducing the blood ammonia concentration, it is possible to contribute to the prevention or improvement of hyperammonemia.

[0021] Hepatic encephalopathy refers to a condition that presents with neuropsychiatric symptoms (disorders of the sleep - wake rhythm, impairment of memory and orientation, abnormal behavior, depressive state, drowsiness, excitement / delirium state, coma, epileptic seizures, flapping tremors), myoclonus, muscle rigidity, etc. along with severe liver dysfunction. The main cause of hepatic encephalopathy is that due to the decline in liver function, the ammonia removal system such as (i) and (iii) above becomes dysfunctional, and high - concentration ammonia reaches the brain, causing a decline in brain function. According to the present invention, by reducing the blood ammonia concentration, it is possible to contribute to the prevention or improvement of hepatic encephalopathy.

Brief Description of the Drawings

[0022]

Figure 1

Modes for Carrying Out the Invention

[0023] Hereinafter, the present invention will be described in detail. The agent for reducing blood ammonia concentration according to the present invention has the following three aspects: The first aspect: containing Clostridium butyricum as an active ingredient. The second aspect: containing 1 - kestose as an active ingredient. The third aspect: containing Clostridium butyricum and 1 - kestose as active ingredients.

[0024] "Blood ammonia concentration" refers to the concentration of ammonia in the blood. The blood ammonia concentration can be measured according to known measurement methods (direct colorimetry, dry chemistry method, enzyme method (GIDH-UV method), enzyme method (NADS-UV method), enzyme cycling method, etc.). In measurement, blood is collected from the subject, and according to the measurement method, the whole blood, or plasma or serum is prepared as a sample. For simplicity, it can be measured using commercially available measurement kits or measurement reagents based on the principles of the above measurement methods.

[0025] For Clostridium butyricum, commercially available products may be used, or those deposited in a culture collection may be used. Examples of commercially available products include probiotics commercially available from Miyarisan Pharmaceutical Co., Ltd. (trade names: Miyarisan BM (registered trademark) fine granules, Miyarisan BM (registered trademark) tablets, Shin Miyarisan Eye (registered trademark) intestinal regulator, Miyarisan (registered trademark) tablets, Strong Miyarisan (registered trademark) tablets).

[0026] Examples of those deposited in a culture collection include Clostridium butyricum Miyairi 588 (FERM BP-2789), Clostridium butyricum Miyairi 585 (FERM BP-6815), Clostridium butyricum Miyairi 595 (FERM BP-6816), Clostridium butyricum Miyairi 630 (FERM BP-6817), Clostridium butyricum NIP1020 (FERM BP-5794), Clostridium butyricum NIP1021 (FERM BP-5795), Clostridium butyricum (FERM P-11868), Clostridium butyricum (FERM P-11869), Clostridium butyricum (FERM P-11870), Clostridium butyricum (ATCC859), Clostridium butyricum (NBRC3315), Clostridium butyricum (ATCC860), Clostridium butyricum (ATCC19398).

[0027] In the present invention, Clostridium butyricum may be used alone as one kind, or two or more kinds may be used in combination. Further, Clostridium butyricum may be used in the form of a culture solution obtained by culturing the same, a residue containing Clostridium butyricum obtained by centrifuging the culture solution, or a dried product thereof.

[0028] The culture of Clostridium butyricum can be carried out by a known culture method, for example, the method disclosed in Japanese Patent Application No. 08-252088. The culture conditions can be appropriately set according to the microbial strain used. For example, Clostridium butyricum is inoculated into a medium (1.0 (w / v)% peptone, 1.0 (w / v)% yeast extract, 1.0 (w / v)% corn starch, and 0.2 (w / v)% precipitated calcium carbonate, pH 6.5 to 7.5) to a concentration of 10 5 ~10 6 cells / mL, and statically cultured at 37°C for 48 hours. Since Clostridium butyricum is an obligate anaerobe, it is preferably cultured under anaerobic conditions by not aerating, or by aerating nitrogen or carbon dioxide gas, or by adding a reducing agent to the medium to lower the redox potential. By centrifuging the culture solution thus obtained to recover the precipitate, a culture solution residue containing Clostridium butyricum can be obtained. Further, by treating this residue by air drying or drying under reduced pressure, a dried product can be obtained. Examples of the air drying conditions include 0 to 80°C, preferably 10 to 20°C, for 1 to 24 hours, preferably 5 to 18 hours. Examples of the drying under reduced pressure conditions include 0 to 80°C, preferably 10 to 20°C, 0.05 to 500 Torr, preferably 1 to 100 Torr, for 1 to 24 hours, preferably 2 to 15 hours.

[0029] 1-Kestose can be produced by performing an enzymatic reaction with an enzyme as disclosed in JP-A-58-201980 using sucrose as a substrate. Specifically, first, β-fructofuranosidase is added to a sucrose solution, and the enzymatic reaction is carried out by allowing it to stand at 37°C to 50°C for about 20 hours to produce 1-kestose. The enzymatic reaction solution containing the produced 1-kestose is subjected to a chromatographic separation method as disclosed in JP-A-2000-232878 to separate and purify 1-kestose from other sugars (glucose, fructose, sucrose, oligosaccharides of 4 sugars or more). After concentrating the solution obtained thereby, which contains high-purity 1-kestose, crystallization is carried out by a crystallization method as disclosed in JP-B-6-70075 to obtain crystals of 1-kestose or a composition containing 1-kestose with a purity of 98% by mass or more.

[0030] In addition, since 1-kestose is contained in commercially available fructooligosaccharides, it may be used as it is, or 1-kestose may be separated and purified from fructooligosaccharides by the above-described method and used. That is, as the 1-kestose of the present invention, a 1-kestose-containing composition such as an oligosaccharide containing 1-kestose may be used. When using a 1-kestose-containing composition, the purity of 1-kestose is preferably 80% or more, more preferably 85% or more, and even more preferably 90% or more. In the present invention, the "purity" of 1-kestose means the mass% of 1-kestose when the total mass of the sugar is 100.

[0031] The agent of the present invention can be used by oral administration to humans or animals. Alternatively, the active ingredient may be added to an enteral nutrient, and this may be used by a method of administration by enteral nutrition via a tube inserted into the digestive tract such as the stomach or small intestine.

[0032] In addition to being composed only of the active ingredients Clostridium butyricum and / or 1-kestose, the agent of the present invention can be used in the forms of pharmaceuticals, quasi-drugs, food additives, supplements, food compositions (such as beverages, seasonings, bread, confectioneries, side dishes, health foods, infant foods, and other processed foods), feeds, feed additives, and the like.

[0033] When the agent of the present invention is in the form of a pharmaceutical, a food additive, or a supplement, examples of its dosage form include solid or liquid dosage forms such as powders, tablets, dragees, capsules, granules, dry syrups, solutions, syrups, drops, and drinks. Each dosage form can be manufactured by methods known to those skilled in the art. For example, in the case of a powder (second aspect), 800 g of 1-kestose and 200 g of lactose are thoroughly mixed, and then 300 mL of 90% ethanol is added to moisten them. Subsequently, after granulating the moist powder, it is dried by ventilation at 60°C for 16 hours, and then sized to obtain 1000 g of a powder (1-kestose content: 800 mg / 1 g) with an appropriate fineness. Also, in the case of a tablet, 300 g of 1-kestose, 380 g of powdered reduced maltose syrup, 180 g of rice starch, and 100 g of dextrin are thoroughly mixed, and then 300 mL of 90 (v / v)% ethanol is added to moisten them to obtain a moist powder. After extrusion granulating this moist powder, it is dried by ventilation at 60°C for 16 hours to obtain granules. After sizing these granules using an 850-μm sieve, 50 g of sucrose fatty acid ester is added to 470 g of the granules and mixed. By subjecting this to a rotary tableting machine (6B-2, Kikusui Seisakusho) for tableting, 5000 tablets (diameter: 8 mm, weight: 200 mg, 1-kestose content: 60 mg / 1 tablet) can be obtained.

[0034] When the agent of the present invention is in the form of a food composition, it may be produced by adding Clostridium butyricum and / or 1-kestose during the production process of ordinary foods and drinks. Clostridium butyricum is spore-forming and has high heat resistance and acid resistance. It can also be dried and processed into a powder form, without showing a strong odor or taste. 1-kestose also has high heat resistance and taste properties, physical properties, and processability similar to those of sugar. Therefore, Clostridium butyricum and / or 1-kestose can be handled in the same way as seasonings such as sugar during the production process of various foods and drinks to produce various foods and drinks.

[0035] When the agent is used for humans or animals, the intake amount (dosage) of the active ingredient can be, for example, 0.04 g / kg body weight or more per day in the case of 1-kestose. In the case of Clostridium butyricum, its culture solution, the residue containing the bacteria obtained by centrifuging the culture solution, or the dried product of the residue can be exemplified as 20 - 360 mg / adult per day, 40 - 200 mg / adult per day, or 60 - 120 mg / adult per day in terms of solid content. Such intake amounts are not limited to once a day and may be taken in multiple divided doses.

[0036] Hereinafter, the present invention will be described based on each example. Note that the technical scope of the present invention is not limited to the features shown by these examples.

Example

[0037] In this example, as the "1-kestose", a composition (produced by Product Food Science Co., Ltd.) containing 1-kestose with a purity of 98% by mass or more was used. As the "Clostridium butyricum", a powder containing Clostridium butyricum Miyairi 588 with an accession number of FERM BP-2789 (number of bacteria: 10 9 ~10 10 / g) was used.

[0038] Melanocortin 4 receptor-deficient mice (MC4R-KO mice) were transferred from Dr. Joel K. Elmquist of the University of Texas Southwestern Medical Center (Texas, USA). MC4R-KO mice are mice that lack the melanocortin 4 receptor (MC4R), which plays an important role in central feeding regulation, with a genetic background of C57BL / 6J mice, and develop non-alcoholic steatohepatitis when fed a high-fat diet (M. Itoh et al., The American Journal of Pathology, Vol. 179, No. 5, 2454-2463, November 2011).

[0039] Eight 8-week-old male MC4R-KO mice were divided into 4 groups, I to IV, with 8 mice in each group. Each mouse in each group was placed in an individual cage and bred for 12 weeks in an environment with a temperature of 22-26°C, humidity of 40-60%, a 12-hour light period and a 12-hour dark period, free access to water, and free access to food. The feed was, as follows, a high-fat diet (D12492; 524 kcal / 100 g, with 60% of the total energy being fat; Research Diets Inc., New Zealand), or a high-fat diet supplemented with Clostridium butyricum and / or 1-kestose was given. Group I: High-fat diet Group II: High-fat diet supplemented with Clostridium butyricum (concentration of Clostridium butyricum 2.0 g / 100 g of feed) Group III: High-fat diet supplemented with 1-kestose (concentration of 1-kestose 2.5 g / 100 g of feed) Group IV: High-fat diet supplemented with Clostridium butyricum and 1-kestose (concentration of Clostridium butyricum 2.0 g / 100 g of feed, concentration of 1-kestose 2.5 g / 100 g of feed)

[0040] Based on the feed intake, the average values of the daily intake of Clostridium butyricum and 1-kestose were calculated for each group. Also, after the end of the breeding period, blood was collected to prepare serum, and the ammonia concentration was measured using the blood ammonia concentration measurement kit "Shikari Kit NH3" (Kanto Chemical) based on the enzyme cycling method. For the measured ammonia concentration, the average value and standard deviation were calculated for each group. The average values of the feed intake, Clostridium butyricum intake, 1-kestose intake, and ammonia concentration are shown in Table 1. Also, the measurement results of the ammonia concentration are shown in Table 2. Also, a bar graph of the average value of the ammonia concentration is shown in Figure 1.

Table 1

[0041]

Table 2

[0042] As shown in Table 1, Table 2, and Figure 1, the serum ammonia concentration was lower in Groups II, III, and IV compared to Group I. In particular, in Group IV, the serum ammonia concentration was significantly lower than that in Group I. From this result, it was revealed that Clostridium butyricum and 1-kestose can reduce the blood ammonia concentration.

Claims

1. An agent for reducing blood ammonia concentration, comprising 1-kestose.

2. An agent for reducing blood ammonia concentration, comprising 1-kestose and Clostridium butyricum.

3. The agent according to claim 1 or 2, which is used for the prevention or improvement of hyperammonemia.

4. The agent according to claim 1 or 2, which is used for the prevention or improvement of hepatic encephalopathy.

5. A method (excluding medical acts) for reducing blood ammonia concentration by administering the agent according to any one of claims 1 to 4 to a human or an animal.

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