WO2016002757A1 - Lactic acid bacterium for suppressing purine absorption and use for same - Google Patents

Abstract

Provided are a lactic acid bacterium for suppressing purine absorption, and a use for said bacterium. The present invention relates to: a method in which lactic acid bacteria are cultured in a solution including adenine, 5-phospho-D-ribose-1-diphosphoric acid, and Mg^2+­, and the conversion activity of adenine into adenylic acid obtained through said culturing is used as an index to screen lactic acid bacteria having an enhanced capacity for converting purine bases into purine nucleotides; an agent for converting purine bases into purine nucleotides, said agent including as an active ingredient the lactic acid bacteria obtained by the abovementioned method; and a use for said agent.

Description

Lactic acid bacteria that suppress purine body absorption and uses thereof

The present invention relates to lactic acid bacteria that suppress purine body absorption and uses thereof.

In Japan, gout patients and hyperuricemia have been increasing year by year due to changes in dietary habits. Hyperuricemia results in decreased uric acid excretion and excessive uric acid production, and increased serum uric acid levels often induce gout that develops acute arthritis with severe pain. There are currently an estimated 1 million gout patients in Japan, and an estimated 10 million hyperuricemia. At present, hyperuricemia is mainly prevented and treated by controlling serum uric acid levels by a combination of diet, exercise, and medication. In diet therapy, by restricting the calorie intake, the intake of dietary purines that are eventually decomposed into uric acid is reduced, but it is not always easy to continue restricting severe calorie intake. Therefore, more effective treatment methods are desired for gout and hyperuricemia. Furthermore, there is a need for the development of foods that are effective in preventing and reducing symptoms of gout and hyperuricemia.

By the way, microorganisms and fermented products that are effective in reducing serum uric acid levels in hyperuricemia have been reported (Patent Documents 1 to 5). For example, Patent Document 1 shows that lactic acid bacteria have high resolution from purine nucleosides to purine bases. For example, Patent Documents 4 and 5 indicate that lactic acid bacteria have purine body resolution. Such microorganisms and fermented products that have a serum uric acid level-reducing action promote the conversion of purine nucleosides into purine bases in the intestine, and convert them from purine nucleosides that are easily absorbed from the intestinal tract to purine bases that are difficult to absorb from the intestinal tract By doing so, it has been considered that purine body absorption is suppressed and excretion is promoted. However, there are few reports on the results of human tests, and an efficient method for obtaining lactic acid bacteria having an effect of reducing serum uric acid levels is not known.

JP 2008-005834 A International publication WO2011 / 102310 International Publication WO2004 / 112809 International Publication WO2008 / 129802 JP 2013-048636

An object of the present invention is to provide a lactic acid bacterium that suppresses purine body absorption and its use.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that certain Lactobacillus species have a high ability to convert a purine base to a purine nucleotide, and complete the present invention. It came to.

That is, the present invention includes the following.
[1] Lactic acid bacteria are cultured in a solution containing adenine, 5-phospho-D-ribose-1-diphosphate, and Mg ²⁺ , and the conversion activity of adenine obtained thereby into adenylate is used as an index. A method for screening a lactic acid bacterium having enhanced ability to convert a purine base to a purine nucleotide as compared with Lactobacillus gasseri ATCC 33323 strain.
[2] The method according to [1], further comprising measuring 5′-nucleotidase activity and selecting a lactic acid bacterium having reduced activity compared to Lactobacillus gasseri ATCC 33323 strain.
[3] The method according to [1] or [2] above, wherein the lactic acid bacterium is a genus Lactobacillus.
[4] The method according to any one of [1] to [3] above, wherein the solution is a buffer solution.

[5] An agent for converting a purine base into a purine nucleotide, comprising as an active ingredient a lactic acid bacterium obtained by the method according to any one of [1] to [4] above.
[6] The conversion agent according to [5] above, for converting adenine to adenylic acid or for converting guanine to guanylic acid.
[7] The conversion agent according to [5] or [6] above, wherein the lactic acid bacteria is Lactobacillus gasseri OLL2959 strain (Accession No. NITE BP-224).

[8] A food or drink or a pharmaceutical comprising the conversion agent according to any one of [5] to [7] above.
[9] The food or drink or medicine according to [8] above, for reducing serum uric acid level.
[10] The food or drink or pharmaceutical according to [9] above, wherein the reduction of serum uric acid level is accompanied by promotion of conversion of adenine to adenylate and promotion of conversion of guanine to guanylate in the intestine.
[11] The food or drink or pharmaceutical according to [9] or [10] above, wherein the subject is a human subject having a serum uric acid level of 6 to 8 mg / dL.
[12] The food or drink or medicine according to any one of [8] to [11] above, wherein the lactic acid bacterium is contained at 1 × 10 ⁸ to 10 ¹⁰ cfu per dose.

[13] By reacting the conversion agent according to any one of [5] to [7] above with a purine base in the presence of 5-phospho-D-ribose-1-diphosphate and Mg ²⁺ , A method of generating a purine nucleotide from a purine base.
[14] The method according to [13] above, wherein adenylic acid is produced from adenine or guanylic acid is produced from guanine.

When the lactic acid bacterium or the conversion agent according to the present invention is used, a purine base can be efficiently converted into a purine nucleotide, thereby reducing the serum uric acid level in the administered subject.
This specification includes the contents of Japanese Patent Application Nos. 2014-134973, 2014-234050, and 2015-064201 which form the basis for claiming priority of the present application.

FIG. 1 is a graph showing the change over time in the conversion rate from adenine to AMP (AMP / adenine conversion rate) by Lactobacillus gasseri OLL2959 strain. FIG. 2 is a graph showing the change over time in the conversion rate of guanine to GMP (GMP / guanine conversion rate) by Lactobacillus gasseri OLL2959 strain. FIG. 3 is a graph showing the purine nucleosidase activity of Lactobacillus gasseri OLL2959 strain. The left bar shows the conversion rate from adenosine to adenine, the middle bar shows inosine to hypoxanthine, and the right bar shows the conversion rate from guanosine to guanine. FIG. 4 is a graph showing the 5′-nucleotidase activity of Lactobacillus gasseri OLL2959 strain. The left bar shows AMP to adenosine, the middle bar shows IMP to inosine, and the right bar shows the conversion rate from GMP to guanosine. FIG. 5 is a graph showing changes over time in serum uric acid levels in human subjects who continuously ingested Lactobacillus gasseri OLL2959 strain. FIG. 6 shows the rate of change in serum uric acid level of each subject after intake of the test food (from the pre-intake test to the 8-week test). FIG. 7 is a graph showing the purine uptake ability of Lactobacillus gasseri OLL2959 strain. FIG. 8 is a graph showing the growth ability of Lactobacillus gasseri OLL2959 strain in the presence of purines. FIG. 9 is a diagram showing the adenine uptake ability of a plurality of lactic acid strains. FIG. 10 is a graph showing the growth ability of a plurality of lactic acid strains in the presence of adenine. FIG. 11 is a graph showing the ability of the Lactobacillus gasseri OLL2959 strain and the purine body to be taken up in the animals to which the purine body was simultaneously administered. FIG. 12 is a graph showing the results of comparison of adenine uptake ability by type of lactic acid strain. FIG. 13 is a graph showing the results of comparing the growth ability of lactic acid strains in the presence of adenine by the type of lactic acid strain.

Hereinafter, the present invention will be described in detail.
Purine is a general term for substances having a purine skeleton, and is classified into purine bases, purine nucleosides, and purine nucleotides. Purine bodies perform various functions mainly in cells of living organisms, and are responsible for transmission of genetic information as, for example, constituents of nucleic acids. Main purine bases include adenine, guanine, hypoxanthine and xanthine. Purine nucleosides are compounds in which a sugar is bound to a purine base, and examples thereof include adenosine, guanosine, inosine and xanthosine combined with ribose, deoxyadenosine, deoxyguanosine, deoxyinosine and deoxyxanthosine combined with deoxyribose. A purine nucleotide is a compound in which a phosphate is bonded to a purine nucleoside, and examples thereof include adenylic acid (AMP), guanylic acid (GMP), inosinic acid (IMP), and xanthylic acid (XMP).

Purine is not only supplied from food to in vivo as a dietary purine via intestinal absorption, but also newly biosynthesized from amino acids and the like through the de novo pathway. Purine bodies are biosynthesized through a salvage pathway that synthesizes purine nucleotides by reusing purine bases generated by degradation of purine nucleotides.

In humans, purine nucleotides are eventually metabolized to uric acid. For example, adenylate is converted to adenosine by 5′-nucleotidase (5′-NT) activity, and adenosine is metabolized to hypoxanthine via inosine. Hypoxanthine becomes xanthine by xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activities. Guanylic acid becomes guanosine by 5'-nucleotidase activity, and further becomes guanine by purine nucleoside phosphorylase (PNP) activity. Guanine is converted to xanthine by guanine deaminase (GDA). Xanthine is metabolized to uric acid by xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activities. On the other hand, each purine nucleoside (adenosine, inosine, xanthosine and guanosine) is converted into a purine base (adenine, hypoxanthine, xanthine and guanine) by purine nucleoside phosphorylase (PNP) activity. Many of adenine, guanine, hypoxanthine and xanthine are reused for biosynthesis of adenylic acid, guanylic acid, inosinic acid and xanthylic acid, respectively, by salvage enzyme activity (salvage pathway).

Lactic acid bacteria also have a purine metabolic pathway similar to that of humans, but there are also differences from human metabolic pathways. For example, most lactic acid bacteria ultimately metabolize purine nucleosides to bases. In the case of Lactobacillus gasseri, purine nucleosides are converted to purine bases by purine nucleosidases.

In the present invention, a lactic acid bacterium having a high conversion ability from a purine base to a nucleotide can be obtained by selecting lactic acid bacteria using the conversion activity of adenine into adenylic acid as an index. The present invention has a conversion ability from a purine base to a nucleotide, using as an index the conversion activity of adenine to adenylate in solution obtained by culturing adenine as a substrate in contact with lactic acid bacteria in a solution. The present invention relates to a method for screening enhanced lactic acid bacteria.

The lactic acid bacterium to be subjected to the screening method of the present invention is not particularly limited, but is preferably a bacterium belonging to the genus Lactobacillus. Lactobacillus genus Lactobacillus gasseri, Lactobacillus delbruecki subspices bulgaricus (Lactobacillus delbrueckii subsp. Burgalicus), Lactobacillus derbrueckii subspices lacdel Lactis), Lactobacillus paraphile (Lactobacillus paraspii caseparacasei), Lactobacillus acidophilus, Lactobacillus ヘ ル acidophilus, Lactobacillus helveticus Lactobacillus helveticus subsp. Jugurti), Lactobacillusobacrispatus, Lactobacillus ラ ク ト amylovorus, Lactobacillus Lactobacillus gallinarum, Lactobacillus oris, Lactobacillus casei subspices rhamnosus, Lactobacillus subsp. fermentum), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus plantarum, Lactobacillus reuteri, and the like, with Lactobacillus reuteri being particularly preferred. Lactic acid bacteria are preferably used for screening after culturing in an appropriate medium (for example, MRS medium) to adjust the concentration.

In the screening method of the present invention, lactic acid bacteria are cultured in a solution containing adenine, 5-phospho-D-ribose-1-diphosphate, and Mg ²⁺ . The conversion reaction from adenine to adenylic acid (AMP-based salvage activity) is based on Mg ² formed by 5-phospho-D-ribose-1-diphosphate (PRPP; also called phosphoribosyl pyrophosphate) in the presence of Mg ^{2+ This} is because it is catalyzed by adenine phosphoribosyltransferase (APRT) using ⁺ PRPP and adenine as substrates. PRPP is added to the solution in the form of any salt of PRPP (eg, any salt such as 5-phospho-D-ribose-1-diphosphate pentasodium salt, potassium salt, calcium salt, etc.) Depending on the situation, it may be added to the solution. Mg ²⁺ can also be incorporated into the solution by adding it to the solution in the form of any salt thereof (eg MgCl ₂ ). The solution used for screening may be any solution as long as lactic acid bacteria can survive and does not inhibit the enzyme activity, but is preferably a buffer solution. Examples of the buffer include, but are not limited to, phosphate buffer, Tris buffer, Tris-HCl buffer, HEPES buffer, and the like. The pH of the solution is not particularly limited, but is usually preferably 6 to 8 in terms of pH.

The concentration of adenine, 5-phospho-D-ribose-1-diphosphate (PRPP), and Mg ²⁺ and lactic acid bacteria in the solution (reaction solution) used for screening depends on the conversion reaction of adenine to adenylic acid by lactic acid bacteria. It can be any suitable amount. For example, the solution has a final concentration of 0.1 mM to 1 mM adenine (preferably 0.1 mM to 0.3 mM), 1 to 15 mM PRPP (preferably 1 mM to 5 mM), and 4 mM to 40 mM Mg ²⁺ ( Preferably, it may contain 4 mM to 15 mM). The reaction solution may also contain, but is not limited to, 5 × 10 ⁸ cfu / ml to 5 × 10 ¹⁰ cfu / ml lactic acid bacteria, for example, 5 × 10 ⁹ cfu / ml lactic acid bacteria. The solution may further contain any substance that does not substantially affect the conversion reaction of adenine into adenylic acid by lactic acid bacteria.

In screening, lactic acid bacteria are preferably suspended and cultured in a solution containing adenine, 5-phospho-D-ribose-1-diphosphate (PRPP), and Mg ²⁺ . Cultivation of lactic acid bacteria is preferably performed by incubating at a temperature suitable for culturing lactic acid bacteria, usually 20 ° C. to 50 ° C., preferably 30 to 40 ° C., for example, approximately 37 ° C. Culture of lactic acid bacteria can be suitably performed by, for example, incubating in a water bath while shaking. Lactic acid bacteria are cultured for a certain period of time, typically at least 15 minutes or more, preferably 30 minutes or more, more preferably 60 minutes or more.

In the above solution, after culturing lactic acid bacteria for a certain period of time, the conversion reaction of adenine to adenylic acid is stopped. This conversion reaction is preferably stopped by adding TFA (trifluoroacetic acid) to the solution. For example, an equal amount of 5% TFA can be added to the solution. In order to examine the transition of the conversion reaction, it is preferable to stop the reaction while gradually changing the length of culture time of lactic acid bacteria.

Subsequently, adenine and adenylic acid in the reaction solution after stopping the conversion reaction are quantified. In addition, other purines (for example, purine nucleosides) in the reaction solution may also be quantified. This quantification may be performed by an arbitrary measurement method, but can be performed by, for example, an HPLC method. It is preferable to collect the supernatant of the reaction solution and subject the filtered filtrate to measurement for quantification.

For example, the measurement by the HPLC method can be performed under the following conditions.
-Mobile phase: A: 20 mM phosphate buffer (pH 7.5)
B: 40 mM phosphate buffer (pH 7.5) / acetonitrile (1: 1)
・ Column: SHISEIDO CAPCELL PAK C18 MG2 (2.0mm id × 150mm)
・ Flow rate: 0.2mL / min ・ Temperature: 40 ℃
・ Injection volume: 5μl
・ Detection wavelength: 254nm (UV)
-Gradient A / B (min): 100/0 (0 min)-100/0 (5 min)-80/20 (20 min) [%]
In the case of quantification by the HPLC method, the amounts of adenine and adenylic acid in the reaction solution can be calculated as relative values by measuring the area under the peak of the HPLC chart. The conversion rate of adenine to adenylic acid (AMP) at each reaction stop time can be calculated by the following formula.
Conversion rate (%) = (Area value under the peak of AMP in the test area−Area value under the peak of the AMP in the control area) / (Area area under the peak of adenine at the 0 minute of the test area−At the 0 minute time point in the control area) Adenine peak area value)

Here, the control group was measured in the same manner with a solution prepared using an equal amount of buffer instead of adding adenine as a substrate.

In the screening method of the present invention, the conversion rate of adenine to adenylic acid (AMP) can be used as a value representing the conversion activity of adenine to adenylic acid. That is, in the screening method of the present invention, the conversion rate of adenine to adenylic acid (AMP) is used as an index to determine whether lactic acid bacteria have the activity to convert adenine to adenylic acid (AMP) and the level of the conversion activity. Can do. For example, in the present invention, if the conversion rate of adenine to adenylic acid (AMP) reaches a maximum of 30% or more by measurement over time, it can be determined that lactic acid bacteria have the activity of converting adenine to adenylic acid (AMP). . In the screening method of the present invention, the conversion rate of adenine to adenylic acid (AMP) is preferably 40% or more, more preferably 50% or more, more preferably 70% or more, and particularly preferably 90%, as measured by time. It is preferable to select lactic acid bacteria that reach% or more. In a preferred embodiment of the screening method of the present invention, a conversion rate of adenine to adenylic acid (AMP) reaches a maximum level (preferably a plateau) as measured over time, for example, a Lactobacillus gasseri reference strain Compared with the ATCC 33323 strain, for example, when increased to 5 times or more, preferably 7 times or more, more preferably 10 times or more, still more preferably 13 times or more, and particularly preferably 20 times or more, from adenine of lactic acid bacteria to adenylic acid It can be determined that “the ability to convert a purine base to a purine nucleotide has been enhanced” as the ability to convert to a remarkably enhanced.

The lactic acid bacteria selected in this way have not only high conversion ability of adenine to adenylic acid (AMP salvage activity) but also high conversion ability of other purine bases to purine nucleotides due to salvage activity. possible. Specifically, the lactic acid bacterium may have an enhanced ability to convert guanine to guanylic acid. Therefore, it is also preferable to examine the conversion activity of other purine bases to purine nucleotides in the same manner for lactic acid bacteria selected as having enhanced ability to convert adenine to adenylate. Specifically, lactic acid bacteria are cultured in a solution containing guanine, 5-phospho-D-ribose-1-diphosphate, and Mg ²⁺ , and the conversion activity of guanine to guanylic acid indicated thereby is indicated as an index. As such, it may be examined whether or not the ability to convert guanine to guanylic acid has been enhanced. Measurement of the conversion activity of guanine to guanylic acid and calculation of the conversion rate are the same as the above conversion of adenine to adenylic acid except that guanine is used instead of adenine and guanylic acid is used instead of adenylic acid. It can be carried out. In the present invention, by the time when the conversion rate of guanine to guanylic acid (GMP) reaches a maximum level (preferably a plateau) as measured over time, for example, compared with ATCC 33323 strain which is a reference strain of Lactobacillus gasseri. For example, when it is increased by 5 times or more, preferably 7 times or more, more preferably 10 times or more, further preferably 13 times or more, particularly preferably 20 times or more, the ability of lactic acid bacteria to convert from guanine to guanylic acid is also significantly enhanced. Can be determined.

In the present invention, lactic acid bacteria having an enhanced ability to convert purine bases into purine nucleotides can be screened in this way. The lactic acid bacteria obtained by the screening method of the present invention have a high conversion ability from a purine base to a purine nucleotide, particularly a high conversion ability from adenine to adenylic acid and a high conversion ability from guanine to guanylic acid.

The lactic acid bacterium obtained by the screening method of the present invention preferably has a higher conversion activity (salvage activity) from a purine base to a purine nucleotide as compared with a degradation activity from a purine nucleotide to a purine nucleoside. Therefore, the screening method of the present invention may further include a step of measuring degradation activity to purine nucleosides using a purine nucleotide as a substrate and selecting lactic acid bacteria having reduced activity. Specifically, the activity of 5′-nucleotidase (5′-NT), which catalyzes the degradation of purine nucleotides into purine nucleosides in lactic acid bacteria, can be measured using a purine nucleotide (eg, adenylic acid) as a substrate.

The activity measurement of 5'-nucleotidase (5'-NT) of lactic acid bacteria can be performed by a conventional method. For example, lactic acid bacteria are cultured in a solution containing purine nucleotides (such as adenylate) and Mg ²⁺ . Mg ²⁺ can be incorporated into the solution by adding it to the solution in the form of any salt thereof (eg MgCl ₂ ). The solution used for screening may be any solution as long as lactic acid bacteria can survive and does not inhibit the enzyme activity, but is preferably a buffer solution. Examples of the buffer include, but are not limited to, phosphate buffer, Tris buffer, Tris-HCl buffer, HEPES buffer, and the like. The pH of the solution is not particularly limited, but is usually preferably 6 to 8 in terms of pH. As a control, it is preferable to use ATCC 33323 strain which is a Lactobacillus gasseri reference strain.

The concentrations of purine nucleotides and Mg ²⁺ and lactic acid bacteria in the reaction solution may be any amount suitable for the conversion reaction of purine nucleotides into purine nucleosides by the ATCC 33323 strain. For example, the reaction solution may contain 0.1 mM to 1 mM purine nucleotides (preferably 0.1 mM to 0.3 mM) and 4 mM to 40 mM Mg ²⁺ (preferably 4 mM to 15 mM) at a final concentration at the start of the reaction. The reaction solution is not particularly limited, and may contain 5 × 10 ⁸ cfu / ml to 5 × 10 ¹⁰ cfu / ml lactic acid bacteria, for example, 5 × 10 ⁹ cfu / ml lactic acid bacteria. The solution may further contain any substance that does not substantially affect the conversion reaction of purine nucleotides into purine nucleosides by lactic acid bacteria. The culture conditions for lactic acid bacteria may be the same conditions as in the conversion test for adenine into adenylic acid.

Measurement of the conversion activity of purine nucleotides into purine nucleosides (ie, 5′-nucleotidase activity) and calculation of the conversion rate are carried out using purine nucleotides (eg, adenic acid) instead of adenine, purine nucleosides (eg, Except for using adenosine in the case of using adenic acid as a substrate, it can be carried out by the same method as the above-described test for the conversion rate of adenine to adenylic acid. However, the reaction time is not particularly limited, but is preferably set longer (for example, 120 minutes) than in the case of conversion of adenine to adenylic acid. In the present invention, the conversion rate of a purine nucleotide into a purine nucleoside, in a preferred example, the conversion rate of adenylate into adenosine is, for example, 1/5 or less compared to, for example, ATCC 33323 strain which is a Lactobacillus gasseri reference strain, Preferably, when it is reduced to 1/7 or less, more preferably 1/10 or less, further preferably 1/13 or less, and particularly preferably 1/20 or less, the 5′-nucleotidase activity of lactic acid bacteria is significantly reduced. Can be determined. In lactic acid bacteria with significantly reduced 5'-nucleotidase activity, the conversion of purine nucleotides to purine nucleotides is superior to the conversion of purine nucleotides to purine nucleosides, which are absorbed from the intestinal tract in the mammal and produce uric acid. A further reduction in purine nucleosides can be achieved.

The present invention also provides an agent for converting a purine base to a purine nucleotide, which can be obtained by the above screening method and contains a lactic acid bacterium having a high ability to convert a purine base to a purine nucleotide as an active ingredient. This conversion agent is also preferably used, for example, for the conversion of adenine to adenylic acid or the conversion of guanine to guanylic acid. By using this conversion agent, it is possible to efficiently convert a purine base as a substrate into a purine nucleotide in an in vivo or in vitro reaction system.

Lactobacillus gasseri OLL2959 strain is a preferred example of a lactic acid bacterium having a high conversion ability from a purine base to a purine nucleotide as described above and having reduced 5′-nucleotidase activity. Lactobacillus gasseri OLL2959 strain is homolactic fermentable and has no gas production ability. The Lactobacillus gasseri OLL2959 strain, dated March 31, 2006 (original deposit date), is the National Institute of Technology and Evaluation of Microorganisms (NPMD) (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture, Japan) 8 Deposited in room 122 (zip code 292-0818) under the deposit number NITE P-224, it was transferred to a deposit under the Budapest Treaty (international deposit) on November 21, 2007, and the deposit number is NITE BP- It has been changed to 224.

Lactobacillus gasseri OLL2959 strain has a high ability to convert purine bases into purine nucleotides (salvage activity) and a low ability to convert purine nucleotides into purine nucleosides. Therefore, when the Lactobacillus gasseri OLL2959 strain is administered to humans, the reuse of purine bases to purine nucleotides by microorganisms is superior to the absorption and metabolism of purine bases and purine nucleosides by humans. It is thought to contribute to the decrease.

The present invention also provides a food or drink or a medicine containing the above-described agent for converting a purine base into a purine nucleotide. The conversion agent of the present invention may be a drug or composition containing lactic acid bacteria having the ability to convert the purine base of the present invention into a purine nucleotide, or a fermented product or culture produced using the lactic acid bacteria. Or a concentrate thereof or a drug or composition containing the same. The lactic acid bacterium according to the present invention contained in the conversion agent of the present invention may be a dead cell or a treated product as long as it shows enzyme activity, but is preferably a living cell. The conversion agent of the present invention can impart a conversion activity of a purine base to a purine nucleotide in a living body (typically in the intestinal tract) to a food or drink or a pharmaceutical product. The food or drink or pharmaceutical comprising the conversion agent of the present invention is a lactic acid bacterium and its enzymatic activity that promotes the conversion of a purine base into a purine nucleotide in the intestinal tract, for example, adenine to adenylate The promotion of the conversion of guanine and the promotion of the conversion of guanine to guanylic acid can effectively reduce the serum uric acid level. That is, the food / beverage products and pharmaceuticals of the present invention may be used for reducing serum uric acid levels accompanied by promotion of conversion of adenine to adenylic acid and promotion of conversion of guanine to guanylic acid. Foods and beverages and pharmaceuticals containing the conversion agent of the present invention can be suitably used for the prevention, treatment, improvement or symptom reduction of gout and hyperuricemia.

In the present specification, “food and drink” is not particularly limited, but includes beverages, foods and functional foods. The kind of food / beverage products containing the conversion agent of this invention is not specifically limited. For example, as a beverage containing the conversion agent of the present invention, fermented milk (drink yogurt etc.), lactic acid bacteria beverage, milk beverage (coffee milk, fruit milk etc.), tea beverage (green tea, black tea, oolong tea etc.), fruit / vegetable Beverages (beverages including fruit juices such as oranges, apples and grapes, tomatoes, carrots and other vegetable juices), alcoholic beverages (beer, sparkling wine, wine, etc.), carbonated beverages, soft drinks, water-based beverages It can be illustrated. Suitable drinks include drink yogurt, lactic acid bacteria drinks, milk drinks, water-based drinks and the like, and particularly preferred drinks include drink yogurt. About the manufacturing method etc. of various drinks, the existing reference books, for example, "Latest soft drinks" (2003) (Kotsu Co., Ltd.) etc. can be referred. Examples of the food include fermented milk (set type yogurt, soft yogurt, cheese, etc.), dairy products, confectionery, and instant food. Suitable foods include yogurts such as set type yogurt and soft yogurt, Confectionery, cheese and the like can be mentioned, and particularly suitable foods include yogurt such as set-type yogurt and soft yogurt. You can refer to existing reference books for the production methods of various foods.

Fermented milk such as yogurt containing lactic acid bacteria that have the ability to convert purine bases to purine nucleotides may include other microorganisms such as lactic acid bacteria that may or may not have the ability to convert purine bases to purine nucleotides. You may manufacture by adding the lactic acid bacteria which have the conversion ability from the purine base of this invention to a purine nucleotide to the dairy product and fermented milk which were manufactured using the starter which can be included. Alternatively, such a starter and a lactic acid bacterium having high conversion ability from the purine base to the purine nucleotide of the present invention may be mixed and used as a starter to produce a dairy product or fermented milk. Dairy products and fermented milk using a starter can be produced according to a conventional method. For example, plain yogurt can be produced by mixing a starter with milk or a dairy product that has been cooled after heating, mixing, homogenizing, and sterilizing, followed by fermentation and cooling. The present invention relates to the use of the lactic acid bacterium having high conversion ability from the purine base to the purine nucleotide of the present invention in the production of dairy products such as yogurt and cheese, and fermented milk (preferably the lactic acid bacterium is used in dairy products, fermented milk or its raw materials. (Addition (mixing)) is preferable, and the use of the lactic acid bacteria in the production of yogurt is particularly preferable. Furthermore, the present invention uses a lactic acid bacterium having a high ability to convert a purine base to a purine nucleotide of the present invention as an active ingredient, and has the ability to convert a purine base to a purine nucleotide in dairy products such as yogurt and cheese and fermented milk. An improvement method is preferred, and a method for improving the ability to convert purine bases into purine nucleotides in yogurt is particularly preferred.

As a food or drink containing the conversion agent of the present invention, a functional food is particularly preferable. The “functional food” of the present invention means a food having a certain functionality for a living body, and includes, for example, food for specified health use (including conditional tokuho [food for specified health use]) and nutritional function food. Health functional foods, functional labeling foods, special purpose foods, nutritional supplements, health supplements, supplements (for example, in various dosage forms such as tablets, coated tablets, dragees, capsules and liquids) and beauty foods (for example, diet foods) And so-called health foods in general. The functional food of the present invention also includes health foods to which health claims based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) are applied.

Specific examples of foods that are preferable as the functional food of the present invention include special-purpose foods such as sick foods, maternal and lactating milk powders, infant formulas, elderly foods, and nursing foods.

The functional food of the present invention is useful for reducing the serum uric acid level. The functional food of the present invention can be suitably used for reducing serum uric acid levels, particularly for reducing serum uric acid levels accompanying promotion of purine base conversion to purine nucleotides. The functional food of the present invention can be suitably used for, for example, reducing serum uric acid level accompanied by promotion of conversion of adenine to adenylic acid and promotion of conversion of guanine to guanylic acid.

Foods and drinks such as the functional food of the present invention (in Japan, preferably a food for specified health use or conditional tokuho [food for specified health use]) suppress or alleviate a decrease in serum uric acid level or an increase in serum uric acid level It may be for this purpose, and may be described or displayed to that effect. The present invention includes the use of a lactic acid bacterium having a high ability to convert a purine base to a purine nucleotide of the present invention in the production of a functional food (preferably adding (compounding) the lactic acid bacterium to a functional food or a raw material thereof. ), And the use of the lactic acid bacteria in the production of the food for specified health use is particularly preferable. Furthermore, the present invention is preferably a method for improving the ability to convert a purine base to a purine nucleotide in a functional food, using the lactic acid bacterium having a high ability to convert the purine base to the purine nucleotide of the present invention as an active ingredient. Particularly preferred is a method for improving the ability to convert a purine base to a purine nucleotide in a food for specified health use.

The functional food of the present invention may be a solid preparation such as a tablet, a granule, a powder, a pill or a capsule, a liquid preparation such as a liquid, a suspension or a syrup, or a gel or a paste. However, it may be in the form of a normal food or drink (for example, beverage, yogurt, confectionery, etc.).

The food and drink of the present invention may contain any food component and is not particularly limited. The food and drink of the present invention may contain water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like in addition to the lactic acid bacteria or the conversion agent of the present invention. Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, their hydrolysates, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose, etc. And various milk-derived components. Examples of the saccharide include general saccharides, processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used alone or in combination of two or more, and may be added using a synthetic product and / or a food containing a large amount thereof.

In addition, the pharmaceutical product (pharmaceutical composition) containing the conversion agent of the present invention may contain a pharmaceutically acceptable carrier or additive in addition to the lactic acid bacterium or the conversion agent of the present invention. Examples of the carrier include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, Arabic Rubber, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, liposomes, etc. Artificial cell structures can be used. Examples of the additive include a binder, an excipient, a lubricant, a disintegrant, a wetting agent, a stabilizer, a buffering agent, a corrigent, a preservative, and a coloring agent. The carrier or additive can be selected appropriately or in combination depending on the dosage form of the preparation. The pharmaceutical product of the present invention may further contain other pharmacological components.

The pharmaceutical product of the present invention is preferably administered orally. The pharmaceutical product of the present invention may be in any dosage form such as a solid preparation such as a tablet, a granule, a powder, a pill and a capsule, a gel, or a liquid preparation such as a liquid, suspension and syrup.

The dose of the food or drink or pharmaceutical product of the present invention is not particularly limited, considering the age and weight of the subject to be administered, the route of administration, the number of administrations, etc., and can be widely changed at the discretion of those skilled in the art. For example, the amount of lactic acid bacteria of the present invention is preferably 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose, more preferably 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu, and 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu is more preferable, for example, 4 × 10 ⁹ to 6 × 10 ¹⁰ cfu is preferable. The food / beverage product or pharmaceutical product of the present invention preferably contains the lactic acid bacterium of the present invention in this amount per dose. The food / beverage product or pharmaceutical product of the present invention is preferably administered to a subject once or more times a day, preferably twice or more times, typically twice a day. The food / beverage product or pharmaceutical product of the present invention is preferably administered continuously, for example, more preferably administered daily. It is preferable that the food or drink or pharmaceutical of the present invention is administered for at least 1 week, preferably 2 weeks or more, for example, 4 weeks or more. The food or drink or pharmaceutical product of the present invention is preferably administered orally. In the present invention, “administration” includes both “intake” of food and drink and “administration” of pharmaceuticals. In the present invention, “oral administration” includes administration by tube feeding via a nasal tube or a gastric fistula tube.

The subject to which the food / beverage product or pharmaceutical product of the present invention is administered is a mammal including humans, domestic animals, pets, experimental (test) animals, and the like. In particular, human subjects are preferred, human subjects with gout and / or hyperuricemia are more preferred, and are not limited to the following: serum uric acid levels of 6 mg / dL or more, for example, 6-10 mg / dL Even more preferred are human subjects exhibiting In one embodiment, a human subject with mild to borderline hyperuricemia with a serum uric acid level of 6-8 mg / dL is preferred. In another embodiment, human subjects exhibiting serum uric acid levels of 7 mg / dL or higher, preferably 7-11 mg / dL (in one example, 7.6 mg / dL to 9.5 mg / dL) are preferred, of which hyperuricemia More preferred are human subjects suffering from symptom and gout. Further, subjects with a correlation between serum uric acid level and purine body intake from food prior to measurement of serum uric acid level (for example, total purine body intake for 3 days) are more preferable as administration (intake) subjects. . A subject who administers the food or drink or pharmaceutical of the present invention has a conversion activity from a purine base to a purine nucleotide (salvage activity), for example, a conversion activity from adenine to adenylic acid, particularly adenine phospho responsible for conversion from adenine to adenylic acid. Suitable for administration to subjects with reduced ribosyltransferase activity.

The present invention also provides a purine nucleotide from a purine base by reacting the conversion agent of the present invention with a purine base in the presence of 5-phospho-D-ribose-1-diphosphate and Mg ²⁺ . A method is also provided. This method may be performed in vivo or may be performed in vitro. When carried out in vivo, this method using the conversion agent of the present invention may use foods and drinks containing the conversion agent of the present invention and may not include medical practice. When carried out in vitro, this method of the present invention is a method for the synthesis (production) of purine nucleotides using a purine base in solution as a substrate. Specifically, this method of the present invention is also preferably a method of generating adenylic acid from adenine. This method of the present invention may also be a method of producing guanylic acid from guanine. Conversion of purine bases by lactic acid bacteria in the presence of 5-phospho-D-ribose-1-diphosphate and Mg ²⁺ can be carried out in the same manner as the above-described test for the conversion of adenine to adenylate. it can.

Furthermore, the present inventors have found that some lactic acid bacteria including Lactobacillus gasseri strains such as Lactobacillus gasseri OLL2959 strain have the ability to take up purines and have high growth ability in the presence of purines. That purine body uptake ability and growth ability in the presence of purine body are correlated, and that such lactic acid bacteria administration (intake) suppresses purine body absorption and contributes to the reduction of serum uric acid level I found. Based on this finding, the action of capturing the purine bodies of the lactic acid bacteria can also be used to reduce the serum uric acid level.

Therefore, in the present invention, by selecting (screening) lactic acid bacteria using as an index the ability of purine bodies to be taken into the cells, lactic acid bacteria having a purine body capturing action can be efficiently obtained (selected). There is provided a method for screening lactic acid bacteria, comprising measuring the amount of purine bodies taken up by a lactic acid bacterium in a medium containing purine bodies, and obtaining (selecting) lactic acid bacteria having a purine body capturing action using the amount as an index. More specifically, the present invention relates to a method of cultivating lactic acid bacteria in a medium containing purine bodies, measuring the amount of purine bodies taken up in the cells, preferably over time, and using this as an index, the lactic acid bacteria having a purine body capturing action. Lactic acid bacteria can be screened by obtaining (selecting). Thus obtained lactic acid bacteria having a purine-capturing action have a high probability of having a serum uric acid level-reducing action. Here, the amount of purine bodies taken up by lactic acid bacteria is large, that is, that the lactic acid bacteria cells have a purine body-capturing action, the large amount of purine bodies in the living body, particularly in the intestinal tract, is captured by the lactic acid bacteria in the intestinal tract. It means that absorption of purines from the intestinal tract is suppressed by being removed from the environment. In addition, since it is known that lactic acid bacteria are excreted without being absorbed from the digestive tract, the purine body captured by the lactic acid bacteria escapes absorption from the intestinal tract and is discharged out of the body together with the lactic acid bacteria. Therefore, in the present invention, lactic acid bacteria are cultured in a medium containing purine bodies, the amount of purine bodies taken up is measured, and lactic acid bacteria having a purine body-capturing action are selected as an index. By acquiring (selecting) a lactic acid bacterium having a body-capturing action as a lactic acid bacterium having a serum uric acid level reducing action, a lactic acid bacterium having a serum uric acid level reducing action can be screened.

In the present invention, a lactic acid bacterium having purine body uptake ability exhibits a high growth ability in the presence of the purine body so as to correlate with the high uptake capacity of the purine body. Therefore, in the present invention, in addition to the selection using the purine body uptake ability as an index, the enhancement of the growth ability in the presence of the purine body of the selected lactic acid bacteria was confirmed, and this was used as an index. It is also possible to acquire (select) lactic acid bacteria having a body-capturing action with higher accuracy. That is, in the present invention, the amount of growth in a medium containing purine bodies of lactic acid bacteria having the ability to take up purine bodies is measured, and this is used as an indicator together with the amount of purine bodies taken up as described above to capture purine bodies. The lactic acid bacteria can be screened by obtaining (selecting) the lactic acid bacteria having. Furthermore, the present invention cultivates lactic acid bacteria in a medium containing purine bodies, measures the growth amount of the bacteria over time, and uses it as an index along with the amount of purine bodies taken up as described above, Lactic acid bacteria having a function of reducing serum uric acid level are obtained by selecting (selecting) lactic acid bacteria having a capturing action of purine bodies obtained as a lactic acid bacterium having a function of reducing serum uric acid level. Can also be screened. However, measurement of the amount of proliferation in the presence of purines and selection using the measurement may be performed, or such selection may not be performed.

The lactic acid bacterium to be subjected to the screening method of the present invention is not particularly limited, but is preferably a bacterium belonging to the genus Lactobacillus. Examples of the genus Lactobacillus include the same as exemplified above, and Lactobacillus gasseri is particularly preferable. Arbitrary strains of lactic acid bacteria to be subjected to the screening method of the present invention are preferably used for screening after culturing in an appropriate medium (for example, MRS medium) to adjust the concentration. The medium used for the screening may be any medium that can grow Lactobacillus gasseri, but a minimal medium or a medium in which purine is added or a part of the components is replaced with purine is preferable. Examples of particularly preferable minimal media are listed in Table 5.

The lactic acid bacterium to be subjected to the screening method of the present invention is not particularly limited, but is preferably a bacterium belonging to the genus Lactobacillus. Examples of the genus Lactobacillus include the same as exemplified above, and Lactobacillus gasseri is particularly preferable. Any strain of these lactic acid bacteria is preferably used for screening after culturing in an appropriate medium (for example, MRS medium) to adjust the concentration. The medium used for the screening may be any medium that allows the lactic acid bacteria to be used, for example, Lactobacillus gasseri, to grow, but a minimal medium or a purine body is added or a part of the components is replaced with the purine body. A medium is preferred. Examples of particularly preferable minimal media are listed in Table 5.

The purine contained in the medium may be a purine base, a purine nucleoside, and / or a purine nucleotide. Examples of purine bases include, but are not limited to, adenine, guanine, hypoxanthine and xanthine, with adenine being particularly preferred. Examples of purine nucleosides include, but are not limited to, adenosine, guanosine, inosine and xanthosine, with adenosine being particularly preferred. Examples of purine nucleotides include, but are not limited to, adenylic acid (AMP), guanylic acid (GMP), inosinic acid (IMP) and xanthylic acid (XMP), with adenylic acid being particularly preferred. The purine may be labeled with a radioisotope.

For the measurement of the amount of purine bodies incorporated in lactic acid bacteria, purine bodies labeled with a label capable of quantitative detection, for example, radioactive isotopes or fluorescent substances, are used as part or all of the purine bodies contained in the medium. A medium using a labeled purine body can be preferably used. As the radioisotope, for example, ¹⁴ C is preferable. The amount of purine bodies taken up by lactic acid bacteria can be determined by, for example, cultivating lactic acid bacteria in a medium containing purine bodies, stopping the reaction by adding TFA (trifluoroacetic acid), etc. after culturing for a certain period of time, The body can be quantified based on the detection of the activity of the labeled substance, and can be measured or determined by comparing with the same activity of the bacterial cells at the start of culture. When the amount of purine bodies taken up by lactic acid bacteria is significantly increased compared to the start of culture, the lactic acid bacteria can be determined to have the ability to take up the purine bodies into the cells (purine body uptake ability). . In the present invention, lactic acid bacteria determined to have purine body uptake ability as described above can be obtained (selected) as lactic acid bacteria having purine body capturing activity. And the lactic acid bacteria selected as a lactic acid bacterium which has a purine body capture | acquisition effect | action can be further acquired (selected) as a lactic acid bacterium which has the effect | action of a serum uric acid value reduction, or its candidate. In addition, it is preferable that the culture | cultivation time of lactic acid bacteria is the time to the arbitrary time in the induction | guidance | derivation phase or logarithmic growth phase of a growth curve. For example, lactic acid bacteria can be cultured 30 minutes and 60 minutes after the start of culture, and purine uptake ability and the like can be measured. At this time, usually, the radioactivity of the purine labeled with a radioisotope may be measured using a liquid scintillation counter.

The amount of growth of lactic acid bacteria in the presence of purine bodies is determined, for example, by culturing lactic acid bacteria in a medium containing purine bodies, and the turbidity (typically absorbance at 650 nm) of the medium after the start of culture and after a certain period of culture. Measurement and determination can be performed by measuring and calculating the difference between the two. When the increase in turbidity when cultured in the presence of purines was significantly increased compared to when cultured in the absence of purines, the lactic acid bacteria were enhanced in the presence of purines. It can be determined that it exhibits proliferative ability. When a lactic acid bacterium has an ability to take up purines and exhibits enhanced proliferation ability in the presence of purines, it means that the lactic acid bacteria can highly assimilate the purines, that is, the lactic acid bacteria The possibility of having a high level of capture and, in turn, the effect of reducing serum uric acid levels is confirmed. In addition, it is preferable that the culture | cultivation time of lactic acid bacteria is the time to the arbitrary time in the logarithmic growth phase of a growth curve. For example, lactic acid bacteria can be cultured until 4 hours and 6 hours after the start of the culture, and the proliferation ability in the presence of purines can be measured.

In the above measurement, lactic acid bacteria are preferably inoculated and cultured at 0.8 × 10 ⁷ to 3 × 10 ⁷ cfu per 1 mL of the medium. The culture conditions for lactic acid bacteria are not particularly limited, but anaerobic culture is preferably performed at 30 to 39 ° C, preferably 36 to 38 ° C.

In the present invention, it is also preferable to further test that the lactic acid bacteria selected as described above have an action of reducing serum uric acid level, for example, according to the method described in Examples below. For example, the lactic acid bacteria selected as described above are administered to a subject once or multiple times, the serum uric acid level is measured, and the presence or absence of a change in serum uric acid level (reduction in serum uric acid level) is determined. It can be determined whether or not the lactic acid bacteria selected as described above have a serum uric acid level reducing action.

The lactic acid bacteria selected as described above have a purine body uptake ability and preferably a high growth ability in the presence of the purine body, that is, a high purine body capturing action. Such lactic acid bacteria typically have an effect of reducing serum uric acid levels. The lactic acid bacteria selected as described above exhibit the purine body uptake ability and high growth ability (that is, high purine body utilization ability) in the presence of purine bodies in the living body (typically in the intestinal tract). As a result, the serum uric acid level can be reduced by capturing and reducing a large amount of purine bodies in the living body (typically in the intestinal tract) and reducing the absorption amount of purine bodies.

Further, it is possible to provide a purine body scavenger containing as an active ingredient, preferably a purine body scavenger for oral administration, using lactic acid bacteria having purine body scavenging action, which can be obtained by the above screening method. it can. This purine capturing agent may contain a carrier or additive that is acceptable for oral administration in addition to lactic acid bacteria having a purine capturing effect, preferably Lactobacillus gasseri. The purine-capturing agent may be a drug or composition containing lactic acid bacteria having purine-capturing activity, or a fermented product, a culture, or a concentrate thereof produced using the fungus. It may be a dried product or a drug or composition containing the same. In addition, it is preferable that the lactic acid bacteria which have a purine body capture | acquisition action contained in this purine body capture | acquisition agent are living cell bodies. As described above, the purine body scavenger has an action of reducing purine bodies in the intestinal tract by taking up the purine bodies of lactic acid bacteria, and is therefore suitable for reducing purine bodies in the intestinal tract, and hence for reducing serum uric acid levels. Can be used.

Preferable examples of the lactic acid bacteria having the purine capturing action as described above include, but are not limited to, Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain, Lactobacillus gasseri P14054ME002 strain and the like. It is done. Lactobacillus gasseri OLL2959 strain is homolactic fermentable and has no gas production ability. The Lactobacillus gasseri OLL2959 strain, dated March 31, 2006 (original deposit date), is the National Institute of Technology and Evaluation of Microorganisms (NPMD) (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture, Japan) 8 Deposited in room 122 (zip code 292-0818) under the deposit number NITE P-224, it was transferred to a deposit under the Budapest Treaty (international deposit) on November 21, 2007, and the deposit number is NITE BP- It has been changed to 224.

The purine body scavenger can also be used in combination with food or drink or medicine. Therefore, the purine body capture | acquisition agent for using it in combination with food-drinks or a pharmaceutical is also provided.

Using the above purine body scavenger, a food or drink or a medicine containing the purine body scavenger can also be provided. These foods and beverages, in the subject who administers (ingested) it, actively incorporates and assimilates purines into the cells, thereby reducing purines in the intestinal tract and effectively increasing serum uric acid levels. A reduction can be brought about. Therefore, these foods and drinks and pharmaceuticals can be used for reducing purines in the intestinal tract. The “purine body in the intestinal tract” here does not include purine bodies retained by bacteria (such as lactic acid bacteria), fungi, viruses, and cells of the subject present in the intestinal tract. These foods and drinks and pharmaceuticals may be used for reducing serum uric acid levels based on the reduction of purines in the intestinal tract. Foods and beverages and pharmaceuticals containing purine-capturing agents can be suitably used, for example, for the prevention, treatment, improvement or reduction of symptoms of gout and hyperuricemia.

"Food and drink" here is not particularly limited, but includes beverages, foods and functional foods. The type of food or drink is not particularly limited. For example, drinks include fermented milk (drink yogurt, etc.), lactic acid bacteria drinks, milk drinks (coffee milk, fruit milk, etc.), tea-based drinks (green tea, tea, oolong tea, etc.) , Fruit / vegetable beverages (beverages containing orange, apple, grape and other fruit juices, tomatoes, carrots and other vegetable juices), alcoholic beverages (beer, sparkling wine, wine, etc.), carbonated beverages, soft drinks, water-based beverages Examples of suitable beverages include drink yogurt, lactic acid bacteria beverages, milk beverages, water-based beverages, etc., and particularly preferred beverages include drink yogurt. As for the production methods of various beverages, existing reference books such as “Latest Soft Drinks” (2003) (Kotsu Co., Ltd.) can be referred to. Examples of the food include fermented milk (set type yogurt, soft yogurt, cheese, etc.), dairy products, confectionery, and instant food. Suitable foods include yogurts such as set type yogurt and soft yogurt, Confectionery, cheese and the like can be mentioned, and particularly suitable beverages include yogurt such as set-type yogurt and soft yogurt. You can refer to existing reference books for the production methods of various foods.

Fermented milk such as yogurt containing a lactic acid bacterium having a purine-capturing action may contain other microorganisms such as lactic acid bacteria that may or may not have a purine-capturing action. You may manufacture by adding the lactic acid bacteria which have the capture | acquisition effect | action of a purine body to the dairy product and fermented milk manufactured using the starter. In addition, the dairy product and fermented milk using a starter can be manufactured in accordance with a conventional method. For example, yogurt can be produced by mixing a starter with milk or a dairy product cooled after heating, mixing, homogenizing, and sterilizing, fermenting and cooling. As a preferred embodiment, the use of lactic acid bacteria having a purine-capturing action in the production of dairy products such as yogurt and cheese and fermented milk (preferably adding (compounding) the lactic acid bacteria to dairy products, fermented milk or its raw materials. And a particularly preferred embodiment provides the use of the lactic acid bacteria in the production of yogurt. Furthermore, there is also provided a method for reducing purine bodies based on the action of capturing purine bodies of lactic acid bacteria in fermented milk and dairy products such as yogurt and cheese, using lactic acid bacteria having purine body capturing action as an active ingredient.

In particular, functional foods are preferred as food and drink. The types and preferred examples of functional foods are the same as those described for functional foods related to foods and drinks using the conversion agent.

This functional food is particularly useful in reducing serum uric acid levels by reducing purine bodies in the intestinal tract. Functional foods are used to reduce serum uric acid levels, especially serum uric acid with the reduction of purine bodies in the intestinal tract by the uptake of purine bodies by lactic acid bacteria and the promotion of growth of lactic acid bacteria, and the resulting absorption of purine bodies in the intestinal tract It can be suitably used for reducing the value.

Foods and drinks such as this functional food (in Japan, preferably food for specified health use, conditional tokuho [food for specified health use] or functional indication food) may be used for reducing purines in the intestinal tract. It may be for suppressing or alleviating a decrease in serum uric acid level or an increase in serum uric acid level, and may be described or displayed to that effect. Use of a lactic acid bacterium having a purine-capturing action in the production of such a functional food (preferably including adding (compounding) the lactic acid bacterium to the functional food or its raw material) is also provided.

The dosage form or shape of this functional food is the same as that described for the functional food relating to foods and drinks using the above-mentioned conversion agent.

The food components other than the lactic acid bacteria contained in the food and drink here are the same as those described for the food and drink using the conversion agent.

In addition to the lactic acid bacteria or purine body capturing agent, the functional food containing the lactic acid bacteria or purine body capturing agent having the purine body capturing action may contain an orally acceptable carrier or additive. Carriers include, for example, water, organic solvents acceptable for oral administration, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, Arabic Examples include gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and surfactants that are acceptable for oral administration. Examples of the additive include a binder, an excipient, a lubricant, a disintegrant, a wetting agent, a stabilizer, a buffering agent, a corrigent, a preservative, and a coloring agent. These carriers or additives can be used singly or in combination of two or more, and can be appropriately used depending on the dosage form of the preparation. In addition, the functional food of the present invention may further contain other functional ingredients.

In addition to the lactic acid bacteria or purine body scavenger of the present invention, the pharmaceutical (pharmaceutical composition) containing a lactic acid bacterium or purine body scavenger having a purine body capturing action is a pharmaceutically acceptable carrier or additive, particularly Orally acceptable carriers or additives may be included. Examples of carriers and additives are the same as those described above for pharmaceutical products using the conversion agent. These carriers or additives can be used singly or in combination of two or more, and can be appropriately used depending on the dosage form of the preparation. In addition, this pharmaceutical may further contain other pharmacological components.

It is preferable to administer the above medicines orally. The pharmaceutical may be in any dosage form such as tablets, granules, powders, pills, capsules and other solid preparations, gels, or liquid preparations such as liquids, suspensions and syrups.

For lactic acid bacteria that have purine-capturing action, purine-capturing agents, foods and drinks or pharmaceuticals, the dose (intake) takes into account the age and weight of the subject to be administered (intake), the route of administration, the number of administrations, etc. However, a wide range of changes can be made at the discretion of those skilled in the art. Therefore, the dose of lactic acid bacteria (Lactobacillus gasseri, etc.) is not particularly limited in lactic acid bacteria having purine body capturing action, purine body capturing agents, foods and drinks or pharmaceuticals, but for example, 1 × 10 per dose ^{An amount of 5} to 1 × 10 ¹¹ cfu is preferable, an amount of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, an amount of 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu is more preferable, for example, 4 An amount of × 10 ⁹ to 6 × 10 ¹⁰ cfu is particularly preferable. The purine-capturing agent, food or drink, or pharmaceutical preferably contains lactic acid bacteria having a purine-capturing action in an amount of 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose, and 1 × 10 ⁸ to 1 More preferably, it is contained in an amount of × 10 ¹⁰ cfu, more preferably 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu, for example, 4 × 10 ⁹ to 6 × 10 ¹⁰ cfu. It is particularly preferred to contain it in an amount.

In one embodiment, a lactic acid bacterium having a purine body-capturing action, a purine body-capturing agent, a food or drink, or a pharmaceutical is once or more a day, preferably twice or more a day, more preferably twice a day. (Or consumed by the consumer). A lactic acid bacterium having a purine body-capturing action, a purine body-capturing agent, a food or drink, or a pharmaceutical product may be continuously administered to a subject, for example, daily. In this case, the lactic acid bacterium, purine capture agent, food or drink or pharmaceutical having a purine capture activity is administered to the subject for at least 1 week, preferably 2 weeks or more, more preferably 4 weeks or more. In the purine capturing agent, food / beverage product or pharmaceutical, when administered continuously to a subject, the dose of the lactic acid bacterium of the present invention is preferably an amount of 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu is more preferable, for example, 4 × 10 ⁹ to 6 × 10 ¹⁰ cfu. Is particularly preferred.

In another embodiment, the lactic acid bacterium having a purine body-capturing action, the purine body-capturing agent, the food or drink, or the pharmaceutical product may be a single dose. In the purine capturing agent, food and drink or pharmaceutical, when administered to a subject once, the dose of lactic acid bacteria having purine capturing activity is 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. The amount is preferably 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu, more preferably 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu, for example, 4 × 10 ⁹ to 6 × 10 ¹⁰ cfu. Is particularly preferred. The lactic acid bacteria having a purine-capturing action, purine-capturing agent, food and drink, or pharmaceutical are preferably administered orally (orally ingested).

Here, “administration” includes both “ingestion” generally used for food and drink and “administration” used for pharmaceutical products. “Oral administration” includes administration by tube feeding via a nasal tube or a gastric fistula tube, in addition to oral administration or ingestion. Accordingly, an oral preparation that can be used for such oral administration is also provided. Therefore, in a preferred embodiment, an oral preparation for reducing purine bodies in the intestinal tract and reducing serum uric acid level is also provided, which contains a lactic acid bacterium or purine body capture agent having a purine body capture action.

In lactic acid bacteria, purine capturing agents, foods and drinks or pharmaceuticals having a purine capturing effect, the subjects to be administered are mammals including humans, domestic animals, pets, experimental (test) animals, etc. The body is preferred and human subjects with gout and / or hyperuricemia are more preferred, including, but not limited to, humans exhibiting serum uric acid levels of 6 mg / dL or more, such as 6-10 mg / dL More preferred are subjects. In one embodiment, a human subject with mild to borderline hyperuricemia with a serum uric acid level of 6-8 mg / dL is preferred. In another embodiment, human subjects exhibiting serum uric acid levels of 7 mg / dL or higher, preferably 7-11 mg / dL (in one example, 7.6 mg / dL to 9.5 mg / dL) are preferred, of which hyperuricemia More preferred are human subjects suffering from symptom and gout. Further, subjects with a correlation between serum uric acid level and purine body intake from food prior to measurement of serum uric acid level (for example, total purine body intake for 3 days) are more preferable as administration (intake) subjects. .

That is, the following aspects are also provided.
[a1] A screening method for lactic acid bacteria, comprising measuring the amount of purine bodies taken up by a lactic acid bacterium in a medium containing purine bodies, and selecting lactic acid bacteria having a purine body capturing action using the amount as an index.
[a2] The method according to [a1], wherein the purine is at least one selected from the group consisting of adenine, adenosine, and adenylic acid.
[a3] The method according to [a1] or [a2], wherein the purine is adenine.
[a4] The method according to any one of [a1] to [a3], wherein the purine is labeled with a radioisotope.
[a5] measuring a growth amount of the lactic acid bacterium in a medium containing a purine body, and selecting a lactic acid bacterium having a purine body capturing action as an index together with the uptake amount of the purine body [a1] to The method according to any one of [a4].
[a6] The method according to any one of [a1] to [a5], wherein the lactic acid bacterium is Lactobacillus gasseri.

[a7] A lactic acid bacterium having a purine-capturing action obtained by the method according to any one of [a1] to [a6].
[a8] A purine trapping agent comprising, as an active ingredient, a lactic acid bacterium having a purine trapping action obtained by the method according to any one of [a1] to [a6].
[a9] The purine body scavenger according to [a8], which is for reducing serum uric acid level.
[a10] The purine capturing agent according to [a8] or [a9], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2959 strain (Accession No. NITE AP-224).

[a11] A food or drink or a pharmaceutical comprising the purine-capturing agent according to any one of [a8] to [a10].
[a12] The food or drink or medicine according to [a11], which is for reducing purines in the intestinal tract.
[a13] The food or drink or pharmaceutical product according to [a11] or [a12], wherein a human subject having a serum uric acid level of 6 to 8 mg / dL is to be administered.
[a14] The food or drink or pharmaceutical according to any one of [a11] to [a13], wherein the lactic acid bacterium is contained at 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu per dose.

Hereinafter, the present invention will be described more specifically using examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
(1) Preparation of lactic acid bacteria Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri ATCC 33323 strain were used as lactic acid bacteria. The ATCC 33323 strain, which is a reference strain of Lactobacillus gasseri, is available from ATCC (American Type Culture Collection) as catalog number ATCC 33323. In addition, RIKEN BRC Microbial Materials Development Office (Japan) Collection of Microorganisms) (RIKEN BRC-JCM) is also available under catalog number JCM 1131T.

Lactic acid bacteria Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri ATCC 33323 strain were each inoculated into MRS medium in two falcon tubes and cultured at 37 ° C. for 16-20 hours. The culture was collected by centrifugation at 6,000 rpm × 10 minutes at 4 ° C., and the resulting lactic acid bacteria were combined into one. This was suspended by adding 10 mL of buffer solution, and then washed by centrifuging at 4 ° C. and 6,000 rpm × 10 minutes to collect the cells twice. Then, a buffer solution was added, and a cell suspension (live cells) diluted to approximately 1 × 10 ¹⁰ cfu / mL was used below.

(2) Measurement of salvage activity for converting adenine to adenylic acid (AMP) About the lactic acid bacteria prepared in (1), the activity to convert adenine to adenylic acid (AMP) was measured using adenine as a substrate.

For each of the Lactobacillus gasseri OLL2959 strain and ATCC 33323 strain, reaction solutions (100 μL each) were prepared with the following composition. Lactic acid bacteria were suspended after preparing a solution (buffer solution) containing MgCl ₂ , PRPP, adenine and Tris-HCl. The concentration of lactic acid bacteria in the reaction solution was approximately 5 × 10 ⁹ cfu / mL.

After the reaction solution was prepared, the lactic acid bacteria were cultured by incubating in a water bath with shaking at 37 ° C. At 0, 15, 30, and 60 minutes, an equal amount of 5% TFA (trifluoro) The conversion reaction was stopped by adding acetic acid).

The reaction solution after stopping the reaction was centrifuged at 4 ° C., 15,000 rpm × 10 minutes. The supernatant was collected, centrifuged again at 4 ° C. and 15,000 rpm × 10 minutes using a centrifugal filter unit, 0.22 μm Ultrafree-MC (micon), and microfiltered.

The obtained filtrate was subjected to HPLC analysis. The HPLC analysis conditions used are as follows.
-Mobile phase: A: 20 mM phosphate buffer (pH 7.5)
B: 40 mM phosphate buffer (pH 7.5) / acetonitrile (1: 1)
・ Column: SHISEIDO CAPCELL PAK C18 MG2 (2.0mm id × 150mm)
・ Flow rate: 0.2mL / min ・ Temperature: 40 ℃
・ Injection volume: 5μl
・ Detection wavelength: 254nm (UV)
-Gradient A / B (min): 100/0 (0 min)-100/0 (5 min)-80/20 (20 min) [%]
Adenine and AMP in the reaction solution were quantified by measuring the area under the peak of the HPLC chart. The conversion rate of adenine to AMP at each reaction stop time was calculated by the following formula.
Conversion rate (%) = (Area value under the peak of AMP in the test area−Area value under the peak of the AMP in the control area) / (Area area under the peak of adenine at the 0 minute of the test area−At the 0 minute time point in the control area) Adenine peak area value)

The change with time of the obtained conversion rate is shown in Table 2 and FIG. When ATCC 33323 strain was used in the reaction, almost no conversion activity from adenine to AMP was shown. On the other hand, when OLL2959 strain was used, the amount of adenine decreased with a rapid increase in the amount of AMP, and most of the adenine was converted to AMP after 60 minutes. As shown in Table 2 and FIG. 1, the OLL2959 strain had an overwhelmingly higher conversion activity from adenine to AMP than the ATCC 33323 strain.

From the above results, it was shown that the Lactobacillus gasseri OLL2959 strain has a very high ability to convert adenine into AMP. That is, it was considered that Lactobacillus gasseri OLL2959 strain has high adenine phosphoribosyltransferase (APRT) activity that catalyzes the conversion of adenine to AMP (salvage pathway).

(3) Measurement of activity to convert guanine to guanosine (GMP) In addition to the pathway to convert adenine to AMP, the salvage pathway includes a pathway to convert guanine to guanylate (GMP), hypoxanthine to inosine acid (IMP) There are also pathways to convert xanthine to xanthylic acid (XMP). In mammals such as humans and lactic acid bacteria, the enzyme APRT that catalyzes the conversion of adenine to AMP is considered to be involved in the conversion of guanine to GMP. On the other hand, hypoxanthine-guanine phosphoribosyltransferase is involved in the conversion of hypoxanthine to IMP and xanthine to XMP in mammals and lactic acid bacteria. In lactic acid bacteria, xanthine phosphoribosyltransferase is also known to be involved in the conversion of xanthine to XMP and guanine to GMP.

Therefore, the conversion activity of guanine to GMP was also measured. At this time, the conversion activity of guanine to GMP was measured in the same manner as in (2) above, except that adenine was changed to guanine and AMP was changed to GMP. The results are shown in Table 3 and FIG. The ATCC 33323 strain showed a very low conversion activity of guanine to GMP, whereas the OLL2959 strain showed a relatively high ability to convert guanine to GMP (Fig. 2).

[Example 2]
(1) Measurement of purine nucleosidase activity As shown in Patent Document 1, the Lactobacillus gasseri OLL2959 strain has high resolution for purine nucleosides such as inosine and guanosine. Therefore, purine nucleosidase activity was measured as the activity of purine nucleoside to decompose into purine bases.

Specifically, the OLL2959 strain was suspended in a final concentration of 0.2 mM adenosine solution prepared using 100 mM phosphate buffer (pH 7.0) to a concentration of approximately 5 × 10 ⁹ cfu / ml. Liquid. Here, the reaction was performed at 37 ° C. for 120 minutes, and adenosine and adenine were measured as area values by HPLC analysis. As a comparative control, ATCC 33323 strain was used and tested in the same manner as described above. The measurement results were expressed as the conversion rate (area comparison) of adenosine (purine nucleoside) to adenine (purine base). This conversion rate was calculated in the same manner as in Example 1 (2).

Furthermore, the same test was performed using inosine and guanosine instead of adenosine. The result is shown in FIG. The Lactobacillus gasseri OLL2959 strain had a markedly higher conversion rate to purine bases, that is, the purine nucleosidase activity was higher than any Lactobacillus gasseri ATCC 33323 strain.

(2) Measurement of 5′-nucleotidase (5′-NT) activity Purine nucleotides converted from purine bases by salvage activity are converted to nucleosides by 5′-nucleotidase activity. Therefore, 5′-nucleotidase activity was measured for Lactobacillus gasseri OLL2959 strain.

Suspend OLL2959 strain cells in a solution of final concentration 5 mM MgCl ₂ , 0.2 mM AMP prepared using 25 mM Tris-HCl buffer (pH 7.5) so that the concentration is approximately 5 × 10 ⁹ cfu / ml. The reaction solution was used (test section). In the control group, a reaction solution was prepared using the above buffer instead of 0.2 mM AMP. Here, the reaction was carried out at 37 ° C. for 120 minutes, and AMP and adenosine were measured as area values by HPLC analysis. As a comparative control, ATCC 33323 strain was used and tested in the same manner as described above. The measurement results were expressed as the conversion rate (area comparison) of AMP (purine nucleotide) to adenosine (nucleoside). This conversion rate was calculated in the same manner as in Example 1 (2).

Furthermore, a similar test was performed using IMP and GMP instead of AMP. The results are shown in Table 4 and FIG. Lactobacillus gasseri OLL2959 strain had a significantly lower conversion rate to purine nucleoside for any purine nucleotide compared to Lactobacillus gasseri ATCCAT33323 strain. That is, the 5′-nucleotidase activity was remarkably low.

From the above measurement results of 5′-nucleotidase activity, the measurement result of salvage activity in Example 1, and the measurement result of (1) purine nucleosidase activity in Example 2, the Lactobacillus gasseri OLL2959 strain It was shown that purine metabolism activity is high, and that the metabolic activity is biased in the direction of positively generating and accumulating nucleotides that can be used as energy.

[Example 3]
Human subjects suspected of mild to borderline hyperuricemia were continuously ingested with Lactobacillus gasseri OLL2959, and the effect on uric acid levels was examined by a placebo-controlled double-blind comparative study (human study) ).

Adult males aged 35 and over who had a uric acid level of 6-8 mg / dL in the test before the start of the study: 14 people (mean age: 44.3 years) were placed in the placebo group so that there was no significant difference between the uric acid level and age And assigned to 2 groups of active group. The placebo group received 2 yogurts (85 g / piece) / day, 4 weeks, without the Lactobacillus gasseri OLL2959 strain. In the active group, yoghurt given to the placebo group was fed with Lactobacillus gasseri OLL2959 strain at 1 × 10 ⁸ cfu / g at 2 (85 g / day) for 4 weeks. In addition, 2 yogurts per day were taken twice after breakfast, either breakfast, lunch, or dinner.

For each subject, blood tests were performed at the start of the test (before the test meal intake), 2 weeks later and 4 weeks later (the test meal intake period), and the serum uric acid level was measured. Calculate the amount of change in serum uric acid value at each time point compared to the value of serum uric acid at the start of the study, and perform a statistical analysis of the change in the amount of serum uric acid value during the study period using repeated measures two-way analysis of variance. It was. The results are shown in FIG.

As shown in FIG. 5, the serum uric acid level was significantly lower in the active group than in the placebo group (p （= 0.042). That is, it was shown that Lactobacillus gasseri OLL2959 strain has an effect of reducing serum uric acid level.

[Example 4]
In this example, patients undergoing outpatient treatment for hyperuricemia and gout were ingested with Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain, and the effect on serum uric acid levels was evaluated. A randomized placebo-controlled double-blind parallel group study was performed.

(1) Production of test food The test food (test food or control food) to be ingested by the subject was produced as follows. The test food containing Lactobacillus gasseri OLL2959 is composed of two types of bacteria, Lactobcillus bulgaricus yogurt starter and Streptococcus thermophilus, and Lactobacillus gasseri OLL2959, which is an active ingredient. A yogurt containing (8.5 × 10 ⁷ cfu / mL) was prepared, and a beverage in a PET bottle filled with 100 g per bottle was produced.

The control food that does not contain Lactobacillus gasseri OLL2959 does not contain Lactobacillus gasseri OLL2959, which contains two types of bacteria, the yogurt starter Lactobacillus bulgaricus and Streptococcus thermophilus Yogurt was prepared in the same manner, and a beverage with a plastic bottle filled with 100 g per bottle was produced. The yogurt of each food was prepared by blending the raw materials, Lactobacillus gasseri OLL2959 strain (test food only), the above-mentioned yogurt starter, dairy product, stabilizer (pectin), flavor, and water. .

After the production of the test food, the number of bacteria was measured for any one product. Specifically, the yogurt of the test food was inoculated into the BL medium, and after 3 days of aerobic culture, the number of colonies on the medium was counted to confirm that the number of bacteria satisfied the above level.

(2) Ingestion test For patients (subjects) over 20 years of age who have developed hyperuricemia and gout who have consented to participate in the study and who are taking uric acid-lowering drugs, a preliminary examination including serum uric acid level measurement is conducted. Yes, the uric acid-lowering drug was administered for 4 weeks.

After 4 weeks of withdrawal, subjects were subjected to a pre-intake test including serum uric acid level measurement. In addition, a dietary survey of the subjects was conducted for 3 days before the pre-intake test.

Subjects with serum uric acid levels after withdrawal were more than 7 mg / dL. Based on serum uric acid levels after withdrawal, serum uric acid levels and ages in pre-intake tests were significant in the test food intake group and the control food intake group The subjects were assigned so that there was no difference. There were no significant differences between groups in height, weight, BMI, blood pressure systole, blood pressure diastole, and pulse measurements.

In accordance with this assignment, after the pre-ingestion test, subjects ingest 2 test foods (test food containing 8.5 x 10 ⁷ cfu / mL of OLL2959 strain or control food not containing OLL2959 strain) at 8 weeks / day. (Test food intake period). During the test food intake period, subjects continued to withdraw from the drug. A test including measurement of serum uric acid level was performed 8 weeks after the start of intake (8-week test). A meal survey was also conducted for 3 days before the 8-week test.

A meal survey was conducted on all subjects for 3 days prior to each of the pre-intake test (week 0) and the 8-week test. Subjects were asked to report the contents of breakfast, lunch, dinner and snacks for 3 days in a diary and / or photographed and reported. Based on this reported dietary content, a registered dietitian calculated energy, protein, lipid, carbohydrate, and salt intake. Moreover, the purine body amount was computed based on the report of each test subject.

Population excluding subjects who dropped out during intake period and less than 90% of test food intake rate, and subjects who were out of correlation with serum uric acid level and purine intake (17; test food intake group: 9; control food) Intake group: Eight patients (serum uric acid level after withdrawal: 7.6 mg / dL to 9.5 mg / dL) were stratified. According to the results up to the time of this 8-week test, the test food intake group showed a tendency for no significant increase in serum uric acid level even during the withdrawal period (FIG. 6). It was shown that the serum uric acid level significantly decreased at p <0.05 compared with the food intake group (p = 0.0342, Mann-Whitney U test).

From the above results, it was shown that ingestion of Lactobacillus gasseri OLL2959 strain suppressed the increase in serum uric acid level caused by food-derived purines even in patients who developed hyperuricemia and gout .

[Example 5] Evaluation test of purine body uptake ability Using purine bodies labeled with a radioisotope (RI), the purine body uptake capacity of Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain was evaluated. .

The Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain, dated March 31, 2006 (original deposit date), is a patent microbiology deposit center (NPMD) of the National Institute for Product Evaluation and Technology (NPMD) (Kisarazu City, Chiba Prefecture, Japan) Kazusa Kamashika 2-5-8 Room 122 Postal Code 292-0818) was deposited under the deposit number NITE P-224, and was transferred to a deposit under the Budapest Treaty (international deposit) on November 21, 2007 The accession number has been changed to NITE BP-224. Lactobacilli gasseri OLL2959 strain was inoculated into MRS medium (Lactobacilli MRS Broth, Difco) and cultured at 37 ° C. for 16-20 hours (4-7 × 10 ⁸ cfu / ml) was used as follows.

Minimal medium (DM medium; Table 5): 0.1 mL of adenylate (AMP), adenosine, or adenine ( ¹⁴ C-AMP, ¹⁴ C-adenosine, ¹⁴ C-adenine, respectively) labeled with radioactive isotope ¹⁴ C Final concentration: 20 μM was added, and then the Lactobacillus gasseri OLL2959 strain culture solution prepared above was inoculated at 2 wt% (0.002 mL: 0.8 to 1.4 × 10 ⁶ cfu) at 37 ° C. And anaerobic culture for 30 minutes.

After that, TFA solution (trifluoroacetic acid, 5%) is added to these media, then the cells are washed with physiological saline, and then irradiated with a liquid scintillation counter (ALOC, LSC-6100). Activity was measured. As a control (0 min), a TFA solution (5%) was added immediately after sample preparation, and then the cells were washed with physiological saline, and then the radioactivity was measured in the same manner as described above. The results are shown in FIG. In FIG. 7, the unit of radioactivity (vertical axis) indicating the amount of ¹⁴ C-labeled purine body in the microbial cells is disintegrations per minute (dpm), which is the number of radioactive materials that disintegrate per minute. In addition, when it culture | cultivated for 60 minutes using DM culture medium, it confirmed that the number of living bacteria at the time of a test start and the end of a test did not change significantly.

From the above results, the Lactobacillus gasseri OLL2959 strain has the ability to take up adenylic acid (AMP), adenosine, and adenine of purines into the cells (uptake of purines), especially uptake of adenine into the cells. It was shown that the ability (purine body uptake ability) was high (FIG. 7).

[Example 6] Evaluation test of proliferation ability in the presence of purine bodies Lactobacillus gasseri OLL2959 strain was cultured in the presence of purine bodies, and the proliferation ability in the presence of purine bodies was evaluated.

DM medium (Table 5): 1 mL of adenylate (AMP), adenosine, or adenine was added as a purine to a final concentration of 400 μM, and then Lactobacillus gaselli OLL2959 strain prepared in Example 5 Was inoculated at 4 wt% (0.04 mL: 1.6 to 2.8 × 10 ⁷ cfu) and anaerobically cultured at 37 ° C. The turbidity (absorbance at 650 nm) of the medium was measured after 0, 4, and 6 hours from the start of the culture. As a comparative control, Lactobacillus gasseri OLL2959 strain was cultured in the same manner except that no purine was added to the minimal medium, and the turbidity of the medium was measured. The results are shown in FIG.

From the above results, the Lactobacillus gasseri OLL2959 strain may be enhanced in the presence of adenylic acid (AMP), adenosine, or adenine, particularly in the presence of adenine. (Figure 8).

[Example 7] Comparative test of adenine uptake ability and growth ability in the presence of adenine Lactobacillus gasseri OLL2959 and other Lactobacillus gasseri strains were cultured in the presence of adenine, and each adenine uptake ability and adenine The proliferative ability in the presence of

Lactobacillus gasseri strains P14054ME001 and P14054ME002 were used as other Lactobacillus gasseri strains. In addition, in Lactobacillus gasseri P14054ME001 strain and P14054ME002 strain, when cultured in MRS medium (Lactobacilli MRS Broth, Difco) without addition of purine bodies for 20 hours, the growth ability of each is Lactobacillus gaselli OLL2959 strain It was equivalent (Table 6).

The adenine uptake ability was evaluated in the same manner as in Example 5 except that only adenine ( ¹⁴ C-adenine) was used as the purine labeled with the radioactive isotope ¹⁴ C. The results are shown in FIG. The Lactobacillus gasseri P14054ME002 strain was not as high in adenine uptake as the Lactobacillus gasseri OLL2959 strain, but the Lactobacillus gasseri P14054ME002 strain was also confirmed to have high adenine uptake capacity (FIG. 9). . Compared with the Lactobacillus gasseri OLL2959 strain and the P14054ME002 strain, it was confirmed that the Lactobacillus gasseri P14054ME001 strain had lower adenine uptake ability (FIG. 9).

In the evaluation of the growth ability in the presence of adenine, DM medium (Table 5): 1 mL of adenine was added to a final concentration of 400 μM, and then culture of Lactobacillus gasseri OLL2959 strain prepared in Example 5 was performed. 4% by weight (0.04 mL: 1.6 to 2.8 × 10 ⁷ cfu) of either P14054ME001 strain or P14054ME002 strain culture solution prepared in the same manner as described in Example 5 at 37 ° C. And anaerobic culture. Then, the turbidity (absorbance at 650 nm) of the medium was measured after 0, 4, and 6 hours from the start of the culture. The results are shown in FIG. Like Lactobacillus gasseri OLL2959, Lactobacillus gasseri P14054ME001 and P14054ME002 showed enhanced proliferation ability in the presence of adenine. In addition, compared with the Lactobacillus gasseri P14054ME001 and P14054ME002 strains, the enhancement of the growth ability of the Lactobacillus gasseri OLL2959 strain was extremely strong. In addition, compared with the Lactobacillus gasseri P14054ME001 strain, the Lactobacillus gasseri P14054ME002 strain had a stronger enhancement of the proliferation ability.

From the above results, it was shown that in Lactobacillus gasseri, the enhancement of the growth ability in the presence of adenine correlates with the high adenine uptake ability. And it was shown that some lactic acid bacteria have high adenine utilization ability.

[Example 8] Purine body uptake ability of Lactobacillus gasseri (animal test)
When the ability of lactic acid bacteria to take up purine bodies is high, administration of lactic acid bacteria and purine bodies to an animal subject at the same time (ingestion) absorbs purine bodies in the subject compared to when the purine bodies are ingested alone. Is considered to be suppressed. Therefore, in order to test the ability of Lactobacillus gasseri to take up purine bodies, an animal experiment was conducted according to the following procedure.

First, 14 of 8 week old Wistar rats (male, 190-210 g) were purchased and acclimated for one week. These rats were fasted approximately 16 hours from the day before the test, and the body weight after this fasting was measured. Based on this fasted body weight, using a grouping program, the rats were classified into a negative group (saline-administered group), AMP (radioisotope ¹⁴ C-AMP) -administered group, and AMP + OLL2959 strain by random sampling. The total of the administration groups (radioisotopes ¹⁴ C-AMP and OLL2959 strain) was divided into 3 groups. At this time, only the negative group was 4 mice and the other group was 5 mice each. All of these rats were placed in a holder without anesthesia, the tail vein was injured with a scalpel, and 60 μL of the exuded blood was collected with a hematocrit tube. This was defined as blood sampling at 0 minutes before administration of the test substance. An equal volume of 2 mg / mL EDTA-2Na solution (EDTA-2Na was dissolved in physiological saline) was added to the collected blood.

Subsequently, the test substance was orally administered by gavage. Here, for these test substances, in the negative group, physiological saline, in the AMP administration group, adenylic acid labeled with the radioisotope ¹⁴ C ( ¹⁴ C-AMP: 57.6 mCi / mmol, 0.1 mCi / ml), In the AMP + OLL2959 strain administration group, ¹⁴ C-AMP and Lactobacillus gasseri OLL2959 strain (1 × 10 ¹⁰ cfu / body) were used. For ¹⁴ C-AMP and OLL2959 strains, those diluted with physiological saline (Otsuka Pharmaceutical) were used. In the AMP group and AMP + OLL2959 group, ¹⁴ C-AMP was administered at 10 μCi / body. In all cases (all groups), the administration volume was 2 mL / body.

15, 30, 45, 60, 90, 120, and 180 minutes after administration of the test substance, all these rats are placed in a holder under anesthesia, the tail vein is injured with a scalpel, and the hematocrit tube is inserted. In use, 60 μL of blood was collected. An equal volume of 2 mg / mL EDTA-2Na solution (EDTA-2Na was dissolved in physiological saline) was added to the collected blood. Immediately after completion of these studies, rats were killed by inhalation of carbon dioxide.

The radioactivity of these collected blood was measured using a liquid scintillation counter (manufactured by Aroka, LSC-6100). The result is shown in FIG. As shown in FIG. 11, significant differences were observed in the amount of purine absorbed at 30, 45 and 60 minutes after administration at which the blood concentration reached a peak (* p <0.05, ** p <0.01, t-test). From this result, it was shown that the amount of purine absorbed from the intestine can be suppressed by ingesting Lactobacillus gasseri OLL2959 strain.

[Example 9] Comparative test of types of lactic acid strains (1) Comparative test of adenine uptake ability In a medium containing radioisotope (RI) -labeled adenine ( ¹⁴ C-adenine), Lactobacillus gaselli OLL2959 strain and Then, Lactobacillus gasseri strain JCM1130 was cultured, and the effect of the type of lactic acid strain on the adenine uptake ability was compared. The Lactobacillus gasseri JCM1130 strain can be obtained as JCM1130 from RIKEN BRC JCM, Tsukuba City, Ibaraki Prefecture, Japan.

Here, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured using MRS medium, respectively, and their proliferation ability was evaluated in advance. That is, Lactobacillus gasseri OLL2959 and Lactobacillus gasseri JCM1130 were each anaerobically cultured at 37 ° C. for 20 hours using MRS medium. At this time, after anaerobic culture for 20 hours, the number of bacteria in the Lactobacillus gasseri JCM1130 strain was 2.5 times or more higher than that in the Lactobacillus gasseri OLL2959 strain. This indicates that when the Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain are cultured in the same medium, basically, the Lactobacillus gasseri JCM1130 strain has a high growth ability (Table 1). 7).

In the comparative test of adenine uptake ability, first, ¹⁴ C-adenine was added to a minimal medium (Table 5) to a final concentration of 20 μM to prepare a medium for this test. Then, using MRS medium, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured, respectively, and these culture solutions were inoculated at 2% by weight in the medium of this test at 37 ° C. And anaerobic culture. In these culture solutions, MRS medium was used to adjust the number of bacteria to the same level.

At the start of these cultures (0 minutes) and 30 or 60 minutes after the start of the culture, 5% TFA solution was added to stop the culture, and then the cells were washed with physiological saline, These radioactivity was measured using a liquid scintillation counter (manufactured by Aroka, LSC-6100). The result is shown in FIG. Here, the radioactivity (vertical axis) in FIG. 12 is represented by the number of disintegrations per minute (dpm) of the radioactive substance.

As shown in FIG. 12, both Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain exhibited the ability to take up adenine. Compared with the Bacillus gasseri JCM1130 strain, the Lactobacillus gasseri OLL2959 strain incorporated adenine more and showed a significant difference in the amount of adenine incorporation (p <0.05, t-test).

From the above results, it was revealed that the Lactobacillus gasseri OLL2959 strain can incorporate significantly more purines than the Lactobacillus gasseri JCM1130 strain, which has a high growth ability in the MRS medium.

(2) Comparative test of growth ability in the presence of adenine Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured in the presence of adenine, and the effect of the type of lactic acid strain on the growth ability of the cells Compared.

A medium for this test was prepared by adding adenine to a minimal medium (Table 5) to a final concentration of 400 μM. Then, using MRS medium, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured, respectively, and these culture solutions were inoculated into the medium of this test at 4% by weight, 37 Anaerobic culture was performed at ℃. Turbidity (absorbance at 650 nm) was measured at the start of culture (0 hour) and 4 and 6 hours after the start of culture. The result is shown in FIG.

As shown in FIG. 13, the Lactobacillus gasseri OLL2959 strain and the Lactobacillus gasseri JCM1130 strain all had enhanced growth ability in the presence of adenine, but Lactobacillus gasseri OLL2959 strain had high growth ability in MRS medium. Compared with the gasseri JCM1130 strain, the Lactobacillus gasseri OLL2959 strain was found to have a significantly higher degree of enhancement of proliferation ability (p <0.05, t-test). Therefore, Lactobacillus gasseri OLL2959 strain was shown to be particularly strongly enhanced in the growth ability in the presence of purines.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

The lactic acid bacterium of the present invention can efficiently convert a purine base into a purine nucleotide as a substrate in vivo or in vitro. Use of the conversion agent for purine nucleotides containing purine bases containing lactic acid bacteria of the present invention promotes the conversion of purine bases to purine nucleotides in the intestinal tract even in subjects with high serum uric acid levels or subjects with reduced salvage activity The serum uric acid level can be reduced. Therefore, the purine base-converting agent containing lactic acid bacteria of the present invention into a purine nucleotide, and foods and drinks and pharmaceuticals containing the same are effective for the prevention and / or treatment of gout and hyperuricemia.

Claims (14)

Lactobacillus culturing lactic acid bacteria in a solution containing adenine, 5-phospho-D-ribose-1-diphosphate, and Mg ²⁺ , and using the resulting conversion activity of adenine to adenylate as an index, A method for screening a lactic acid bacterium having an enhanced ability to convert a purine base into a purine nucleotide as compared to the gasseri ATCC 33323 strain. The method according to claim 1, further comprising measuring 5′-nucleotidase activity and selecting a lactic acid bacterium having reduced activity compared to Lactobacillus gasseri ATCC 33323 strain. The method according to claim 1 or 2, wherein the lactic acid bacterium is a genus Lactobacillus. The method according to any one of claims 1 to 3, wherein the solution is a buffer solution. An agent for converting a purine base into a purine nucleotide, comprising as an active ingredient the lactic acid bacterium obtained by the method according to any one of claims 1 to 4. The conversion agent according to claim 5, for conversion of adenine into adenylic acid or conversion of guanine into guanylic acid. The conversion agent according to claim 5 or 6, wherein the lactic acid bacterium is Lactobacillus gasseri OLL2959 strain (Accession No. NITE BP-224). A food or drink or a pharmaceutical comprising the conversion agent according to any one of claims 5 to 7. The food or drink or medicine according to claim 8 for reducing serum uric acid level. 10. The food or drink or pharmaceutical product according to claim 9, wherein the reduction of serum uric acid level is accompanied by promotion of conversion of adenine to adenylate and promotion of conversion of guanine to guanylate in the intestinal tract. The food or drink or pharmaceutical according to claim 9 or 10, wherein the subject is a human subject exhibiting a serum uric acid level of 6 to 8 mg / dL. The food or drink or pharmaceutical according to any one of claims 8 to 11, comprising 1 × 10 ⁸ to 10 ¹⁰ cfu of the lactic acid bacterium per dose. The purine base is purified from the purine base by reacting the conversion agent according to any one of claims 5 to 7 with the purine base in the presence of 5-phospho-D-ribose-1-diphosphate and Mg ^2+. A method of generating nucleotides. The method according to claim 13, for producing adenylic acid from adenine or guanylic acid from guanine.

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