JPWO2016080371A1 - Lactic acid bacteria having purine uptake ability and uses thereof - Google Patents

Abstract

A method for obtaining lactic acid bacteria having an action of reducing serum uric acid level is provided. The present invention provides a screening method for lactic acid bacteria, which includes measuring the amount of purine bodies taken up by a lactic acid bacterium in a medium containing purine bodies, and screening lactic acid bacteria having a purine body capturing action using the amount as an index. The present invention relates to a purine capturing agent for reducing serum uric acid level using lactic acid bacteria.

Description

  The present invention relates to a lactic acid bacterium having an ability to take up purines 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, the intake of dietary purines that are ultimately decomposed into uric acid is reduced by limiting the calorie intake, but it is not always easy to continue the severe restriction of calorie intake. Therefore, more effective treatment methods are desired for gout and hyperuricemia. Furthermore, it is desired to develop foods that are effective in preventing or 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. These conventional microorganisms and fermented products that have a serum uric acid level-reducing action promote the conversion of purine nucleosides into purine bases in the intestinal tract. It has been thought that the conversion leads to suppression of purine body absorption and promotion of excretion. 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 WO2009 / 069704 JP 2013-048636

  This invention makes it a subject to provide the efficient acquisition method (selection method) of the lactic acid bacteria which has a reduction | decrease effect of a serum uric acid level. Another object of the present invention is to provide a lactic acid bacterium having an ability to take up purines and use thereof.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that lactic acid bacteria such as Lactobacillus gasseri have high purine capacity in the presence of purine bodies and high purine capacity in the presence of purine bodies. The presence of strains, the ability to take up purine bodies in such lactic acid bacteria and the growth ability in the presence of purine bodies are correlated, and administration (intake) of such lactic acid bacteria reduces serum uric acid levels. The present invention has been found and the present invention has been completed.

That is, the present invention includes the following.
[1] 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.
[2] The method according to [1] above, wherein the purine body in the medium is a purine base.
[3] The method according to [1] or [2] above, wherein purines in the medium are labeled with a radioisotope.
[4] measuring the growth amount of the lactic acid bacteria in a medium containing purine bodies, and selecting the lactic acid bacteria having a purine body capturing action as an index together with the uptake amount of the purine bodies. The method according to any one of [3].
[5] The method according to any one of [1] to [4] above, wherein the lactic acid bacterium is Lactobacillus gasseri.
[6] A lactic acid bacterium having a purine-capturing action obtained by the method according to any one of [1] to [5] above.
[7] 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 [1] to [5].
[8] The purine body scavenger according to [7] above, which is for reducing serum uric acid level.
[9] The purine capturing agent according to [7] or [8] above, wherein the lactic acid bacterium is Lactobacillus gaselli OLL2959 strain (Accession No. NITE BP-224).
[10] A food or drink or a pharmaceutical comprising the purine body scavenger according to any one of [7] to [9].
[11] The food or drink or medicine according to [10] above, which is for reducing purines in the intestinal tract.
[12] The food or drink or medicine according to [10] or [11] above, wherein the subject is a human subject having a serum uric acid level of 6 to 8 mg / dL.
[13] The food or drink or medicine according to any one of [10] to [12] above, wherein the lactic acid bacterium is contained at 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu per dose.

According to the present invention, it is possible to efficiently obtain (select) lactic acid bacteria having a purine body capturing action and a serum uric acid level reducing action. If the lactic acid bacteria or the capturing agent according to the present invention is used, the purine body can be captured efficiently.
This specification includes the contents of Japanese Patent Application Nos. 2014-234050 and 2015-064201 which form the basis of the priority claim of the present application.

It is a graph which shows the uptake | capture ability of the Lactobacillus gasseri OLL2959 strain purine body. It is a graph which shows the growth ability of the Lactobacillus gasseri OLL2959 strain | stump | stock in presence of a purine body. It is a figure which shows the uptake | capture ability of adenine of several lactic acid strains. It is a graph which shows the growth ability of several lactic acid strains in presence of adenine. It is a graph which shows a time-dependent change of the serum uric acid level in the human subject who orally administered (oral intake) Lactobacillus gasseri OLL2959 strain continuously. It is a graph which shows the uptake | capture capacity | capacitance of the Lactobacillus gasseri OLL2959 strain and the purine body of the animal which administered the purine body simultaneously. It is a graph which shows the result of having compared the uptake ability of adenine with the kind of lactic acid strain. It is a graph which shows the result of having compared the growth ability of the lactic acid strain in presence of adenine with the kind of lactic acid strain. It is a graph which shows the time-dependent change of turbidity (OD650) and the amount of nucleic acids in Lactobacillus gasseri OLL2959 strain. It is a graph which shows the time-dependent change of the nucleic acid amount and the radioactivity in a nucleic acid in Lactobacillus gasseri OLL2959 strain. It is a graph which shows the IMP absorption reduction effect of OLL2959 strain in the animal which administered Lactobacillus gasseri OLL2959 strain and ¹⁴ C-IMP. Radioactivity (dpm): Mean value ± SD. * p <0.1. A: 15 minutes after administration, B: 30 minutes after administration. From the left, the results of the negative group (negative control group), IMP group (IMP administration group), and IMP + OLL2959 group (IMP + OLL2959 strain administration group) are shown. It is a graph which shows the IMP absorption reduction effect of OLL2959 strain in the animal which administered Lactobacillus gasseri OLL2959 strain and ¹⁴ C-IMP. Radioactivity (dpm): Mean value ± SD. * p <0.1. A: 45 minutes after administration, B: 60 minutes after administration. From the left, the results of the negative group (negative control group), IMP group (IMP administration group), and IMP + OLL2959 group (IMP + OLL2959 strain administration group) are shown. It is a graph which shows the hypoxanthine absorption reduction effect of OLL2959 strain | stump | stock in the animal which administered Lactobacillus gasseri OLL2959 strain | stump | stock and ¹⁴ C-hypoxanthine. Radioactivity (dpm): Mean value ± SD. ## p <0.01. A: 15 minutes after administration, B: 30 minutes after administration. From the left, the results of the negative group (negative control group), hypoxanthine group (hypoxanthine administration group), hypoxanthine + OLL2959 group (hypoxanthine + OLL2959 strain administration group) are shown. It is a graph which shows the hypoxanthine absorption reduction effect of OLL2959 strain | stump | stock in the animal which administered Lactobacillus gasseri OLL2959 strain | stump | stock and ¹⁴ C-hypoxanthine. Radioactivity (dpm): Mean value ± SD. ## p <0.01, # p <0.05. A: 45 minutes after administration, B: 60 minutes after administration. From the left, the results of the negative group (negative control group), hypoxanthine group (hypoxanthine administration group), hypoxanthine + OLL2959 group (hypoxanthine + OLL2959 strain administration group) are shown. It is a graph which shows the uptake ability (capture action) of hypoxanthine and inosine of Lactobacillus gasseri OLL2959 strain. It is a graph which shows the survival rate in a yogurt of Lactobacillus gasseri OLL2959 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 bodies are not only supplied from food to the body as dietary purine bodies through 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 ultimately 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 purine metabolic pathways similar to humans, but are different 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, lactic acid bacteria having a purine-capturing action can be efficiently obtained (selected) by selecting (screening) lactic acid bacteria using as an index the ability of purine bodies to be taken into the cells. The present invention relates to a method for screening lactic acid bacteria, which comprises 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. It is related with the screening method of lactic acid bacteria including acquiring (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 intestine 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, the present invention cultivates lactic acid bacteria in a medium containing purine bodies, measures the amount of purine bodies taken up in the bacterial bodies, and selects lactic acid bacteria having a purine body capturing action as an index. The present invention also relates to a screening method for lactic acid bacteria having a serum uric acid level reducing action, including obtaining (selecting) lactic acid bacteria having a body capturing action as a lactic acid bacterium having a serum uric acid level reducing action.

  In the present invention, a lactic acid bacterium having purine body uptake ability exhibits 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 above selection using the purine body uptake ability as an index, by confirming the enhancement of the growth ability of the selected lactic acid bacteria in the presence of the purine body, the purine body has a capturing action. Lactic acid bacteria can be obtained (selected) with higher accuracy. That is, the present invention measures the amount of growth in a medium containing purine bodies of lactic acid bacteria having the ability to take up purine bodies, and uses this as an index together with the amount of purine bodies taken up as described above to capture purine bodies. The present invention also relates to a method for screening for lactic acid bacteria, which comprises obtaining (selecting) lactic acid bacteria having a pH. 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, Selecting lactic acid bacteria having a capturing action, and obtaining (selecting) lactic acid bacteria having a capturing action of the obtained purine bodies as lactic acid bacteria having a serum uric acid value reducing action, and having a serum uric acid value reducing action It also relates to a screening method for lactic acid bacteria. 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. Purine bodies (for example, purine bases) taken up in large quantities by lactic acid bacteria are used for nucleic acid synthesis necessary for growth, which brings about high growth ability of 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 Lactobacillus genus. Examples of the genus Lactobacillus include Lactobacillus gasseri, Lactobacillus delbrueckii subspice burgalicus, Lactobacillus delbrueckii subsp. Burgalicus, and Lactobacillus delbrueckii subspeckis lactis), Lactobacillus paracasei subsp. paracasei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus helveticus Lactobacillus helveticus subsp. Jugurti), Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus galovorus Rum (Lactobacillus gallinarum), Lactobacillus oris (Lactobacillus oris), Lactobacillus casei subspices rhamnosus (Lactobacillus casei subsp. Rhamnosus), Lactobacillus johnsonii, Lactobacillus johnsonii Strains such as Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus reuteri, and the like, with Lactobacillus gasseri bacteria being particularly preferred. 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 preferred minimal media are listed in Table 1.

  The purine body to be included in the medium may be a purine base, a purine nucleoside, and / or a purine nucleotide. In a preferred embodiment, the purine body to be included in the medium is a purine base. 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. In another embodiment, preferred examples of purine bases, purine nucleosides, and purine nucleotides include hypoxanthine, inosine (IMP), and inosinic acid, respectively, with hypoxanthine being particularly preferred.

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 that at the start of culture (at 0 minutes after the start of culture), the ability of the lactic acid bacteria to take up the purines into the cells (purine body uptake ability) Can be determined. When the amount of purine bodies taken up by lactic acid bacteria is significantly increased compared to that at the start of culture (at 0 minutes after the start of culture), the ability to take the lactic acid bacteria into the cells (purine body uptake capacity) It can be determined to be high. Alternatively, when the amount of purine bodies taken up by lactic acid bacteria is significantly increased compared to Lactobacillus gasseri strain JCM1130, the ability of the lactic acid bacteria to take the purine bodies into the cells (purine body uptake ability) is high. It can also be determined. 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. Alternatively, if the increase in turbidity when cultured in the presence of purines is significantly enhanced compared to Lactobacillus gasseri strain JCM1130, the lactic acid bacteria are significantly enhanced in the presence of the purines. It can also be determined that it exhibits the increased proliferation 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, the lactic acid bacteria are preferably inoculated and cultured at 1.0 × 10 ^{6 to} 1.0 × 10 ¹¹ cfu, for example, 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. Purine bodies that can be taken in, that is, captured by the selected lactic acid bacteria are, for example, purine bases, purine nucleosides, and / or purine nucleotides. The purine bodies that can be taken up by the selected lactic acid bacteria are not necessarily limited to the purine bodies included in the medium in the screening. Examples of purine bases include, but are not limited to, adenine, guanine, hypoxanthine and xanthine. Examples of purine nucleosides include, but are not limited to, adenosine, guanosine, inosine and xanthosine. Examples of purine nucleotides include, but are not limited to, adenylic acid (AMP), guanylic acid (GMP), inosinic acid (IMP), and xanthylic acid (XMP). In one embodiment, the purine bodies that can be taken up by the selected lactic acid bacteria include at least one selected from the group consisting of adenine, adenosine, adenylic acid, hypoxanthine, inosine, and inosinic acid, preferably all of them. .

  The present invention also provides lactic acid bacteria selected by the above screening method and having a purine capturing action. This lactic acid bacterium typically has an action of reducing serum uric acid level.

  The present invention also provides a purine body scavenger, preferably for oral administration, which can be obtained by the screening method described above and contains, as an active ingredient, a lactic acid bacterium having a purine body capturing action (hereinafter also referred to as the lactic acid bacterium of the present invention). A purine body scavenger is also provided. The purine capture agent of the present invention may contain a carrier or additive that is acceptable for oral administration in addition to the Lactobacillus gasseri bacterium of the present invention. The purine capturing agent of the present invention may be a drug or composition containing the lactic acid bacteria of the present invention, or a fermented product, a culture, or a concentrate / dried product produced using the bacteria. Or a drug or composition containing the product. The lactic acid bacterium according to the present invention contained in the purine capturing agent of the present invention is preferably a living microbial cell. As described above, the purine capturing agent of the present invention has an action of reducing purine bodies in the intestinal tract by taking up the purine bodies of lactic acid bacteria. Therefore, the purine body capturing agent for reducing purine bodies in the intestinal tract and thus reducing the serum uric acid level. Can be suitably used. The lactic acid bacteria of the present invention and the purine capturing agent of the present invention also incorporate purine bodies that are umami components of fish and meat, for example, purine nucleotides such as inosinic acid (IMP), adenylic acid (AMP), etc. To reduce its absorption into. Therefore, the lactic acid bacteria of the present invention and the purine capturing agent of the present invention can also be used to reduce the absorption of such umami components from the intestinal tract. The present invention also provides an absorption reducing agent for umami components (in this case, purine bodies or purine nucleotides that are umami components of fish and meat, such as inosinic acid), comprising the lactic acid bacterium of the present invention or the purine body capture agent of the present invention. . This umami component absorption reducing agent is also preferably for oral administration.

  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 No. 122 Postcode 292-0818) was deposited under the deposit number NITE P-224 and transferred to the deposit under the Budapest Treaty (international deposit) on November 21, 2007. The deposit number is NITE BP- It has been changed to 224. Alternatively, in another embodiment, the lactic acid bacterium having the purine-capturing action as described above may be a lactic acid bacterium or a Lactobacillus gasseri bacterium excluding Lactobacillus gasseri OLL2959 strain.

  The present invention also contemplates using the purine body scavenger according to the present invention in combination with a food or drink or a pharmaceutical product. Therefore, this invention also provides the purine body capture | acquisition agent which concerns on this invention for using it in combination with food-drinks or a pharmaceutical.

  This invention also provides food-drinks or a pharmaceutical containing the purine body capture | acquisition agent which concerns on this invention. In the subject who administers (ingests) the food / beverage product or pharmaceutical product of the present invention, the purine body is actively taken up and assimilated into the fungus body, thereby reducing the purine body in the intestinal tract, and the effect of serum uric acid level Reduction can be brought about. Therefore, the food / beverage products and pharmaceuticals of the present invention may be used for reducing purines in the intestinal tract. The “purine body in the intestinal tract” referred to in the present invention does not include purine bodies retained by bacteria (such as lactic acid bacteria), fungi, viruses, and subject cells present in the intestinal tract. The food / beverage products and pharmaceuticals of the present invention may be for reducing serum uric acid levels based on the reduction of purines in the intestinal tract. The food / beverage products and pharmaceuticals containing the purine-capturing agent of the present invention can be suitably used, for example, for prevention, treatment, improvement or reduction of symptoms of gout and hyperuricemia.

  In the present specification, the “food or drink” is not particularly limited, but includes beverages, foods and functional foods. The type of the food or drink according to the present invention is not particularly limited. For example, as beverages, fermented milk (drink yogurt and the like), lactic acid bacteria beverages, milk beverages (coffee milk, fruit milk, etc.), tea-based beverages (green tea, black tea) , Oolong tea, etc.), fruit / vegetable beverages (beverages containing fruit juices such as oranges, apples and grapes, tomatoes, carrots, etc.), alcoholic beverages (beer, sparkling wine, wine, etc.), carbonated beverages, soft drinks Examples of suitable beverages include drink yogurt, lactic acid bacteria beverages, milk beverages, water-based beverages, and the like. Particularly suitable 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 food include fermented milk (set type yogurt, soft yogurt, cheese, etc.), dairy products, confectionery, and instant food. Suitable foods include set type yogurt, soft yogurt, and confectionery. Particularly preferred drinks include set type yogurt, soft yogurt and the like. 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. The present invention uses a lactic acid bacterium 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 bacterium to dairy products, fermented milk or its raw materials. And the use of the lactic acid bacteria in the production of yogurt is particularly preferable. Furthermore, the present invention also provides a method for reducing purine bodies based on the action of capturing purine bodies of lactic acid bacteria in the production of fermented milk and dairy products such as yogurt and cheese, which uses lactic acid bacteria having a purine body capturing action as an active ingredient. provide. Lactic acid bacteria (for example, Lactobacillus gasseri OLL2959 strain) having a purine capturing action according to the present invention can exhibit good survival in fermented milk such as yogurt and dairy products.

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

  More specific and preferred examples of 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 particularly useful in reducing serum uric acid levels by reducing purine bodies in the intestinal tract. The functional food of the present invention is used for reducing serum uric acid levels, particularly reducing purine in the intestinal tract by taking up purine bodies by lactic acid bacteria and promoting the growth of lactic acid bacteria, and consequently reducing absorption of purine bodies in the intestinal tract. It can be suitably used for reducing the accompanying serum uric acid level.

  The food and drink such as the functional food of the present invention (preferably, food for specified health use or conditional tokuho [food for specified health use]) may be used for reducing purine bodies in the intestinal tract, It may be for suppressing or alleviating a decrease or an increase in serum uric acid level, and may be described or displayed to that effect. The present invention is the 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) preferable.

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

  The food / beverage products of this invention may contain arbitrary food components, and are not specifically limited. The food and drink of the present invention may contain water, protein, carbohydrate, lipid, vitamins, minerals, organic acid, organic base, fruit juice, flavors and the like. 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-lacto Globulin, α-lactalbumin, lactoferrin, soy protein, egg protein, meat protein and other animal and vegetable proteins, hydrolysates thereof, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose 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 thereof 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, tartaric acid, and the like. These components can be used alone or in combination of two or more thereof, and may be added using a synthetic product and / or a food containing a large amount thereof.

  Moreover, the functional food containing the lactic acid bacterium or purine body scavenger of the present invention may contain an orally acceptable carrier or additive in addition to the lactic acid bacterium or purine body scavenger of the present invention. 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, the pharmaceutical (pharmaceutical composition) containing the lactic acid bacterium or purine body scavenger of the present invention is a pharmaceutically acceptable carrier or additive, particularly for oral use, in addition to the lactic acid bacterium or purine body scavenger of the present invention. Acceptable carriers or additives may be included. 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. These include artificial cell structures. 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. The pharmaceutical product of the present invention may further contain other pharmacological components.

  The pharmaceutical agent of the present invention is preferably administered orally. The pharmaceutical product of the present invention may be in any dosage form such as solid preparations such as tablets, granules, powders, pills and capsules, gels, or liquid preparations such as liquids, suspensions and syrups. .

In the lactic acid bacteria, purine capture agents, foods and drinks or pharmaceuticals of the present invention, the dose (intake) is determined based on the age and weight of the subject to be administered (intake), the administration route, the number of administrations, etc. A wide range of changes can be made at the discretion of the supplier. Therefore, the dose of the lactic acid bacterium (Lactobacillus gasseri bacterium, etc.) of the present invention is not particularly limited in the lactic acid bacterium, purine capturing agent, food or drink or pharmaceutical of the present invention, but for example, 1 × 10 ⁵ per dose The amount of ˜1 × 10 ¹¹ cfu is preferable, the amount of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, the 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 preferred. The purine-capturing agent, food or drink or pharmaceutical of the present invention preferably contains the lactic acid bacterium of the present invention 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 in an amount of 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu, for example, an amount of 4 × 10 ^{9 to} 6 × 10 ¹⁰ cfu. It is particularly preferable to contain.

In one embodiment of the present invention, the lactic acid bacterium, purine capturing agent, food or drink or pharmaceutical of the present invention is applied to the subject at least once a day, preferably at least twice a day, more preferably twice a day. Administered (or taken by the consumer). The lactic acid bacteria, purine-capturing agent, food or drink or pharmaceutical of the present invention may be continuously administered to a subject, for example, may be administered daily. In this case, the lactic acid bacteria, purine-capturing agent, food / beverage product or pharmaceutical of the present invention 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 or drink or pharmaceutical of the present invention, when continuously administered to a subject, the dose of the lactic acid bacterium of the present invention is an amount that is 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. The amount of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, the amount of 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 of the present invention, the lactic acid bacterium, purine-capturing agent, food or drink, or pharmaceutical of the present invention may be a single dose. In the purine body capturing agent, food or drink or pharmaceutical of the present invention, when administered to a subject once, the dose of the lactic acid bacteria of the present invention is 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. Preferably, the amount is 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. The amount is particularly preferred. The lactic acid bacteria, purine-capturing agent, food and drink or pharmaceutical of the present invention is preferably administered orally (orally ingested).

  In the present invention, “administration” includes both “ingestion” generally used for foods and drinks and “administration” used for pharmaceutical products. In the present invention, “oral administration” includes administration by tube feeding via a nasal tube or a gastric fistula tube, in addition to oral administration or ingestion. Therefore, the present invention also provides an oral preparation that can be used for such oral administration. Therefore, in a preferred embodiment of the present invention, an oral preparation for reducing purine bodies in the intestinal tract and containing serum uric acid levels is also provided, which contains the lactic acid bacterium or purine capture agent of the present invention.

  The subject to be administered with the lactic acid bacterium, purine capture agent, food or drink or pharmaceutical of the present invention is a mammal including humans, domestic animals, pets, experimental (test) animals, etc., preferably human subjects, More preferred are human subjects with gout and / or hyperuricemia, and even more preferred are human subjects exhibiting serum uric acid levels of 6 mg / dL or more, for example 6-10 mg / dL. In one embodiment, a human subject with mild to borderline hyperuricemia with a serum uric acid level of 6-8 mg / dL is more preferred as an administration subject. The present invention also administers (ingests) the purine body capturing agent, food or drink or pharmaceutical of the present invention containing the lactic acid bacterium of the present invention or the lactic acid bacterium of the present invention in an effective amount as described above to the subject. By this, the purine body is capture | acquired in the microbial cell of lactic acid bacteria, and the method of reducing the purine body in an intestinal tract and reducing a serum uric acid level is also provided. The present invention also provides a method of capturing a purine body in the lactic acid bacteria by bringing the lactic acid bacterium or purine body capture agent of the present invention into contact with the purine body. The present invention also provides use of the lactic acid bacterium or purine body capturing agent of the present invention for imparting purine body capturing action to foods and drinks, pharmaceuticals or other drugs. The present invention also provides the use of the lactic acid bacteria, purine scavengers, foods and drinks, or pharmaceuticals of the present invention to reduce the risk of developing gout or hyperuricemia.

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

[Example 1] Evaluation test of purine body uptake ability In this example, a purine body labeled with a radioisotope (RI) was used to purify the purine body of Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain. The uptake ability 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. The culture (4-7 * 10 < ⁸ > cfu / ml) which inoculated Lactobacillus gasseri OLL2959 strain into MRS culture medium (Lactobacilli MRS Broth, Difco), and was cultured at 37 degreeC for 16 to 20 hours was used below.

Minimal medium (DM medium; Table 1): 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. 1, the unit of radioactivity (vertical axis) indicating the amount of ¹⁴ C-labeled purine in the microbial cell is disintegrations per minute (dpm), which is the number of radioactive substances that disintegrate per minute. In addition, it was confirmed that there was no significant change in the viable cell count at the start of the test and at the end of the test in the culture for 60 minutes using the DM medium.

  As a result, the Lactobacillus gasseri OLL2959 strain has the ability to take adenylic acid (AMP), adenosine, and adenine, which are purines, into the cells (purine incorporation ability), particularly adenine. It was shown that it has a high ability to be taken in (purine body uptake ability) (FIG. 1).

[Example 2] Evaluation test of proliferation ability in the presence of purine bodies In this example, 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 1): 1 mL of adenylate (AMP), adenosine, or adenine was added as a purine to a final concentration of 400 μM, and then the Lactobacillus gasseri OLL2959 strain prepared in Example 1 was used. 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 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.

  As a result, it was shown that the Lactobacillus gasseri OLL2959 strain is enhanced in the presence of adenylic acid (AMP), adenosine, or adenine, particularly in the presence of adenine. (FIG. 2).

[Example 3] Comparative test of adenine uptake ability and proliferation ability in the presence of adenine In this example, Lactobacillus gasseri OLL2959 strain and other Lactobacillus gasseri strains were cultured in the presence of adenine, and each adenine was cultured. The ability of uptake and the ability to grow in the presence of adenine were compared.

  As other Lactobacillus gasseri strains, Lactobacillus gasseri strains P14054ME001 and P14054ME002 were used. In addition, in Lactobacillus gasseri P14054ME001 and P14054ME002 strains, when cultured in MRS medium (Lactobacilli MRS Broth, Difco) without addition of purines for 20 hours, the respective growth ability is It was equivalent (Table 2).

Evaluation of adenine uptake ability was carried out in the same manner as in Example 1 except that only adenine ( ¹⁴ C-adenine) was used as the purine labeled with the radioisotope ¹⁴ C. The results are shown in FIG. Although not as high as the Lactobacillus gasseri OLL2959 strain, the Lactobacillus gasseri P14054ME002 strain was also confirmed to have high adenine uptake capacity (FIG. 3). Compared with the Lactobacillus gasseri OLL2959 strain and the P14054ME002 strain, it was confirmed that the Lactobacillus gasseri P14054ME001 strain had low adenine uptake ability (FIG. 3).

In the evaluation of the growth ability in the presence of adenine, DM medium (Table 1): 1 mL of adenine was added to a final concentration of 400 μM, and then the Lactobacillus gasseri OLL2959 strain prepared in Example 1 was cultured. One of the P14054ME001 strain and the P14054ME002 strain culture solution prepared in the same manner as described in Example 1 was inoculated at 4% by weight (0.04 mL: 1.6 to 2.8 × 10 ⁷ cfu), and the mixture was incubated at 37 ° C. And anaerobic culture. 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 to 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 these results, it was shown that the enhancement of the growth ability in the presence of adenine correlates with the high uptake ability of adenine in Lactobacillus gasseri. And it was shown that some lactic acid bacteria have high adenine utilization ability.

[Example 4] Evaluation test of effect of reducing serum uric acid level of Lactobacillus gaselli OLL2959 strain A human subject suspected of mild to borderline hyperuricemia was allowed to continuously ingest Lactobacillus gaselli OLL2959 strain, A placebo-controlled double-blind comparative study examined the effect on uric acid levels (human study).

14 adult males aged 35 and over (mean age 44.3 years old) who had uric acid levels of 6-8 mg / dL in the pre-start study were active with the placebo group so that there was no significant difference between uric acid levels and age Assigned to 2 groups. The placebo group received 2 yogurts (85 g / piece) / day for 4 weeks without Lactobacillus gasseri OLL2959 strain. In the active group, yoghurt fed to the placebo group containing Lactobacillus gasseri OLL2959 strain at 1 × 10 ⁸ cfu / g was ingested at 2 (85 g / piece) / day for 4 weeks. In addition, 2 yogurts / day were taken twice after 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 by a conventional method. 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 5] Single dose test of Lactobacillus gasseri (animal test)
Unlike humans, rats have uricase, a urate-degrading enzyme, so it is necessary to administer potassium oxonate, a uricase inhibitor, to increase serum uric acid levels. For this reason, potassium oxonate 0.5 g / kg is forcibly administered orally to Wistar rats (male) fasted for 16 hours. To the lactic acid bacteria group, dry yeast (suspended in water for injection) and Lactobacillus gasseri OLL2959 (suspended in physiological saline) are forcibly orally administered 60 minutes after administration of potassium oxonate due to purine loading. For the negative group, water for injection is administered instead of dry yeast, and physiological saline is administered instead of Lactobacillus gasseri. In the control group, physiological saline is administered together with dry yeast instead of Lactobacillus gasseri.

  After forced oral administration, blood is collected over time (30 minutes, 60 minutes, 90 minutes, 120 minutes, and 150 minutes later), and the blood uric acid level of each blood sample is measured by a conventional method. Examine the transition. If the blood uric acid level in the lactic acid bacteria group remained low compared to the control group, the effect of purine body uptake of Lactobacillus gasseri OLL2959 even after a single administration showed that the amount of purine body absorption from the intestinal tract decreased. It is.

[Example 6] Uptake ability of purine bodies 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 8-star Wistar rats (male, 190-210 g) were purchased and acclimatized for a week. These rats were fasted approximately 16 hours from the day before the test, after which they were weighed. Based on the body weight after fasting, rats were divided into negative groups (saline-administered group), AMP (radioisotope ¹⁴ C-AMP) -administered group, and AMP + OLL2959 strain using a grouping program. (Radioisotopes ¹⁴ C-AMP and OLL2959 strain) were administered to a total of 3 groups (only 4 in the negative group and 5 in the other groups). All of these rats were placed in a holder without anesthesia, the tail vein was injured with a scalpel, and 60 μL of the swelled 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 a hematocrit tube is used. Then, 60 μL of the blood that had come out was collected. An equal amount of 2 mg / mL EDTA-2Na solution (EDTA-2Na was dissolved in physiological saline) was added to these collected blood. At the end of these studies, the rats were immediately sacrificed by inhalation of carbon dioxide.

The radioactivity of these collected blood was measured using a liquid scintillation counter (ALOC, LSC-6100). The result is shown in FIG.
As shown in FIG. 6, 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 7] Single dose test of Lactobacillus gasseri (human test)
A healthy male over 20 years old ingested 498 mg of purine body preparation (mixture of 5′-adenylic acid, disodium 5′-inosinate and disodium 5′-guaninate) once, 30 minutes, 60 minutes Blood samples are taken after minutes, 120 minutes, and 150 minutes, and changes in blood uric acid levels are examined. Ten subjects with similar transitions are selected and are the subjects of this study. In this study, 112 mL of yogurt containing Lactobacillus gasseri OLL2959 strain (active group; containing 8.5 × 10 ⁷ cfu / mL of Lactobacillus gasseri OLL2959 strain) or yogurt not containing the bacteria (placebo group) Ingest, collect blood 30 minutes, 60 minutes, 120 minutes, and 150 minutes after ingestion, measure blood uric acid levels for each blood sample in the usual way, examine the transition of blood uric acid levels, and perform a crossover test . If the increase in uric acid levels was suppressed in the active group compared to the placebo group, it was shown that the amount of purine absorbed from the intestinal tract decreased due to the effect of purine incorporation of Lactobacillus gaselli OLL2959 even in a single administration. It is.

[Example 8] Comparative test of types of lactic acid strains (1) Comparative test of adenine uptake ability In a medium containing adenine ( ¹⁴ C-adenine) labeled with a radioisotope (RI), 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). 3).

In a comparative test of adenine uptake, first, ¹⁴ C-adenine was added to a minimal medium (Table 1) 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. 7 is represented by the number of disintegrations per minute (dpm) of radioactive materials per minute.

As shown in FIG. 7, 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 these results, it was clarified that the Lactobacillus gasseri OLL2959 strain incorporated significantly more purines than the Lactobacillus gasseri strain JCM1130, 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.
To the minimal medium (Table 1), adenine was added to a final concentration of 400 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 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. 8, 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.

[Example 9] Utilization of Purine for Nucleic Acid Synthesis A test was conducted to verify how Lactobacillus gasseri OLL2959 uses adenine in the process of growing after taking up adenine.

Lactobacillus gasseri OLL2959 strain in culture is recovered and prepared to 1.0 × 10 ¹⁰ (1.0E + 10) cfu / ml, and 6% in a minimal medium containing a final concentration of 400 μM adenine. Inoculated. Thereafter, ¹⁴ C-adenine was added, and the medium was collected after 0, 1, 2, 3, and 4 hours, and turbidity measurement (OD at 650 nm) was performed. Furthermore, the cells are collected by centrifuging the medium at 3,000 rpm for 10 minutes at 4 ° C, washed twice with distilled water, and then extracted with a DNA extraction kit ISOPLANT II (Nippon Gene). went. The concentration of the extracted nucleic acid was measured using a spectrophotometer. Thereafter, the entire amount of the extracted nucleic acid was added to a vial to which a liquid scintillation cocktail was added, and the radioactivity was measured with a liquid scintillation counter.

  As a result, turbidity increased depending on the culture time (OLL2959 strain proliferated), and the amount of nucleic acid (DNA concentration) also increased depending on the culture time (FIG. 9). Furthermore, as the amount of nucleic acid increased, the radioactivity in the nucleic acid also increased (FIG. 10), indicating that OLL2959 strain incorporated adenine and utilized it for the synthesis of nucleic acids necessary for cell growth.

[Example 10] Evaluation of Lactobacillus gasseri uptake ability of hypoxanthine and IMP (inosinic acid) (animal test)
28 Wistar rat males (8 weeks old) were acclimated for about one week after purchase. Rats were fasted for about 16 hours from the day before the test, and body weight was measured after fasting. Based on body weight after fasting rats, negative control (saline) group, IMP (radioisotope ¹⁴ C-IMP) administration group, and IMP + OLL2959 strain (radioisotope ¹⁴ C-IMP and OLL2959 strain) administration group Were divided into a total of 3 groups (8 for the negative control group only, 10 for the other group).

  In all cases, 60 μL of blood was collected from the tail vein of rats without anesthesia. This was defined as blood sampling at 0 minutes before administration of the test substance. An equal amount of 2 mg / mL EDTA-2Na solution (EDTA-2Na dissolved in physiological saline) was added to the collected blood and ice-cooled.

Then, the test substance was orally administered to the rats. In the IMP administration group, ¹⁴ C-IMP was diluted with physiological saline and administered at 2 mL / body. To the IMP + OLL2959 strain administration group, 2 mL / body of a mixture prepared by mixing ¹⁴ C-IMP and OLL2959 strain before administration was administered. In these groups, ¹⁴ C-IMP was administered at 10 μCi / body. To the negative control group, physiological saline was administered at 2 mL / body.

  In all cases, 60 μL of blood was collected from the tail vein of rats under no anesthesia at 15, 30, 45, 60, 90, 120 and 180 minutes after administration of the test substance. An equal amount of 2 mg / mL EDTA-2Na solution was added to the collected blood and ice-cooled.

  The collected blood was measured for radioactivity using a liquid scintillation counter.

Furthermore, the same animal test was conducted using ¹⁴ C-hypoxanthine instead of ¹⁴ C-IMP. In this test using ¹⁴ C-hypoxanthine, a total of 3 groups (4 groups for the negative control group, 4 groups for the negative control group, 4 groups for the negative control group, 4 groups for the negative control group) 5) were used.

  The results are shown in FIGS. As shown in FIGS. 11 and 12, before and after the time when the blood concentration of the test substance reached the peak, the IMP absorption amount was significantly reduced in the rats to which Lactobacillus gasseri OLL2959 was administered once (* p <0.1, hypoxanthine administration group vs. hypoxanthine + OLL2959 strain administration group, Student's t test). Similarly, as shown in FIGS. 13 and 14, hypoxanthine absorption was significantly reduced in rats that were administered a single dose of Lactobacillus gasseri OLL2959 (## p <0.01, # p <0.05, hypo Xanthine administration group vs. hypoxanthine + OLL2959 strain administration group, Student's t-test). From these results, the effect of reducing purine body absorption by single administration of Lactobacillus gasseri OLL2959 strain was further shown. Since IMP is also an umami component of fish and meat, it has great significance to confirm the effect of reducing absorption of IMP and its base hypoxanthine.

[Example 11] Evaluation of purine uptake ability in vitro
Add ¹⁴ C-hypoxanthine and ¹⁴ C-inosine at 1 μCi / ml to Lactobacillus gaselli OLL2959 strain prepared in PBS to about 1.0 × 10 ⁸ cfu / ml, and incubate at 37 ° C. for 15 or 30 minutes did. The cells after incubation were collected, and the radioactivity was measured using a liquid scintillation counter.

  The result is shown in FIG. Lactobacillus gasseri OLL2959 strain was shown to take up (capture) both inosine and hypoxanthine into its cells in vitro (FIG. 15). The amount of incorporation was higher for hypoxanthine, a purine base, than inosine, a nucleoside. This result was similar to that in the relationship between adenosine and adenine.

[Example 12] Evaluation of survival of OLL2959 strain in yogurt Lactobacillus gasseri OLL2959 strain was added to drink yogurt or physiological saline to a concentration of 2.5 × 10 ⁸ ± 0.1 × 10 ⁸ cfu / ml. The viable cell count was measured on day 0, day 7, day 21 and day 28. The survival rate at each time point was expressed as a ratio (%) of the viable cell count to the viable cell count on the 0th day.

  As a result, the survival rate of the Lactobacillus gasseri OLL2959 strain added to the drink yogurt was 72.6% on the 7th day, 58.5% on the 21st day, and 61.0% on the 28th day (FIG. 16). In contrast, the survival rate of Lactobacillus gasseri OLL2959 strain added to physiological saline was 10.6% on the 7th day and 0.0% on the 21st and 28th days (FIG. 16).

  From these results, it was shown that the Lactobacillus gasseri OLL2959 strain has higher survival in yogurt than saline.

  ADVANTAGE OF THE INVENTION According to this invention, the lactic acid bacteria which have the purine body capture | acquisition effect | action with high purine body uptake | capture ability and the proliferation ability in purine body presence can be acquired efficiently. Such lactic acid bacteria can reduce the amount of purine bodies in the administered intestinal tract, thereby reducing the amount of purine bodies absorbed in the intestinal tract, and as a result, the serum uric acid level can be reduced. Therefore, this invention is useful also in developing the oral preparation for reducing the purine body in an intestinal tract and reducing a serum uric acid level.

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

Claims (13)

  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 selecting lactic acid bacteria having a purine body-capturing action using the amount as an index.   The method according to claim 1, wherein the purine body in the medium is a purine base.   The method according to claim 1 or 2, wherein the purine in the medium is labeled with a radioisotope.   The method comprises any one of claims 1 to 3, comprising 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. The method according to item.   The method according to any one of claims 1 to 4, wherein the lactic acid bacterium is Lactobacillus gasseri.   A lactic acid bacterium having a purine-capturing action obtained by the method according to any one of claims 1 to 5.   The purine body capture | acquisition agent which contains the lactic acid bacteria which have the capture | acquisition effect | action of a purine body obtained by the method of any one of Claims 1-5 as an active ingredient.   The purine body capturing agent according to claim 7, which is used for reducing serum uric acid level.   The purine body capturing agent according to claim 7 or 8, wherein the lactic acid bacterium is Lactobacillus gasseri OLL2959 strain (Accession No. NITE BP-224).   Food-drinks or a pharmaceutical containing the purine body trapping agent of any one of Claims 7-9.   The food or drink or pharmaceutical product according to claim 9, which is for reducing purine bodies in the intestinal tract.   The food-drinks or pharmaceutical product of Claim 10 or 11 which makes a human subject which shows the serum uric acid level of 6-8 mg / dL a subject of administration. The food-drinks or pharmaceutical of any one of Claims 10-12 which contain the said lactic acid bacteria by 1 * 10 < ⁸ > -1 * 10 < ¹⁰ > cfu per dose.

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