JP6782166B2 - Lactic acid bacteria capable of taking up purines and their uses - Google Patents

Description

The present invention relates to a lactic acid bacterium having an ability to take up purines and its use.

In recent years, the number of gout patients and hyperuricemia patients has been increasing year by year due to changes in eating habits in Japan. In hyperuricemia, decreased uric acid excretion and excessive uric acid production occur, and increased serum uric acid levels often induce gout, which causes severely painful acute arthritis. Currently, there are an estimated 1 million gout patients and an estimated 10 million hyperuricemia patients in Japan. At present, hyperuricemia is mainly prevented and treated by controlling serum uric acid levels by combining diet therapy, exercise therapy, and medication. In the diet, the intake of dietary purines that are finally decomposed into uric acid is reduced by limiting the calorie intake, but it is not always easy to continue the strict calorie intake restriction. Therefore, more effective treatment methods for gout and hyperuricemia are desired. Furthermore, it is desired to develop foods that are effective in preventing gout and hyperuricemia and alleviating the symptoms.

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 a high resolution from purine nucleosides to purine bases. For example, Patent Documents 4 and 5 show that lactic acid bacteria have the resolution of purines. Such conventional microorganisms and fermented products having a function of reducing serum uric acid level promote the conversion of purine nucleosides to purine bases in the intestinal tract, and change from purine nucleosides that are easily absorbed from the intestinal tract to purine bases that are difficult to be absorbed from the intestinal tract. It has been thought that the conversion suppresses the absorption of purines and promotes their 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.

Japanese Unexamined Patent Publication No. 2008-005834 International release WO2011 / 102310 International release WO2004 / 112809 International release WO 2009/06970 No. Japanese Unexamined Patent Publication No. 2013-048636

An object of the present invention is to provide an efficient method (selection method) for obtaining lactic acid bacteria having an effect of reducing 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 its use.

As a result of diligent studies to solve the above problems, the present inventors have found that lactic acid bacteria such as Lactobacillus gasseri have a purine uptake ability and a high proliferative ability in the presence of purines. The presence of a strain having a strain, the correlation between the ability to take up purines and the ability to grow in the presence of purines in such lactic acid bacteria, and the administration (ingestion) of such lactic acid bacteria reduce serum uric acid levels. We have found that we can obtain it, and have completed the present invention.

That is, the present invention includes the following.
[1] A method for screening lactic acid bacteria, which comprises measuring the amount of purine uptake of lactic acid bacteria in a medium containing purines and selecting lactic acid bacteria having a purine-capturing action using the amount as an index.
[2] The method according to [1] above, wherein the purine in the medium is a purine base.
[3] The method according to [1] or [2] above, wherein the purines in the medium are labeled with a radioisotope.
[4] The above [1] to [4], which comprises measuring the growth amount of the lactic acid bacterium in a medium containing a purine and selecting the lactic acid bacterium having a purine-capturing action as an index together with the uptake amount of the purine. The method described in any of 3].
[5] The method according to any one of the above [1] to [4], wherein the lactic acid bacterium is Lactobacillus gasseri.
[6] A lactic acid bacterium having a purine-capturing effect obtained by the method according to any one of the above [1] to [5].
[7] A purine scavenger containing lactic acid bacteria having a purine scavenging action obtained by the method according to any one of the above [1] to [5] as an active ingredient.
[8] The purine scavenger according to [7] above, which is used for reducing serum uric acid levels.
[9] The purine scavenger according to the above [7] or [8], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2959 strain (accession number NITE BP-224).
[10] A food or drink or a drug containing the purine scavenger according to any one of the above [7] to [9].
[11] The food or drink or drug according to the above [10], which is used for reducing purines in the intestinal tract.
[12] The food or drink or drug according to the above [10] or [11], which is administered to a human subject having a serum uric acid level of 6 to 8 mg / dL.
[13] The food or drink or drug according to any one of the above [10] to [12], which contains the lactic acid bacterium in a dose of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu.

According to the present invention, lactic acid bacteria having an action of capturing purines and an action of reducing serum uric acid level can be efficiently obtained (selected). By using the lactic acid bacterium or the scavenger according to the present invention, purines can be efficiently captured.
This specification includes the contents of Japanese Patent Application No. 2014-234050 and Japanese Patent Application No. 2015-064201, which are the basis of the priority claim of the present application.

It is a graph which shows the purine uptake ability of the Lactobacillus gasseri OLL2959 strain. It is a graph which shows the proliferative ability of the Lactobacillus gasseri OLL2959 strain in the presence of purines. It is a figure which shows the uptake ability of adenine of a plurality of lactic acid bacteria strains. It is a graph which shows the growth ability of a plurality of lactic acid bacteria strains in the presence of adenine. It is a graph which shows the time-dependent change of the serum uric acid level in the human subject who continuously orally administered (orally ingested) the Lactobacillus gasseri OLL2959 strain. It is a graph which shows the purine uptake ability of the Lactobacillus gasseri OLL2959 strain in the animal which co-administered the Lactobacillus gasseri OLL2959 strain and the purine body. It is a graph which shows the result of having compared the uptake ability of adenine by the type of a lactic acid bacterium strain. It is a graph which shows the result of having compared the growth ability of a lactic acid bacterium strain in the presence of adenine by the type of a lactic acid bacterium strain. It is a graph which shows the turbidity (OD650) and the change with time of the amount of nucleic acid in the Lactobacillus gasseri OLL2959 strain. It is a graph which shows the time-dependent change of the amount of nucleic acid and the radioactivity in the nucleic acid in the Lactobacillus gasseri OLL2959 strain. It is a graph which shows the IMP absorption reduction effect of the Lactobacillus gasseri OLL2959 strain and the OLL2959 strain in the animal to which ¹⁴ C-IMP was administered. Radioactivity (dpm): mean ± 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 the Lactobacillus gasseri OLL2959 strain and the OLL2959 strain in the animal to which ¹⁴ C-IMP was administered. Radioactivity (dpm): mean ± 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 reducing effect of the Lactobacillus gasseri OLL2959 strain and the OLL2959 strain in the animal to which ¹⁴ C-hypoxanthine was administered. Radioactivity (dpm): mean ± 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), and hypoxanthine + OLL2959 group (hypoxanthine + OLL2959 strain administration group) are shown. It is a graph which shows the hypoxanthine absorption reducing effect of the Lactobacillus gasseri OLL2959 strain and the OLL2959 strain in the animal to which ¹⁴ C-hypoxanthine was administered. Radioactivity (dpm): mean ± 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), and hypoxanthine + OLL2959 group (hypoxanthine + OLL2959 strain administration group) are shown. It is a graph which shows the uptake ability (capture action) of hypoxanthine and inosin of Lactobacillus gasseri OLL2959 strain. It is a graph which shows the survival rate in yogurt of the 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. Purines perform various functions mainly in cells of the living body, and are responsible for transmitting genetic information as a component of nucleic acid, for example. The main purine bases are 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 bound to ribose, deoxyadenosin, deoxyguanosine, deoxyguanosine and deoxyxanthosine bound to deoxyribose. Purine nucleotides are compounds in which phosphoric acid is bound to purine nucleoside, and examples thereof include adenylic acid (AMP), guanylic acid (GMP), inosinic acid (IMP), and xanthylic acid (XMP).

Purines are supplied in vivo from food as dietary purines via intestinal absorption, and are newly biosynthesized from amino acids and the like by the de novo pathway. In addition, purines are biosynthesized via a salvage pathway in which purine bases produced by decomposition of purine nucleotides are reused to synthesize purine nucleotides.

In humans, purine nucleotides are ultimately metabolized to uric acid. For example, adenylic acid becomes adenosine by 5'-nucleotidase (5'-NT) activity, and adenosine is metabolized to hypoxanthine via inosine. Hypoxanthine is converted to xanthine by xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activities. Guanilic 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, xanthine and guanine) is converted to 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 by salvage enzyme activity (salvage pathway).

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

In the present invention, lactic acid bacteria having a purine-capturing action can be efficiently obtained (selected) by selecting (screening) lactic acid bacteria using the ability of purines to be taken up into the cells as an index. The present invention relates to a method for screening lactic acid bacteria, which comprises measuring the amount of purine taken up by lactic acid bacteria in a medium containing purines and obtaining (selecting) lactic acid bacteria having a purine-capturing action using the amount as an index. More specifically, in the present invention, lactic acid bacteria are cultured in a medium containing purines, the amount of purines taken up in the cells is preferably measured over time, and the lactic acid bacteria having a purine capture action is used as an index. The present invention relates to a method for screening lactic acid bacteria, including obtaining (selecting) the medium. The lactic acid bacterium having a purine-capturing action thus obtained is highly likely to have a serum uric acid level reducing action. Here, the fact that the amount of purines taken up by lactic acid bacteria is large, that is, that the purine cells of the lactic acid bacteria have a purine capture action, means that a large amount of purines in the living body, particularly in the intestinal tract, is captured by the lactic acid bacteria and in the intestinal tract. By being removed from the environment of, it means that the absorption of purines from the intestinal tract is suppressed. Since it is known that lactic acid bacteria are excreted without being absorbed from the digestive tract, purines captured by the lactic acid bacteria escape absorption from the intestinal tract and are excreted from the body together with the lactic acid bacteria. Therefore, in the present invention, lactic acid bacteria are cultured in a medium containing purines, the amount of uptake of purines in the cells is measured, and lactic acid bacteria having a purine-capturing action are selected using this as an index, and the obtained purines are obtained. It also relates to a method for screening a lactic acid bacterium having a serum uric acid level reducing action, which comprises acquiring (selecting) a lactic acid bacterium 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 a purine uptake ability exhibits a high proliferative ability in the presence of a purine body so as to correlate with the high purine uptake ability. Therefore, in the present invention, in addition to the above-mentioned selection using the purine uptake ability as an index, the purine capture action is obtained by confirming the enhancement of the proliferative ability of the selected lactic acid bacterium in the presence of the purine. It is also possible to acquire (select) lactic acid bacteria with higher accuracy. That is, the present invention measures the amount of growth of lactic acid bacteria having the ability to take up purines in a medium containing purines, and uses it as an index together with the amount of uptake of purines measured as described above to capture purines. It also relates to a method for screening lactic acid bacteria, including obtaining (selecting) lactic acid bacteria having. Further, in the present invention, lactic acid bacteria are cultured in a medium containing purines, the amount of growth of the bacteria is measured over time, and the amount of uptake of purines measured as described above is used as an index of the purines. It has a serum uric acid level reducing action, including selecting a lactic acid bacterium having a capturing action and acquiring (selecting) the obtained lactic acid bacterium having a purine trapping action as a lactic acid bacterium having a serum uric acid level reducing action. It is also related to the screening method for lactic acid bacteria. However, the amount of growth in the presence of purines may be measured and selection may be performed using the same as an index, or such selection may not be performed. Purines (for example, purine bases) taken up in large quantities by lactic acid bacteria are used for nucleic acid synthesis required for growth, which results in high growth ability of lactic acid bacteria.

The lactic acid bacterium used in the screening method of the present invention is not particularly limited, but is preferably a bacterium belonging to the genus Lactobacillus. Lactobacillus gasseri, Lactobacillus delbrueckii subsp. Burgalicus, Lactobacillus delbrueckii subsp. Burgalicus, Lactobacillus delbrueckii subsp. .lactis), Lactobacillus paracasei subsp. Paracasei, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus subsp. Paracasei Lactobacillus helveticus subsp. Jugurti), Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gallinarum, Lactobacillus subsp.・ Lactobacillus casei subsp. Rhamnosus, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus plantalum -Strains such as Lactobacillus reuteri can be mentioned, but Lactobacillus gasseri is particularly preferable. It is preferable that any strain of lactic acid bacteria used in the screening method of the present invention is used for screening after culturing in an appropriate medium (for example, MRS medium) to adjust the concentration. The medium used for screening may be any medium in which Lactobacillus gasseri can grow, but a minimum medium or a medium based on which purines are added or some components are replaced with purines is preferable. Table 1 shows an example of a particularly preferable minimum medium.

The purine contained in the medium may be a purine base, a purine nucleoside, and / or a purine nucleotide. In a preferred embodiment, the purine contained 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, inosin (IMP), and inosinic acid, respectively, with hypoxanthine being particularly preferred.

To measure the amount of purines taken up by lactic acid bacteria, purines labeled with a label that can be quantitatively detected, for example, radioisotopes or fluorescent substances, are used as part or all of the purines contained in the medium. A medium using a labeled purine can be preferably used. As the radioisotope, for example, ¹⁴ C is preferable. To determine the amount of purine uptake of lactic acid bacteria, for example, cultivate lactic acid bacteria in a medium containing purines, add TFA (trifluoroacetic acid) or the like after culturing for a certain period of time to stop the reaction, and label the purines after culturing. 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 cells at the start of culture. When the amount of purines taken up by lactic acid bacteria is significantly increased compared to the start of culture (0 minutes after the start of culture), the lactic acid bacteria have the ability to take up the purines into the cells (the ability to take up purines). Can be determined to have. When the amount of purines taken up by lactic acid bacteria increases significantly compared to the start of culture (0 minutes after the start of culture), the ability of the lactic acid bacteria to take up the purines into the cells (purine uptake ability) It can be judged to be high. Alternatively, when the amount of purine uptake of lactic acid bacteria is significantly increased as compared with the Lactobacillus gasseri JCM1130 strain, it is said that the ability of the lactic acid bacteria to take up the purines into the cells (purine uptake ability) is high. It can also be determined. In the present invention, the lactic acid bacterium thus determined to have the purine uptake ability can be obtained (selected) as the lactic acid bacterium having the purine capture action. Then, the lactic acid bacterium selected as the lactic acid bacterium having the purine capture action can be further acquired (selected) as the lactic acid bacterium having the serum uric acid level reducing action or a candidate thereof. The culture time of lactic acid bacteria is preferably the time up to an arbitrary time point in the induction period or the logarithmic growth period of the growth curve. For example, lactic acid bacteria can be cultured until 30 minutes and 60 minutes after the start of culture, and the purine uptake ability and the like can be measured. At this time, usually, the radioactivity of the purine labeled with the radioisotope may be measured using a liquid scintillation counter.

The amount of growth of lactic acid bacteria in the presence of purines is determined by, for example, the turbidity (typically, the absorbance at 650 nm) of the medium at the start of culturing and after culturing for a certain period of time when the lactic acid bacteria are cultured in a medium containing purines. Measurement and judgment can be made by measuring and calculating the difference between the two. When the amount of 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 the 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 the Lactobacillus gasseri JCM1130 strain, the lactic acid bacteria are significantly enhanced in the presence of the purines. It can also be determined to indicate the increased proliferative capacity. When a lactic acid bacterium has a purine uptake ability and exhibits an enhanced proliferative ability in the presence of the purine, it means that the lactic acid bacterium can highly assimilate the purine, that is, the lactic acid bacterium can assimilate the purine. It is supported that it can be highly captured, and that it may have an effect of reducing serum uric acid level. The culture time of lactic acid bacteria is preferably the time up to an arbitrary time point in the logarithmic growth phase of the growth curve. For example, lactic acid bacteria can be cultured until 4 hours and 6 hours after the start of culturing, and the growth ability and the like in the presence of purines can be measured.

In the above measurement, it is preferable that the lactic acid bacterium is inoculated and cultured in 1.0 × 10 ^{6 to} 1.0 × 10 ¹¹ cfu, for example 0.8 × 10 ⁷ to 3 × 10 ⁷ cfu, with respect to 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 effect of reducing serum uric acid level, for example, according to the method described in Examples described later. 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 the serum uric acid level (decrease in the serum uric acid level) is examined. It can be determined whether or not the lactic acid bacteria selected as described above have an effect of reducing the serum uric acid level.

The lactic acid bacteria selected as described above have a high purine uptake ability and preferably a high proliferative ability in the presence of purines, that is, have a high purine capture action. Such lactic acid bacteria typically have the effect of reducing serum uric acid levels. The lactic acid bacteria selected as described above exhibit purine uptake ability and high proliferative ability (that is, high purine assimilation ability) in the presence of purines in vivo (typically in the intestinal tract). As a result, the serum uric acid level can be reduced by capturing and reducing a large amount of purines in the living body (typically in the intestinal tract) and reducing the amount of purines absorbed. Purines that the selected lactic acid bacteria can or can capture are, for example, purine bases, purine nucleosides, and / or purine nucleotides. The purines that can be taken up by the selected lactic acid bacteria are not necessarily limited to the purines contained 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 purines that the selected lactic acid bacteria can take up 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 a lactic acid bacterium having a purine-capturing action, which is selected by the above screening method. This lactic acid bacterium typically has an action of reducing serum uric acid level.

In addition, the present invention is a purine scavenger containing a lactic acid bacterium having a purine scavenging action (hereinafter, also referred to as the lactic acid bacterium of the present invention) as an active ingredient, which can be obtained by the above screening method, preferably for oral administration. Purine scavengers are also provided. The purine scavenger of the present invention may contain a carrier or additive acceptable for oral administration in addition to the Lactobacillus gasseri bacterium of the present invention. The purine scavenger of the present invention may be a drug or composition containing the cells of the lactic acid bacterium of the present invention, or a fermented product, a culture, or a concentrate / dried thereof produced using the bacterium. It may be a substance or a drug or composition containing the same. The lactic acid bacterium according to the present invention contained in the purine scavenger of the present invention is preferably a viable cell. As described above, the purine scavenger of the present invention has an action of reducing purines in the intestinal tract by taking up purines of lactic acid bacteria, and therefore, for reducing purines in the intestinal tract, and thus in reducing serum uric acid levels. Can be suitably used for use. The lactic acid bacterium of the present invention and the purine body capturing agent of the present invention also take in purine bodies such as inosinic acid (IMP) and adenylic acid (AMP), which are umami components of fish and meat, into the cells and enter the body from the intestinal tract. Reduce its absorption into. Therefore, the lactic acid bacterium of the present invention and the purine scavenger 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 a umami component (here, a purine or purine nucleotide that is a umami component of fish or meat such as inosinic acid), which comprises the lactic acid bacterium of the present invention or the purine body capturing agent of the present invention. .. This umami component absorption reducing agent is also preferably for oral administration.

Preferable examples of the lactic acid bacterium having a purine-capturing action as described above include, but are not limited to, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri P14054ME002 strain. Be done. Lactobacillus gasseri OLL2959 strain is homolactic fermentable and has no gas-producing ability. Lactobacillus gasseri OLL2959 strain was dated March 31, 2006 (original deposit date), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan) After being deposited at Room 122, Postal Code 292-0818) with the deposit number NITE P-224, it was transferred to the deposit (international deposit) based on the Budapest Treaty on November 21, 2007, and the deposit number is NITE BP- It has been changed to 224. Alternatively, in another embodiment, the lactic acid bacterium having a purine-capturing action as described above may be a lactic acid bacterium or a Lactobacillus gasseri bacterium excluding the Lactobacillus gasseri OLL2959 strain.

The present invention also intends to use the purine scavenger according to the present invention in combination with foods and drinks or pharmaceuticals. Therefore, the present invention also provides a purine scavenger according to the present invention for use in combination with foods and drinks or pharmaceuticals.

The present invention also provides foods and drinks or pharmaceuticals containing the purine scavenger according to the present invention. The food or drink or pharmaceutical product of the present invention positively takes up purines into the cells and assimilate them in the subject to whom the drug is administered (ingested), thereby reducing the purines in the intestinal tract and the effect of serum uric acid level. Reduction can be achieved. Therefore, the foods and drinks and pharmaceuticals of the present invention may be used for reducing purines in the intestinal tract. The "purine body in the intestinal tract" as used in the present invention does not include purines held by bacteria (lactic acid bacteria, etc.), fungi, viruses, subject cells, etc. existing in the intestinal tract. The food and drink and pharmaceutical products of the present invention may be used for reducing serum uric acid levels based on the reduction of purines in the intestinal tract. The food or drink or pharmaceutical product containing the purine scavenger of the present invention can be suitably used for, for example, prevention, treatment, improvement or alleviation of symptoms of gout and hyperuricemia.

In the present specification, the “food and drink” is not particularly limited, but includes beverages, foods and functional foods. The type of food or drink according to the present invention is not particularly limited, and examples of the beverage include fermented milk (drink yogurt, etc.), lactic acid bacteria beverage, dairy beverage (coffee milk, fruit milk, etc.), and tea-based beverage (green tea, tea). , Karyu tea, etc.), fruit / vegetable beverages (beverages containing fruit juices such as oranges, apples, grapes, vegetable juices such as tomatoes, carrots), alcoholic beverages (beer, sparkling liquor, wine, etc.), carbonated beverages, soft drinks , Water and the like, suitable beverages include drink yogurt, lactic acid bacteria beverage, dairy beverage, water-based beverage and the like, and particularly suitable beverages include drink yogurt. For the manufacturing method of various beverages, existing reference books such as "Latest Soft Drinks" (2003) (Korin Co., Ltd.) can be referred to. Further, for example, examples of foods include fermented milk (set type yogurt, soft yogurt, cheese, etc.), dairy products, confectionery, instant foods, etc., and suitable foods include set type yogurt, soft yogurt, confectionery, etc. Examples of particularly suitable beverages include set-type yogurt and soft yogurt. Existing reference books can be referred to for manufacturing methods of various foods.

Fermented milk such as yogurt containing lactic acid bacteria having a purine-capturing effect may contain, for example, other microorganisms such as lactic acid bacteria that may or may not have a purine-capturing effect. It may be produced by adding lactic acid bacteria having a purin-capturing action to dairy products or fermented milk produced using a starter. Dairy products and fermented milk using a starter can be produced according to a conventional method. For example, yogurt can be produced by mixing a starter with milk or a dairy product cooled after heating, mixing, homogenization, and sterilization, and fermenting and cooling. The present invention uses a lactic acid bacterium having a purin-capturing action in the production of dairy products such as yogurt and cheese and fermented milk (preferably, the lactic acid bacterium is added (blended) to the dairy product, fermented milk or its raw material). Included), and it is particularly preferable to use the lactic acid bacterium in the production of yogurt. Furthermore, the present invention also comprises a method for reducing purines based on the purine-capturing action of lactic acid bacteria in the production of fermented milk such as yogurt and cheese and dairy products using lactic acid bacteria having a purine-capturing action as an active ingredient. provide. The lactic acid bacterium having a purine-capturing action according to the present invention (for example, Lactobacillus gasseri OLL2959 strain) can exhibit good survivability in fermented milk such as yogurt and dairy products.

As the food and drink according to the present invention, functional foods are particularly preferable. The "functional food" of the present invention means a food having a certain functionality with respect to the living body, for example, food for specified health use (including conditional food [food for specified health use]) and nutritional function in Japan. Health functional foods including foods, functional foods, special purpose foods, nutritional supplements, health supplements, supplements (for example, various dosage forms such as tablets, coated tablets, sugar-coated tablets, capsules and liquids) and It includes all so-called health foods such as beauty foods (for example, diet foods). In addition, 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 preferable examples of the functional food of the present invention include foods for the sick, powdered milk for pregnant and lactating women, prepared powdered milk for infants, foods for the elderly, foods for nursing care, and other special purpose foods. ..

The functional food of the present invention is particularly useful in reducing serum uric acid levels by reducing purines in the intestinal tract. The functional food of the present invention is used for reducing serum uric acid levels, particularly reducing purines in the intestinal tract by taking up purines by lactic acid bacteria and promoting the growth of lactic acid bacteria, and as a result, reducing the absorption of purines in the intestinal tract. It can be suitably used for reducing the accompanying serum uric acid level.

Foods and drinks such as functional foods of the present invention (preferably foods for specified health use or conditional foods [foods for specified health use]) may be used for reducing purines in the intestinal tract, or have serum uric acid levels. It may be for suppressing or alleviating a decrease or an increase in serum uric acid level, and may be described or indicated to that effect. The present invention may be the use of a lactic acid bacterium having a purine-capturing action in the production of such a functional food (preferably including adding (blending) the lactic acid bacterium to a functional food or a raw material thereof). preferable.

The functional food of the present invention may be a solid preparation such as tablets, granules, powders, pills, capsules, a liquid preparation such as a liquid preparation, a suspension preparation, a syrup preparation, or a gel preparation or a paste preparation. However, it may be in the shape of a normal food or drink (for example, beverage, yogurt, confectionery, etc.).

The food or 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, sugars, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Examples of proteins include whole fat powder, defatted milk powder, partially defatted milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lacto Animal and vegetable proteins such as globulin, α-lactoalbumin, lactoferrin, soybean protein, chicken egg protein, meat protein, these hydrolysates, butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, lactose Various milk-derived components and the like can be mentioned. Examples of carbohydrates include general sugars, modified starches (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber and the like. Examples of lipids include animal fats and oils such as lard, fish oil, and other separated oils, hydrogenated oils, and ester exchange oils; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, and these separated oils. Examples thereof include vegetable oils and fats such as hydrogenated oils and ester exchange 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, and choline. , Folic acid and the like, and examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, milky minerals and the like. 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, and may be added using a synthetic product and / or a food containing a large amount thereof.

In addition, the functional food containing the lactic acid bacterium or purine scavenger of the present invention may contain an orally acceptable carrier or additive in addition to the lactic acid bacterium or purine scavenger of the present invention. Carriers include, for example, water, organic solvents acceptable for oral administration, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, Arabia. Examples thereof include rubber, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, 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 buffer, a flavoring agent, a preservative, a coloring agent and the like. These carriers or additives can be used alone or in combination of two or more, and can be appropriately used depending on the dosage form of the preparation. The functional food of the present invention may further contain other functional ingredients.

Further, the pharmaceutical product (pharmaceutical composition) containing the lactic acid bacterium or purine scavenger of the present invention is, in addition to the lactic acid bacterium or purine scavenger of the present invention, a pharmaceutically acceptable carrier or additive, particularly for oral use. It may contain an acceptable carrier or additive. Carriers include, for example, water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, 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, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, liposome, etc. Artificial cell structures and the like. Examples of the additive include a binder, an excipient, a lubricant, a disintegrant, a wetting agent, a stabilizer, a buffer, a flavoring agent, a preservative, a coloring agent and the like. These carriers or additives can be used alone 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 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 tablets, granules, powders, pills and capsules, a gel preparation, or a liquid preparation such as a liquid preparation, a suspension preparation and a syrup preparation. ..

In the lactic acid bacterium, purine capture agent, food and drink or pharmaceutical product of the present invention, the dose (intake) thereof shall be determined in consideration of the age and body weight of the subject to be administered (intake), the route of administration, the number of administrations, and the like. It can be changed extensively at the discretion of the vendor. Therefore, in the lactic acid bacterium, purine body capturing agent, food and drink or pharmaceutical product of the present invention, the dose of the lactic acid bacterium of the present invention (Lactobacillus gasseri, etc.) is not particularly limited, 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, and 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 preferable. The purine scavenger, food or drink or pharmaceutical product 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, preferably 1 × 10 ⁸ to 1 ×. more preferably contains in an amount of 10 ¹⁰ cfu, more preferably contained in an amount of ^{1 × 10 9 ~1 × 10 10} cfu, for example, a ^{4 × 10 9 ~6 × 10 10} cfu amount It is particularly preferable to contain in.

In one embodiment of the present invention, the lactic acid bacterium, purine scavenger, food or drink or pharmaceutical product of the present invention is applied to a subject at least once a day, preferably at least twice a day, more preferably twice a day. Administered (or ingested by the consumer). The lactic acid bacterium, purine scavenger, food or drink or pharmaceutical product of the present invention may be continuously administered to a subject, for example, daily. In this case, the lactic acid bacterium, purine scavenger, food or drink or pharmaceutical product 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 scavenger, food and drink or pharmaceutical product of the present invention, when continuously administered to a subject, the dose of the lactic acid bacterium of the present invention is 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. Is preferable, the amount of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, and 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 scavenger, food or drink or pharmaceutical product of the present invention may be administered in a single dose. In the purine scavenger, food and drink or pharmaceutical product of the present invention, when administered once to a subject, the dose of the lactic acid bacterium of the present invention is 1 × 10 ⁵ to 1 × 10 ¹¹ cfu per dose. Preferably, the amount of 1 × 10 ⁸ to 1 × 10 ¹⁰ cfu is more preferable, and the amount of 1 × 10 ⁹ to 1 × 10 ¹⁰ cfu is more preferable, for example, 4 × 10 ^{9 to} 6 × 10 ¹⁰ cfu. The amount is particularly preferred. The lactic acid bacterium, purine scavenger, food or drink or pharmaceutical product of the present invention is preferably orally administered (orally ingested).

In the present invention, "administration" includes both "ingestion" generally used for foods and drinks and "administration" used for pharmaceuticals. In the present invention, "oral administration" includes administration or ingestion by mouth, as well as administration by tube feeding via a nasal tube, gastric fistula tube, or the like. 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 purines in the intestinal tract and reducing serum uric acid levels, which comprises the lactic acid bacterium or purine scavenger of the present invention, is also provided.

The subject to which the lactic acid bacterium, purine body capture agent, food or drink or pharmaceutical product of the present invention is administered is a mammal including humans, domestic animals, pet animals, experimental (test) animals and the like, and a human subject is preferable. Human subjects with gout and / or hyperuricemia are more preferred, and human subjects with serum uric acid levels of 6 mg / dL or higher, eg 6-10 mg / dL, are even more preferred. In one embodiment, a human subject with mild to borderline hyperuricemia showing a serum uric acid level of 6-8 mg / dL is more preferred as the subject of administration. The present invention also administers (ingests) the purine-capturing agent, food or drink or drug 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. Also provided is a method of capturing purines in the cells of lactic acid bacteria, thereby reducing purines in the intestinal tract and reducing serum uric acid levels. The present invention also provides a method for capturing purines in the cells of lactic acid bacteria by contacting the lactic acid bacterium or purine capture agent of the present invention with the purines. The present invention also provides the use of the lactic acid bacterium or purine scavenger of the present invention for imparting a purine scavenging effect 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 in more detail with reference to Examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Evaluation test of purine uptake ability In this example, a purine body of Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain was used in a purine body labeled with a radioisotope (RI). The uptake ability was evaluated.

Lactobacillus gasseri (Lactobacillus gasseri) OLL2959 strain was dated March 31, 2006 (original deposit date), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (Kisarazu City, Chiba Prefecture, Japan) After being deposited at Kazusakamatari Room 2-5-8 122, postal code 292-0818) with a deposit number of NITE P-224, it was transferred to a deposit (international deposit) based on the Budapest Treaty on November 21, 2007. , The accession number has been changed to NITE BP-224. Lactobacillus gasseri OLL2959 strain was inoculated into MRS medium (Lactobacilli MRS Broth, Difco), and the culture (4 to 7 × 10 ⁸ cfu / ml) cultured at 37 ° C. for 16 to 20 hours was used below.

Minimal medium (DM medium; Table 1): in 0.1 mL, radioisotopes ¹⁴ C labeled with adenylate (AMP), adenosine, or adenine (respectively ¹⁴ C-AMP, ¹⁴ C- adenosine, ¹⁴ C-adenine) and Add to a final concentration of 20 μM, then inoculate the culture medium of the Lactobacillus gasseri OLL2959 strain prepared above at 2% by weight (0.002 mL: 0.8 to 1.4 × 10 ⁶ cfu) at 37 ° C. , Anaerobic culture for 30 minutes.

Then, a TFA solution (trifluoroacetic acid, 5%) was added to these media, and then the cells were washed with physiological saline and then radiated with a liquid scintillation counter (Aroca, LSC-6100). The activity was measured. As a control (0 minutes), TFA solution (5%) was added immediately after sample preparation, then the cells were washed with saline and then radioactivity was measured in the same manner as above. The result is shown in FIG. In FIG. 1, the unit of radioactivity (vertical axis) indicating the amount of ¹⁴ C-labeled purines in the cells is disintegrations per minute (dpm), which is the number of radioactive substances that decay 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 60-minute culture using the DM medium.

As a result, the Lactobacillus gasseri OLL2959 strain has the ability to take up purines adenylic acid (AMP), adenosine, and adenine into the cells (purine uptake ability), especially adenine into the cells. It was shown that the ability to take up purines (purine uptake ability) is high (Fig. 1).

[Example 2] Evaluation test of proliferative ability in the presence of purines In this example, the Lactobacillus gasseri OLL2959 strain was cultured in the presence of purines, and the proliferative ability in the presence of purines was evaluated.

DM medium (Table 1): Adenylic acid (AMP), adenosine, or adenine as a purine was added to 1 mL to a final concentration of 400 μM, and then Lactobacillus gasseri OLL2959 strain prepared in Example 1 was added. The culture medium was inoculated at 4% by weight (0.04 mL: 1.6 to 2.8 × 10 ⁷ cfu) and anaerobically cultured at 37 ° C. Then, 0 hours, 4 hours and 6 hours after the start of this culture, the turbidity of the medium (absorbance at 650 nm) was measured. As a control, the Lactobacillus gasseri OLL2959 strain was cultured by the same method except that purines were not added to the minimum medium, and the turbidity of the medium was measured. The result is shown in FIG.

As a result, it was shown that the Lactobacillus gasseri OLL2959 strain has enhanced proliferative ability in the presence of adenylic acid (AMP), adenosine, or adenine, and in particular, the proliferative ability is further enhanced in the presence of adenine. (Fig. 2).

[Example 3] Comparative test of uptake ability of adenine 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 uptake ability of Adenine and the ability to proliferate in the presence of adenine were compared.

As other Lactobacillus gasseri strains, Lactobacillus gasseri P14054ME001 strain and P14054ME002 strain were used. Lactobacillus gasseri P14054ME001 strain and P14054ME002 strain had the same proliferative capacity as Lactobacillus gasseri OLL2959 strain when cultured in MRS medium (Lactobacilli MRS Broth, Difco) without purines for 20 hours. It was equivalent (Table 2).

The evaluation of the uptake ability of adenine was carried out in the same manner as in Example 1 except that only adenine ( ¹⁴ C-adenine) was used as a purine labeled with the radioisotope ¹⁴ C. The result is shown in FIG. Although not as high as the Lactobacillus gasseri OLL2959 strain, it was confirmed that the Lactobacillus gasseri P14054ME002 strain also has a high adenine uptake ability (Fig. 3). Compared with the Lactobacillus gasseri OLL2959 strain and the P14054ME002 strain, it was confirmed that the Lactobacillus gasseri P14054ME001 strain had a lower ability to take up adenine (Fig. 3).

In the evaluation of proliferative ability in the presence of adenine, adenine was added to DM medium (Table 1): 1 mL so that the final concentration was 400 μM, and then the culture of the Lactobacillus gasseri OLL2959 strain prepared in Example 1 was cultured. Inoculate the solution and the culture medium of P14054ME001 strain or P14054ME002 strain prepared in the same manner as in Example 1 at 4% by weight (0.04 mL: 1.6 to 2.8 × 10 ⁷ cfu) at 37 ° C. Was anaerobically cultured in. Then, 0 hours, 4 hours and 6 hours after the start of this culture, the turbidity of the medium (absorbance at 650 nm) was measured. The result is shown in FIG. Similar to the Lactobacillus gasseri OLL2959 strain, the Lactobacillus gasseri P14054ME001 and P14054ME002 strains also showed enhanced proliferative capacity in the presence of adenine. In addition, the degree of enhancement of the proliferative ability of the Lactobacillus gasseri OLL2959 strain was extremely stronger than that of the Lactobacillus gasseri P14054ME001 strain and the P14054ME002 strain. Compared with the Lactobacillus gasseri P14054ME001 strain, the Lactobacillus gasseri P14054ME002 strain had a stronger degree of enhancement of its proliferative ability.

From these results, it was shown that in Lactobacillus gasseri, the enhancement of the proliferative ability in the presence of adenine correlates with the high uptake ability of adenine. It was shown that some lactic acid bacteria have a high ability to assimilate adenine.

[Example 4] Evaluation test of reducing serum uric acid level of Lactobacillus gasseri OLL2959 strain Human subjects suspected of having mild to borderline hyperuricemia were allowed to continuously ingest Lactobacillus gasseri OLL2959 strain. A placebo-controlled, double-blind, controlled trial was used to examine the effect on uric acid levels (human study).

14 adult males aged 35 years or older (mean age 44.3 years) with uric acid levels of 6-8 mg / dL at pre-study test were active with the placebo group so that there was no significant difference in uric acid levels and age. It was assigned to 2 groups. The placebo group was fed 2 yogurts (85 g / piece) / day without the Lactobacillus gasseri OLL2959 strain for 4 weeks. The active group was ingested 2 (85 g / piece) / day of yogurt given to the placebo group containing Lactobacillus gasseri OLL2959 strain at 1 × 10 ⁸ cfu / g for 4 weeks. Two yogurts / day were ingested twice after breakfast, lunch, or supper.

Blood tests were performed on each subject at the start of the test (before the test meal intake), 2 weeks and 4 weeks later (test meal intake period), and the serum uric acid level was measured by a conventional method. The amount of change in serum uric acid level at each time point compared to the serum uric acid level at the start of the test was calculated, and the change in serum uric acid level during the test period was statistically analyzed by repeated measurement two-way ANOVA. It was. The results are shown in FIG.

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

[Example 5] Single-dose test of Lactobacillus gasseri (animal test)
Unlike humans, rats have uricase, which is a uric acid-degrading enzyme, so it is necessary to administer potassium oxonate, which is a uricase inhibitor, in order to raise serum uric acid levels. Therefore, 0.5 g / kg of potassium oxonate is orally administered to Wistar rats (male) fasted for 16 hours. 60 minutes after administration of potassium oxonate, dry yeast (suspended in water for injection) and Lactobacillus gasseri OLL2959 strain (suspended in physiological saline) are orally administered to the lactic acid bacteria group due to purine loading. In the negative group, water for injection is administered instead of dry yeast, and physiological saline is administered instead of Lactobacillus gasseri. The control group is administered saline along with dry yeast in place of Lactobacillus gasseri.

After forced oral administration, blood is collected over time (30 minutes, 60 minutes, 90 minutes, 120 minutes, and 150 minutes), and the blood uric acid level of each blood sample is measured by a conventional method to determine the blood uric acid level. Examine the transition. If the blood uric acid level in the lactic acid bacteria group remained lower than that in the control group, it was shown that the amount of purines absorbed from the intestinal tract decreased due to the effect of purine uptake of the Lactobacillus gasseri OLL2959 strain even after a single administration. Is done.

[Example 6] Ability to take up purines of Lactobacillus gasseri (animal test)
When the ability of lactic acid bacteria to take up purines is high, when an animal subject is simultaneously administered (ingested) with lactic acid bacteria and purines, the absorption of purines in the subject is compared with the case where the purines are ingested alone. Is thought to be suppressed. Therefore, in order to test the purine uptake ability of Lactobacillus gasseri, an animal experiment was conducted according to the following procedure.

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

Then, the test substance was orally administered by gavage. Here, these test substances include physiological saline in the negative group and adenylic acid ( ¹⁴ C-AMP: 57.6 mCi / mmol, 0.1 mCi / ml) labeled with the radioisotope ¹⁴ C in the AMP-administered group. 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 Co., Ltd.) were used. In the AMP group and the 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 were placed in a holder under anesthesia, the tail vein was injured with a scalpel, and a hematocrit tube was used. Then, 60 μL of these spouted blood was collected. An equal volume of 2 mg / mL EDTA-2Na solution (EDTA-2Na dissolved in physiological saline) was added to these collected blood. Rats were immediately killed by inhalation of carbon dioxide after completion of these tests.

The radioactivity of these collected blood was measured using a liquid scintillation counter (LSC-6100 manufactured by Aloka). The result is shown in FIG.
As shown in FIG. 6, significant differences were observed in the amount of purines absorbed 30, 45 and 60 minutes after the administration when the blood concentration reached its peak (* p <0.05, ** p <0.01, t-test). From this result, it was shown that the amount of purines absorbed from the intestinal tract can be suppressed by ingesting the Lactobacillus gasseri OLL2959 strain.

[Example 7] Single-dose test of Lactobacillus gasseri (human test)
A healthy male aged 20 years or older was given a single dose of 498 mg of a purine preparation (mixture of 5'-adenylic acid, 5'-disodium inosinate and 5'-disodium guanate) for 30 minutes, 60. Blood is collected after minutes, 120 minutes, and 150 minutes, and the transition of blood uric acid level is examined. Ten subjects showing similar transitions will be selected and targeted for this study. In this study, the selected subjects received 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 containing no such bacteria (placebo group). Blood is collected 30 minutes, 60 minutes, 120 minutes, and 150 minutes after ingestion, the blood uric acid level is measured for each blood sample by a conventional method, the transition of the blood uric acid level is examined, and a crossover test is performed. .. It was shown that if the increase in uric acid level was suppressed in the active group compared with the placebo group, the amount of purine absorbed from the intestinal tract decreased due to the effect of purine uptake of the Lactobacillus gasseri OLL2959 strain even in a single dose. Is done.

[Example 8] Comparative test of types of lactic acid bacteria strain (1) Comparative test of uptake ability of adenine In a medium containing adenine ( ¹⁴ C-adenine) labeled with a radioisotope (RI), with Lactobacillus gasseri OLL2959 strain , Lactobacillus gasseri JCM1130 strain was cultivated, and the influence of the type of lactic acid bacterium strain was compared on the uptake ability of adenine. The Lactobacillus gasseri JCM1130 strain can be obtained as JCM1130 from the Japan Collection of Microorganisms (RIKEN BRC JCM; Tsukuba City, Ibaraki Prefecture, Japan), RIKEN BioResource Center.
Here, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured using MRS medium, respectively, and their proliferative ability was evaluated in advance. That is, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were anaerobically cultured at 37 ° C. for 20 hours using MRS medium, respectively. 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. From this, it was shown that when the Lactobacillus gasseri OLL2959 strain and the Lactobacillus gasseri JCM1130 strain were cultured in the same medium, the growth ability of the Lactobacillus gasseri JCM1130 strain was basically high (Table). 3).

In the comparative test of adenine uptake ability, first, ¹⁴ C-adenine was added to the minimum medium (Table 1) so as to have a final concentration of 20 μM to prepare the medium for this test. Next, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured using MRS medium, respectively, and then these cultures were inoculated into the medium of this test at 37% by weight and 37 ° C. Was anaerobically cultured in. These cultures were adjusted to have the same number of bacteria using MRS medium.

At the start of these cultures (0 minutes) and 30 or 60 minutes after the start of the culture, a 5% TFA solution was added to stop the culture, and then the cells were washed with physiological saline. These radioactivitys were measured using a liquid scintillation counter (LSC-6100, manufactured by Aloka). The result is shown in FIG. Here, the radioactivity (vertical axis) in FIG. 7 is represented by the number of radioactive substances disintegrating per minute (dpm).

As shown in FIG. 7, both Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain exhibited the ability to take up adenin, but lacto was observed 30 and 60 minutes after the start of these cultures. Compared with the Bacillus gasseri JCM1130 strain, the Lactobacillus gasseri OLL2959 strain took up a large amount of adenin, and a significant difference was observed in the amount of adenin uptake (p <0.05, t-test).
From this result, it was clarified that the Lactobacillus gasseri OLL2959 strain can take up a significantly larger amount of purines than the Lactobacillus gasseri JCM1130 strain, which has a high proliferative ability in the MRS medium.

(2) Comparative test of proliferative ability in the presence of adenine In the presence of adenine, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultivated, and the influence of the type of lactic acid strain on the proliferative ability of the cells. Was compared.
Adenine was added to the minimum medium (Table 1) to a final concentration of 400 μM to prepare the medium for this test. Next, Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain were cultured using MRS medium, respectively, and then these cultures were inoculated into the medium of this test at 4% by weight to 37. It was anaerobically cultured at ° C. The turbidity (absorbance at 650 nm) was measured at the start of these cultures (0 hours) and 4 and 6 hours after the start of the culture. The result is shown in FIG.

As shown in FIG. 8, in both Lactobacillus gasseri OLL2959 strain and Lactobacillus gasseri JCM1130 strain, the proliferative ability was enhanced in the presence of adenine, but the proliferative ability in the MRS medium was high. Compared with the Lactobacillus gasseri JCM1130 strain, the Lactobacillus gasseri OLL2959 strain was found to have a significantly higher degree of enhancement of proliferative capacity (p <0.05, t-test). Therefore, it was shown that the Lactobacillus gasseri OLL2959 strain was particularly strongly enhanced in its proliferative capacity in the presence of purines.

[Example 9] Utilization of purines for nucleic acid synthesis A test was conducted to verify how the Lactobacillus gasseri OLL2959 strain utilizes adenine in the process of proliferating after taking up adenine.

The cells of the Lactobacillus gasseri OLL2959 strain in culture were collected, prepared to 1.0 × 10 ¹⁰ (1.0E + 10) cfu / ml, and in a minimum medium containing 400 μM adenine at a final concentration of 6%. Inoculated. After that, 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 were collected by centrifuging the medium at 3,000 rpm for 10 minutes at 4 ° C, washed twice with distilled water, and then the nucleic acid was extracted from the cells using the DNA extraction kit ISOPLANT II (Nippon Gene). went. The concentration of the extracted nucleic acid was measured using a spectrophotometer. Then, the whole amount of the extracted nucleic acid was added to the vial containing the liquid scintillation cocktail, and the radioactivity was measured with a liquid scintillation counter.

As a result, the turbidity increased in a culture time-dependent manner (OLL2959 strain proliferated), and the nucleic acid amount (DNA concentration) also increased in a culture time-dependent manner (Fig. 9). Furthermore, as the amount of nucleic acid increased, the radioactivity in the nucleic acid also increased (Fig. 10), indicating that the OLL2959 strain takes up adenine and uses it for the synthesis of nucleic acid necessary for cell proliferation.

[Example 10] Evaluation of uptake ability of hypoxanthine and IMP (inosinic acid) of Lactobacillus gasseri (animal test)
Twenty-eight male Wistar rats (8 weeks old) were acclimatized for about one week after purchase. Rats were fasted for about 16 hours from the day before the test, and weight was measured after the fast. Negative control (physiological saline) group, IMP (radioisotope ¹⁴ C-IMP) administration group, and IMP + OLL2959 strain (radioisotope ¹⁴ C-IMP and OLL2959 strain) administration group based on the body weight of rats after fasting. (8 animals only in the negative control group, 10 animals each in the other groups).

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

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

In all cases, 60 μL of blood was collected from the tail vein of rats without anesthesia at 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes and 180 minutes after administration of the test substance. An equal volume of 2 mg / mL EDTA-2Na solution was added to the collected blood, and the mixture was ice-cooled.

The radioactivity of the collected blood was measured with a liquid scintillation counter.

A similar animal study was performed using ¹⁴ C-hypoxanthine instead of ¹⁴ C-IMP. In this test using ¹⁴ C-hypoxanthine, a total of 3 groups (negative control group only 4 animals, other groups each) were negative control (saline administration) group, hypoxanthine administration group, and hypoxanthine + OLL2959 strain administration group. 5 animals) were used.

The results are shown in FIGS. 11 to 14. As shown in FIGS. 11 and 12, the amount of IMP absorption was significantly reduced in rats given a single dose of Lactobacillus gasseri OLL2959 strain before and after the peak blood concentration of the test substance (*). p <0.1, IMP administration group vs. IMP + OLL2959 strain administration group, Student's t-test). Similarly, as shown in FIGS. 13 and 14, hypoxanthine absorption was significantly reduced in single-dose rats of the Lactobacillus gasseri OLL2959 strain (## p <0.01, # p <0.05, hippo). Xanthine-administered group vs. hypoxanthine + OLL2959 strain-administered group, Student's t-test). From these results, the effect of reducing purine absorption by a single administration of Lactobacillus gasseri OLL2959 strain was further shown. Since IMP is also an umami component of fish and meat, it is of great significance to confirm the absorption-reducing effect on 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 gasseri OLL2959 strain prepared with PBS to 1.0 × 10 ⁸ cfu / ml, and incubate at 37 ° C for 15 minutes or 30 minutes. did. After incubation, the cells were collected and their radioactivity was measured using a liquid scintillation counter.

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

[Example 12] Evaluation of viability 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. Viable cell counts were measured on (0th day), 7th, 21st and 28th days. The survival rate at each time point was expressed as the 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 the Lactobacillus gasseri OLL2959 strain added to saline was 10.6% on the 7th day and 0.0% on the 21st and 28th days (Fig. 16).

These results indicate that the Lactobacillus gasseri OLL2959 strain has higher survivability in yogurt than saline.

According to the present invention, it is possible to efficiently obtain a lactic acid bacterium having a purine-capturing action, which has a high purine uptake ability and a proliferative ability in the presence of a purine. Such lactic acid bacteria can reduce the amount of purines administered in the intestinal tract, thereby reducing the amount of purines absorbed in the intestinal tract, resulting in a reduction in serum uric acid levels. Therefore, the present invention is also useful in developing an oral preparation for reducing purines in the intestinal tract and reducing serum uric acid levels.

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

Claims (4)

By measuring the amount of purine base taken up by lactic acid bacteria in a medium containing purine base and comparing it with the amount of uptake of purine base of Lactobacillus gasseri JCM1130 strain, lactic acid bacteria having higher purine base uptake ability. A method for screening lactic acid bacteria, which comprises selecting a lactic acid bacterium, wherein the lactic acid bacterium is Lactobacillus gasseri. The method according to claim 1, wherein the purine base in the medium is labeled with a radioisotope, and the amount of purine base taken up by lactic acid bacteria is measured based on the radioactivity of the lactic acid bacteria cells. The method according to claim 1 or 2, wherein the growth amount of the lactic acid bacterium in a medium containing a purine base is measured, and the lactic acid bacterium having a higher growth ability is further selected using the lactic acid bacterium as an index. Lactic acid bacteria are selected by the method according to any one of claims 1 to 3, and the purine base in the intestinal tract is obtained by incorporating the purine base into the cells of the lactic acid bacterium containing the obtained lactic acid bacterium as an active ingredient. A method for producing a food or drug, which comprises producing a food or drug for reduction.

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