Fermented tea composition for intestinal regulation and its manufacturing method

Abstract

To provide a novel fermented tea composition for controlling an intestinal function such as control of intestinal bacterial flora and shortening of a digestive tract passage time, and a method of producing the same.SOLUTION: A fermented tea composition for controlling an intestinal function includes as active principle a fermented tea substance by at least one type of lactic acid bacteria selected from a group consisting of Lactobacillus pentosus, Lactobacillus vaccinostercus, Lactobacillus coryniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a fermented tea composition for intestinal regulation and a method for producing the same, and more specifically to a fermented tea composition for intestinal regulation, such as for regulating intestinal flora and shortening digestive tract transit time, and a method for producing the same. [Background technology]

[0002] Constipation is caused by various factors such as poor eating habits, lack of exercise, and stress, and is a symptom experienced by many people, so it tends to be overlooked. However, constipation not only reduces QOL, but has also been reported to increase the risk of developing coronary artery disease and ischemic stroke, as well as the risk of death. In recent years, it has been considered important to maintain normal bowel movements. On the other hand, constipation is closely related to the intestinal flora, and it has also been reported that changes in the intestinal flora contribute to the improvement of constipation. For example, Xiaolong Ge et al., 2017, Scientific Reports 7:441 (Non-Patent Document 1) reported that the Shannon-Wiener diversity index (H') value of bacteria in the feces of constipated patients was significantly higher than that of healthy subjects, and Kimura Osamu et al., 1971, Effect of wheat germ on the intestinal flora of constipated adults, Japanese Journal of Bacteriology 26:5-6, p.222-227 (Non-Patent Document 2) reported that, along with improvement of constipation, there was an increase in the number of bacteria of the genus Lactobacillus and Bifidobacterium and a decrease in the number of bacteria of the genus Catenabacterium in the feces. In addition, the intestinal flora is thought to play various other roles such as digestive support, immune response regulation, and anti-inflammatory properties.

[0003] In addition, lactic acid bacteria such as Lactobacillus genus inhabit the human intestinal tract and are considered to be useful intestinal bacteria that exert various physiological activities such as infection prevention, prevention of intestinal putrefaction, and promotion of intestinal motility. For example, JP 2018-85953 A (Patent Document 1) describes a specific Lactobacillus plantarum as a lactic acid bacterium that is intended to be established in the intestinal tract by oral ingestion.

[0004] On the other hand, it is known that lactic acid bacteria can be used to prepare fermentation products from various substrates. For example, Japanese Patent Application Laid-Open No. 2016-167984 (Patent Document 2) describes a fermentation product produced by inoculating Lactobacillus pentosus, which produces pyrogallol from a substrate, into a food material and fermenting it. Japanese Patent Application Laid-Open No. 2011-72217 (Patent Document 3) describes a tea lactic acid bacteria fermentation product obtained by fermenting a hydrolyzate of tea leaves or a tea leaf extract with a specific Lactobacillus brevis.

[0005] In addition, as for the uses of the fermentation products of the lactic acid bacteria, for example, in Patent Document 2, it is described that the produced pyrogallol has an anti-allergic effect. Also, in International Publication No. 2006 / 090729 (Patent Document 4), it is described that a tea-fermented beverage containing a tea fermentation broth of a specific lactic acid bacterium belonging to the genus Lactobacillus can exhibit an IgA production-enhancing effect and a mucosal immune activation effect. However, regarding the tea fermentation products of lactic acid bacteria that enable intestinal regulation such as regulating the intestinal flora and shortening the gastrointestinal transit time for the prevention and improvement of the above-mentioned constipation, research and development are still insufficient.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

[0008] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technology, and aims to provide a novel fermented tea composition for intestinal regulation, which has intestinal regulating effects such as regulating the intestinal flora and shortening the digestive tract transit time, and a method for producing the same. [Means for solving the problem]

[0009] The present inventors have conducted intensive research to achieve the above object, and first examined the effect of a tea fermentation composition containing a tea fermentation product prepared by fermenting tea extract with Lactobacillus pentosus (L. pentosus OLL203984, accession number: NITE BP-01988) on improving constipation in Ncx / Hox11L.1 gene knockout mice (NcxKO mice, introduced from Chiba University, National University Corporation). The Ncx / Hox11L.1 gene is a transcription factor that controls differentiation and cell death of enteric nerve cells, and NcxKO mice have an excess of enteric nerve cells. Therefore, it has been confirmed that NcxKO mice have reduced intestinal motility, take longer for digestive contents to pass through the digestive tract than wild-type mice (WT mice), and develop chronic constipation. When the fermented tea composition was administered to the NcxKO mice, it was confirmed that the digestive transit time was significantly shorter and the expression level of the iNOS gene tended to decrease in the NcxKO mice administered the fermented tea composition compared to the mice administered water, and that the intestinal flora changed in a direction toward improving constipation compared to before administration. From these results, the present inventors found that the fermented tea product has an intestinal regulating effect, such as shortening the digestive transit time and regulating the intestinal flora, which was not previously known for the fermented tea product of Lactobacillus pentosus.

[0010] Furthermore, the present inventors have discovered that since the Lactobacillus pentosus is a lactic acid bacterium that produces pyrogallol and the fermented tea composition contains pyrogallol as a characteristic component not contained in unfermented tea, a tea fermentation product from lactic acid bacteria having pyrogallol-producing activity has an intestinal regulating effect similar to that of the Lactobacillus pentosus, and have thus completed the present invention.

[0011] The present invention, based on these findings, has the following features. [1] A tea fermentation composition for intestinal regulation, comprising as an active ingredient a tea fermentation product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostelcus, Lactobacillus coryniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri. [2] The fermented tea composition for intestinal regulation described in [1], wherein the lactic acid bacteria are pyrogallol-producing lactic acid bacteria. [3] The fermented tea composition for intestinal regulation described in [1] or [2], wherein the fermented tea product contains pyrogallol. [4] The fermented tea composition for intestinal regulation described in any one of [1] to [3], wherein the fermented tea product contains at least one species selected from the group consisting of the lactic acid bacteria and processed products thereof. [5] The fermented tea composition for intestinal regulation described in any one of [1] to [4], which is a composition for regulating intestinal flora. [6] The fermented tea composition for intestinal regulation according to any one of [1] to [5], which is a composition for shortening digestive tract transit time. [7] The fermented tea composition for intestinal regulation according to any one of [1] to [6], which is a composition for reducing the expression level of the iNOS gene in the intestine. [8] The fermented tea composition for intestinal regulation according to any one of [1] to [7], which is a composition for preventing or improving constipation. [9] The intestinal regulating tea fermentation composition according to any one of [1] to [8], which is a food or drink composition.

[10] A method for producing an intestinal regulating tea fermentation composition, a step of preparing a fermentation mix containing a tea extract and at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostercus, Lactobacillus colliniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri; a step of fermenting the tea extract with the lactic acid bacterium in the fermentation mix to obtain a tea fermented product; A method for producing an intestinal regulating tea fermentation composition, comprising the above steps.

[11] The method for producing an intestinal regulating tea fermentation composition according to

[10] , wherein the lactic acid bacterium is a pyrogallol-producing lactic acid bacterium.

[12] The method for producing an intestinal regulating tea fermentation composition according to

[10] or

[11] , wherein the tea fermented product contains pyrogallol.

[13] The method for producing an intestinal regulating tea fermentation composition according to any one of

[10] to

[12] , wherein the tea fermented product contains at least one selected from the group consisting of the lactic acid bacterium and its processed product.

[14] The method for producing an intestinal regulating tea fermentation composition according to any one of

[10] to

[13] , wherein the intestinal regulating tea fermentation composition is a composition for regulating the intestinal flora.

[15] The method for producing an intestinal regulating tea fermentation composition according to any one of

[10] to

[14] , wherein the intestinal regulating tea fermentation composition is a composition for shortening the gastrointestinal transit time.

[16] The method for producing an intestinal regulating tea fermentation composition according to any one of

[10] to

[15] , wherein the intestinal regulating tea fermentation composition is a composition for reducing the expression level of the iNOS gene in the intestine.

[17] The method for producing a fermented tea composition for intestinal regulation according to any one of

[10] to

[16] , wherein the fermented tea composition for intestinal regulation is a composition for preventing or improving constipation.

[18] The method for producing a fermented tea composition for intestinal regulation according to any one of

[10] to

[17] , wherein the fermented tea composition for intestinal regulation is a food or beverage composition.

[19] Use of a tea fermentation product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostelcus, Lactobacillus coryniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri for the manufacture of a tea fermentation composition for intestinal regulation.

[20] A composition for use in intestinal regulation, comprising a tea fermentation product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostelcus, Lactobacillus coryniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri. [twenty one] A method for regulating the intestines in a subject, comprising administering to the subject a composition containing a tea fermentation product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostelcus, Lactobacillus coeliformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri. to provide. Effect of the Invention

[0012] According to the present invention, it is possible to provide a novel fermented tea composition for intestinal regulation, which has intestinal regulating effects such as regulating the intestinal flora and shortening the digestive tract transit time, and a method for producing the same. Furthermore, since the fermented tea composition for intestinal regulation of the present invention can be made into a tea beverage as a food and beverage composition, it is easy to ingest (drink), and it is easy to make drinking a habit. [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the average body weight of mice in each group from the start of administration of water or the fermented tea composition obtained in Example 1 (before administration) until 4 weeks later. [Diagram 2] 1 is a graph showing the average gastrointestinal transit time of mice in each group 4 weeks after the start of administration of water or the fermented tea composition obtained in Example 1. [Diagram 3] 1 is a graph showing the difference (Δ number of species) in the number of bacterial species in each stool before and after administration of water or the fermented tea composition obtained in Example 1. [Figure 4] 1 is a graph showing the difference in Shannon-Wiener diversity index (ΔShannon diversity index) of bacteria in each feces before and after administration of water or the fermented tea composition obtained in Example 1. [Diagram 5] 1 is a graph showing the amount of p-cresol per gram (dry weight) of cecal content 4 weeks after the start of administration of water or the fermented tea composition obtained in Example 1. [Figure 6] 1 is a graph showing the amount of skatole per gram (dry weight) of cecal content 4 weeks after the start of administration of water or the fermented tea composition obtained in Example 1. [Figure 7] 1 is a graph showing the expression level of the iNOS gene (relative iNOS mRNA level) in each colon 4 weeks after the start of administration of water or the fermented tea composition obtained in Example 1. [Figure 8] 1 is a graph showing the average body weight of mice in each group from the start of administration (before administration) of water, unfermented tea, or the fermented tea composition obtained in Example 1 until 4 weeks later. [Figure 9] 1 is a graph showing the amount of skatole per gram (dry weight) of cecal content 4 weeks after the start of administration of water, unfermented tea, or the fermented tea composition obtained in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below based on preferred embodiments thereof.

[0015] The tea fermentation composition for intestinal regulation of the present invention contains, as an active ingredient, a tea fermented product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostercus, Lactobacillus colliniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri. Further, the method for producing the tea fermentation composition for intestinal regulation of the present invention comprises a step of preparing a fermentation mix containing a tea extract and at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus, Lactobacillus vaccinostercus, Lactobacillus colliniformis, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri; a step of fermenting the tea extract with the lactic acid bacterium in the fermentation mix to obtain a tea fermented product; and includes.

[0016] (Tea fermented product) The tea fermentation composition for intestinal regulation of the present invention contains, as an active ingredient, a tea fermented product of the following specific lactic acid bacterium. In the present invention, the tea fermented product of lactic acid bacterium refers to a product (fermentation product) obtained by fermenting a tea extract with lactic acid bacterium using the tea extract as a substrate.

[0017] [Lactic acid bacterium] The fermented tea product of the present invention is selected from the group consisting of Lactobacillus pentosus (also called Lactiplantibacillus pentosus), Lactobacillus vaccinostercus (also called Paucilactobacillus vaccinostercus), Lactobacillus coryniformis (also called Loigolactobacillus coryniformis), Lactobacillus fermentum (also called Limosilactobacillus fermentum), Lactobacillus acidophilus (also called Lactobacillus fermentum), Lactobacillus fermentum, ... The present invention relates to a tea fermentation product of at least one lactic acid bacterium selected from the group consisting of Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus buchneri (also called Lentilactobacillus buchneri).

[0018] Among the above lactic acid bacteria, the lactic acid bacteria according to the present invention are preferably pyrogallol-producing lactic acid bacteria having pyrogallol-producing activity. The present inventors believe that pyrogallol, which is not contained in unfermented tea extract, is specifically produced in the fermented tea product according to the present invention having an intestinal regulating effect, and this mainly contributes to the intestinal regulating effect. Pyrogallol is also known as 1,2,3-trihydroxybenzene.

[0019] In the present invention, the fact that the lactic acid bacterium has the pyrogallol-producing activity can be confirmed, for example, by the production of pyrogallol from the following tea extract (substrate). As the production amount of pyrogallol at this time, for example, the tea extract is used as a mixture of the total amount of 4 mL of the hot water extract (100 °C, 3 minutes) of the tea leaves (for 5 leaves) of Abukuma sencha and the pulverized product of the tea leaf pieces (for 5 leaves) after the hot water extraction. With respect to this total amount, the number of lactic acid bacteria (live bacteria) is 1×10 6 ~×1×10 10 cfu / mL, and when fermented under anaerobic conditions at 30 °C, the production amount of pyrogallol is preferably 0.01 μg / mL or more, more preferably 0.01 to 200 μg / mL, even more preferably 0.01 to 100 μg / mL, and even more preferably 0.01 to 50 μg / mL.

[0020] Examples of such lactic acid bacteria having pyrogallol-producing activity include, for example, lactic acid bacteria having a pyrogallol production amount of 1 μg / mL or more in Table 1 described in JP-A-2016-167984 (Patent Document 2). More specifically, as the lactic acid bacterium having pyrogallol-producing activity, for example, as Lactobacillus pentosus, OLL203969 strain (Accession No.: NITE BP-01986), OLL203982 strain (Accession No.: NITE BP-01987), and OLL203984 strain (Accession No.: NITE BP-01988) can be mentioned. In addition, as long as each of these strains has pyrogallol-producing activity, it also includes subcultured strains, bred strains, artificially mutated strains, naturally mutated strains, and genetically recombinant strains of each of the above strains.

[0021] As the lactic acid bacterium according to the present invention, one kind may be used alone or two or more kinds may be used in combination. From the viewpoint that the flavor of the tea fermented product and the tea fermentation composition tends to be better, Lactobacillus pentosus is more preferable, and at least one selected from the group consisting of OLL203969 strain, OLL203982 strain, and OLL203984 strain is more preferable, and OLL203984 strain is even more preferable.

[0022] The method for culturing the lactic acid bacteria according to the present invention is not particularly limited, and the bacteria can be cultured by a known method or a method similar thereto depending on the type of lactic acid bacteria, and the desired number of bacteria can be adjusted.

[0023] [Tea extract] In the present invention, the tea extract refers to a mixture extracted from tea, and more specifically, refers to a mixture obtained by extracting tea leaves and / or stems, etc. (e.g., Camellia siensis var. sinensis, Camellia siensis var. assamica, Yabukita species, etc., of the Camellia genus) with an extraction solvent. The tea leaves and stems include raw leaves and stems, as well as processed teas such as green tea, bancha, gyokuro, and tencha; semi-fermented tea called Choryu tea; and fermented tea called black tea. The tea leaves and stems may be one of these alone or two or more of them in combination. In addition, the extraction solvent may be water (including warm water and hot water), organic solvent (e.g., aqueous organic solvent such as ethanol), etc., and one of these may be one of these alone or two or more of them in combination. The extraction method and conditions can be appropriately determined depending on the type of tea used, the type of the extraction solvent, etc.

[0024] The tea extract of the present invention may be one obtained by concentrating and purifying the above-mentioned mixture by a known method or a method similar thereto, or a commercially available tea extract or tea beverage may be used as appropriate.

[0025] The tea extract according to the present invention preferably contains catechins, more preferably gallate-type catechins. In the present invention, catechins refer to epigallocatechin (EGC), epicatechin (EC), catechin (C), gallocatechin (GC), epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), and gallocatechin gallate (GCg), and may be one of these alone or a mixture of two or more of them. In the present invention, gallate-type catechins refer to catechins having a galloyl group, and may be epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), and gallocatechin gallate (GCg), and may be one of these alone or a combination of two or more of them.

[0026] The content of the catechins in the tea extract according to the present invention is not particularly limited, but is preferably 0.01 to 1.0 w / v%, and more preferably 0.02 to 0.2 w / v%, in terms of the total volume of the tea extract (when the catechins are a mixture of two or more kinds, the total of the catechins, the same applies below). The content of the gallate-type catechins in the tea extract according to the present invention is preferably 0.005 to 0.5 w / v%, and more preferably 0.01 to 0.1 w / v%, in terms of the total volume of the tea extract (when the gallate-type catechins are a mixture of two or more kinds, the total of the catechins, the same applies below). In the present invention, "w / v%" refers to the ratio of mass (g) to a total volume of 100 mL.

[0027] The tea extract of the present invention may contain, in addition to the catechins, other components derived from tea, within the scope that does not impair the effects of the present invention. Examples of the other components include sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, and organic acids. The tea extract of the present invention may contain one of these alone or two or more of them in combination.

[0028] (Method of producing fermented tea) The tea fermented product of the present invention can be obtained by contacting the tea extract with the lactic acid bacteria and fermenting it, and more specifically, it can be obtained by a method including a step of preparing a fermentation mix containing the tea extract and the lactic acid bacteria (preparation step), and a step of fermenting the tea extract in the fermentation mix with the lactic acid bacteria to obtain the tea fermented product (fermentation step).

[0029] The fermentation mix is ​​preferably an aqueous solution. Examples of the solvent for the aqueous solution include water, phosphate buffer, physiological saline, liquid medium, and generally extracted tea, and one of these may be used alone or two or more may be used in combination. Among these, water or generally extracted tea is preferred from the viewpoint that the fermentation mix (fermented tea product) after fermentation can be used as the tea fermentation composition of the present invention as it is.

[0030] The tea extract contained in the fermentation mix may be in the form of a solid such as a dry powder; an aqueous solution such as a concentrate or dilution (which generally contains extracted tea); or a slurry.

[0031] The concentration of the tea extract in the fermentation mix is ​​not particularly limited, but is preferably 0.01 to 1.0 w / v% in terms of the content of the catechins, more preferably 0.02 to 0.4 w / v%, and even more preferably 0.02 to 0.2 w / v% in terms of the total volume of the fermentation mix. Even more preferably, the content of the gallate-type catechins is preferably 0.005 to 0.5 w / v%, more preferably 0.01 to 0.2 w / v%, and even more preferably 0.01 to 0.1 w / v% in terms of the total volume of the fermentation mix. If the concentration of the tea extract is less than the lower limit, the amount of the substrate is reduced, and the tea fermentation product according to the present invention tends not to be sufficiently obtained. On the other hand, if the concentration exceeds the upper limit, the pyrogallol-producing activity is reduced, making it difficult to efficiently obtain pyrogallol, and the intestinal regulating effect of the obtained tea fermentation product tends to be reduced. In the present invention, when the solvent of the fermentation mix contains the above-mentioned generally extracted tea, the concentration of the tea extract (catechins content) also includes the content of catechins derived from such tea.

[0032] The lactic acid bacteria contained in the fermentation mix may be in the form of a lactic acid bacteria composition containing the lactic acid bacteria. In the present invention, the lactic acid bacteria composition includes a culture containing medium components after the completion of the cultivation of lactic acid bacteria; a concentrate, dilution, dry product, frozen product, etc. of the culture, and one of these may be used alone or two or more may be used in combination. Among these, the lactic acid bacteria is preferably contained in the fermentation mix in the form of the lactic acid bacteria composition, more preferably in the form of a culture or a concentrate of the culture. In addition, the lactic acid bacteria contained in the fermentation mix may be a lactic acid bacteria or a lactic acid bacteria composition that has been activated and cultured by a known method.

[0033] The concentration of the lactic acid bacteria in the fermentation mix is ​​not particularly limited, but is preferably 1×10 in terms of the number of lactic acid bacteria (viable bacteria) relative to the total volume of the fermentation mix. 6 ~1×10 10 cfu / mL is preferred, and 1×107 ~1×10 9 If the concentration of the lactic acid bacteria is less than the lower limit, fermentation does not proceed sufficiently, and the fermented tea product according to the present invention tends not to be obtained sufficiently, whereas if the concentration of the lactic acid bacteria is more than the upper limit, pyrogallol production activity decreases, making it difficult to efficiently obtain pyrogallol, and the intestinal regulating effect of the obtained fermented tea product tends to decrease.

[0034] The pH of the fermentation mix can be appropriately selected depending on the growth conditions of the lactic acid bacteria, the amount of the fermentation mix, etc., and is preferably, for example, pH 4.8 to 8.3, and more preferably pH 5.3 to 7.8. If the pH is outside the range, the fermentation does not proceed sufficiently, and the fermented tea product according to the present invention tends not to be obtained sufficiently.

[0035] The fermentation mix may contain other components in addition to the tea extract, the lactic acid bacteria, and the solvent, within the range that does not inhibit the effects of the present invention. Examples of the other components include the components contained in the lactic acid bacteria composition, carbonates, sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, and pH adjusters. The fermentation mix may contain one of these alone or two or more of them in combination.

[0036] In the fermentation step, the fermentation method can be appropriately selected depending on the growth conditions of the lactic acid bacteria, the amount of the fermentation mix, etc., and is not particularly limited, but for example, it is preferable to leave the fermentation mix stationary or agitate (preferably leave stationary) under aerobic conditions for 12 to 72 hours, more preferably 18 to 48 hours, at a temperature of 25 to 40° C., more preferably 30 to 37° C. Fermentation under nitrogen aeration conditions can also be adopted instead of aerobic conditions.

[0037] A tea fermented product, which is an active ingredient of the tea fermentation composition of the present invention, can be obtained from the fermentation mix after fermentation. The fermentation mix after fermentation may further be subjected to crushing and / or heat treatment of the lactic acid bacteria. Therefore, the tea fermented product according to the present invention preferably contains at least one selected from the group consisting of the lactic acid bacteria (live bacteria and / or killed bacteria) and a processed product thereof (crushed product and / or heat treated product of the lactic acid bacteria). In the tea fermented product according to the present invention, the content of the lactic acid bacteria and / or a processed product thereof is 1×10 in terms of viable bacteria count. 6 ~1×10 10 cfu / mL is preferred, and 1×10 7 ~1×10 9 More preferably, 1×10 cfu / mL. 8 ~1×10 9 If the content of the lactic acid bacteria and / or a processed product thereof is less than the lower limit, the intestinal regulating effect of the fermented tea product tends to decrease, whereas if the content of the lactic acid bacteria and / or a processed product thereof is more than the upper limit, the flavor tends to decrease.

[0038] From the viewpoint of a more excellent intestinal regulating effect, the fermented tea product according to the present invention preferably contains pyrogallol. In the fermented tea product according to the present invention, the content of pyrogallol is preferably 0.01 to 200 μg / mL, more preferably 0.01 to 100 μg / mL, even more preferably 0.01 to 55 μg / mL, and even more preferably 0.01 to 50 μg / mL. If the content of pyrogallol is less than the lower limit, the intestinal regulating effect of the fermented tea product tends to decrease.

[0039] Furthermore, from the same viewpoint, the content of pyrogallol in the fermented tea product of the present invention is preferably 0.001 to 15 mass%, and more preferably 0.001 to 10 mass%, relative to the total content of pyrogallol, gallic acid, and catechins that are optionally contained in the fermented tea product.

[0040] As for the tea fermented product according to the present invention, from the viewpoint of excellent intestinal regulating effect, it is preferably contained gallic acid. In the tea fermented product according to the present invention, the content of the gallic acid is preferably 5 to 60 μg / mL, more preferably 10 to 50 μg / mL, and still more preferably 15 to 45 μg / mL. When the content of the gallic acid is less than the lower limit, the intestinal regulating effect by the tea fermented product tends to decrease.

[0041] Also, in the tea fermented product according to the present invention, from the same viewpoint, with respect to the total content of pyrogallol, gallic acid, and catechins contained in the tea fermented product as needed, the content of the gallic acid is preferably 1 to 10% by mass, and more preferably 3 to 8% by mass.

[0042] As for the tea fermented product according to the present invention, from the viewpoint of excellent intestinal regulating effect, it is preferably contained at least one of the catechins. In the tea fermented product according to the present invention, the content of the catechins is preferably 100 to 10000 μg / mL, and more preferably 200 to 2000 μg / mL. When the content of the catechins is less than the lower limit, the intestinal regulating effect by the tea fermented product tends to decrease. On the other hand, when it exceeds the upper limit, there is a risk of generating bitterness and affecting the flavor of the tea fermented composition.

[0043] Also, in the tea fermented product according to the present invention, from the same viewpoint, with respect to the total content of pyrogallol, gallic acid, and catechins contained in the tea fermented product as needed, the content of the catechins is preferably 70 to 98% by mass, and more preferably 80 to 96% by mass.

[0044] Furthermore, from the viewpoint of achieving a more excellent intestinal regulating effect, the fermented tea product according to the present invention preferably has a good balance between the gallate-type catechins and other catechins in the catechins, and the content of the gallate-type catechins in the total catechins is preferably 5 to 35% by mass, more preferably 10 to 30% by mass. When the content of the gallate-type catechins is within the above range, the fermented tea product tends to achieve a more excellent intestinal regulating effect.

[0045] The fermented tea product of the present invention may further contain the fermentation mix and other components derived therefrom, in addition to the above. Examples of the other components include sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, and organic acids, and the fermented tea product of the present invention may contain one of these alone or two or more of them in combination.

[0046] (Fermented tea composition) The tea fermented composition of the present invention contains the tea fermented product and can be obtained by using the tea fermented product. The tea fermented composition may consist of the tea fermented product alone, or may consist of the tea fermented product that has been concentrated, dried, frozen, or the like. The tea fermented composition of the present invention may further contain components other than the tea fermented product within a range that does not impair the effects of the present invention. In this case, the content of the tea fermented product is not particularly limited, and is appropriately adjusted depending on the form of the composition described below, and the purpose, subject, method, dosage, etc. of administering the composition.

[0047] The fermented tea composition of the present invention can be used for intestinal regulation by containing the fermented tea according to the present invention as an active ingredient. More specifically, it is preferably used as a composition for regulating the intestinal flora (composition for regulating the intestinal flora), a composition for shortening the digestive tract transit time (composition for shortening the digestive tract transit time), or a composition for decreasing the expression level of the iNOS gene in the intestine (composition for decreasing the expression level of the iNOS gene), and can also be used as a composition for preventing or improving constipation in which at least one of the following is effective: regulating the intestinal flora, shortening the digestive tract transit time, and decreasing the expression level of the iNOS gene.

[0048] Furthermore, the tea fermented composition of the present invention can be used for a method for regulating the intestinal flora of a human or non-human animal, a method for shortening the digestive tract transit time, a method for reducing the expression level of iNOS gene in the intestine, and a method for preventing or improving constipation, and the present invention also provides these methods. These methods comprise a step of administering an effective amount of the tea fermented composition of the present invention to a subject (a human or non-human animal, preferably a mammal), more preferably a step of administering to a subject suffering from constipation.

[0049] In the present invention, the intestinal flora regulation refers to changing the intestinal flora, preferably changing it to a flora that can improve constipation. More specifically, such changes include, for example, an increase in the occupancy rate of at least one species selected from the group consisting of Lactobacillales and Bifidobacteriales, more preferably Lactobacillales, in the intestine, a decrease in the number of at least one species selected from the group consisting of Clostridiales and Bacteroidales, a decrease in the number of bacterial species in the intestine, a decrease in the Shannon-Wiener diversity index (H') of bacteria in the intestine, a decrease in the concentration of p-cresol and / or skatole in the intestine, etc., based on the level before administration of the tea fermentation composition.

[0050] In the present invention, the gastrointestinal transit time refers to the time it takes for the gastrointestinal contents to pass through the gastrointestinal tract (from the mouth to the anus), and the shortening of the gastrointestinal transit time can be confirmed, for example, by a shortening of the gastrointestinal transit time compared to the administration of water instead of the composition (water group). It can also be confirmed by a decrease in the expression level of the iNOS gene in the intestine compared to the water group.

[0051] The tea fermented composition of the present invention can be administered orally or enterally to humans or non-human animals (preferably mammals). In the present invention, oral administration includes ingestion of a food or drink composition or a feed composition. The tea fermented composition of the present invention can be, for example, a pharmaceutical composition, a quasi-drug composition, a food or drink composition, a feed composition, etc., depending on the purpose, subject, method, dosage, etc. of administration of the composition.

[0052] The pharmaceutical composition and quasi-drug composition according to the present invention can be, for example, a preparation, the form of which is not particularly limited, and examples thereof include solid preparations such as tablets, pills, granules, powders, powders, and capsules; liquid preparations such as general liquid preparations, suspensions, emulsions, and syrups; jellies; preparations for enteral administration; and suppositories. The preparations can be produced, for example, by adding one or more of the preparation auxiliary agents such as solvents, dispersants, emulsifiers, thickeners, gelling agents, surfactants, buffers, stabilizers, preservatives, excipients, binders, disintegrants, dissolution aids, lubricants, colorants, flavorings, sweeteners, coating agents, and flavors to the fermented tea product according to the present invention or its concentrate, dried product, or frozen product according to the present invention, according to a known method or a method similar thereto.

[0053] Furthermore, the pharmaceutical compositions and quasi-drug compositions may each further contain an appropriate amount of one or a combination of two or more of the following additives, within the range that does not impair the effects of the present invention: water, lipids, carbohydrates, proteins, sugar alcohols, minerals (calcium, magnesium, sodium, potassium, iron, copper, zinc, etc.), vitamins (vitamins A, B1, B2, B6, B12, C, D, E, K, etc.), peptides, amino acids, organic acids, and pH adjusters.

[0054] The form of the food and drink composition according to the present invention is not particularly limited, and examples thereof include solid forms such as bars, liquid forms such as beverages and liquid foods, paste forms, semi-liquid forms, gel forms (jelly forms), gel-like oils and fats (semi-solid oils and fats), and powder forms. The food and drink compositions can also be used as liquid foods, powdered liquid foods, nutritional pastes, oral and tube-fed nutritional supplements, beverages, gel foods, etc., for nutritional management of oral and enteral nutrition patients, elderly people, infants, etc.

[0055] Examples of the food and drink composition according to the present invention include, but are not limited to, beverages (tea, carbonated beverages, cocoa, coffee, lactic acid bacteria beverages, soy milk beverages, fruit and vegetable juice beverages, soft drinks, nutritional beverages, alcoholic beverages, etc.), processed foods (chocolate, gum, gummy candy, jelly, baked goods (bread, cake, cookies, biscuits, etc.), candy, etc.), dairy products (modified milk powder (powdered milk), modified milk, milk beverages, fermented milk, yogurt, ice cream, cheese, cream, butter, margarine, condensed milk, etc.), seasonings (sauces, soups, dressings, mayonnaise, mayonnaise-type seasonings, cream, etc.), supplements, edible oils, functional edible oils and fats, etc. Such food and drink compositions can be produced, for example, by a method of blending the fermented tea product according to the present invention; its concentrate, dried product, or frozen product; or the preparation according to the present invention into an existing food or drink or its production process.

[0056] The food and drink composition according to the present invention may further contain various ingredients that can be contained in food and drink within the range that does not impair the effects of the present invention. Such ingredients are not particularly limited, and may contain, for example, one of the formulation adjuvants and additives listed in the above-mentioned pharmaceutical compositions and quasi-drug compositions, dietary fiber (resistant dextrin, etc.), fruits and vegetables and their processed products, animal and plant herbal extracts, and naturally derived polymers (collagen, hyaluronic acid, chondroitin, etc.) alone or in combination of two or more of them in an appropriate amount.

[0057] The food and beverage composition according to the present invention may be, for example, a general food, a health food, a functional food, a health functional food (e.g., a food for specified health uses, a nutrient functional food, a nutritional supplement, a food with functional claims, etc.), a food for special dietary uses (e.g., a food for infants, a food for pregnant women, a food for the sick, etc.), a medical food (a food prescribed under the supervision of a doctor as defined by the U.S. Food and Drug Administration (FDA) and the Orphan Drug Act), a therapeutic food (a food that serves the purpose of treatment and is prepared based on a menu prepared by a nutritionist or the like in accordance with a doctor's diet prescription), or a dietary therapy food, and the food and beverage composition may be labeled with the action or efficacy brought about by the fermented tea product according to the present invention (e.g., intestinal regulation, shortening of digestive transit time, regulation of intestinal flora, reduction of iNOS gene expression level, prevention or improvement of constipation, etc.).

[0058] The feed composition according to the present invention may be the above-mentioned food and drink composition appropriately modified depending on the purpose, subject, method, dose, etc. of feeding the feed composition.

[0059] Furthermore, the dosage of the fermented tea composition of the present invention can be appropriately determined for each individual case, taking into consideration the subject's species, age, weight, sex, type of disease, severity of symptoms, etc. For example, the dosage for humans (adults) is preferably 1 to 100 mg, more preferably 2 to 50 mg, and even more preferably 2 to 10 mg, of gallic acid.

[0060] The fermented tea composition of the present invention is preferably packaged (preferably enclosed) in a packaging container from the time of production until administration. The packaging container is not particularly limited, but examples thereof include wrapping paper, packaging bags, soft bags, tubes, cheer packs, paper containers, cans, bottles, capsules, etc. EXAMPLES

[0061] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples. In the following, the symbol "%" indicates mass / volume (w / v) percent (g / 100 mL) unless otherwise specified.

[0062] <Lactic acid bacteria> In each of the following preparation examples, the following lactic acid bacteria strains were used as lactic acid bacteria 1: Lactic acid bacteria 1: Lactobacillus pentosus (L. pentosus) OLL203984 strain (Accession number: NITE BP-01988) Lactic acid bacteria 1 is a strain isolated by the applicant of the present invention, and has the above-mentioned pyrogallol-producing activity.

[0063] Example 1 <Preparation of fermented tea composition> Lactic acid bacteria 1 was activated twice in MRS medium at 37° C. under anaerobic conditions for 18 hours, and then the activated culture solution was inoculated into the MRS medium at 1% (volume %) and cultured at 37° C. under anaerobic conditions for 18 hours. The culture solution was centrifuged at 5000 rpm and 4° C. for 15 minutes, washed once with physiological saline, and then centrifuged again at 5000 rpm and 4° C. for 15 minutes. The precipitate (culture product) was suspended in an equal amount of tea extract and used as Lactic Acid Bacteria Composition 1 (1.9×10 11 cfu / mL). As the tea extract, "Meiji Deep-Steamed Shizuoka Tea" manufactured by Meiji Co., Ltd. was used. Lactic acid bacteria composition 1 was added at 10% (volume %, 2 × 10 in terms of lactic acid bacteria count) to the tea extract. 8 The culture solution was inoculated at a concentration of 0.01 cfu / mL and cultured at 30° C. under aerobic conditions for 48 hours. The pH of the culture solution after 48 hours of culture was 4.7. This culture solution was sterilized by heating at 122° C. for 10 minutes to obtain a fermented tea composition (lactic acid bacteria fermented tea).

[0064] <Measurement of pyrogallol, gallic acid, and catechin concentrations> The obtained fermented tea composition was centrifuged at 13000 rpm, 4°C for 3 minutes, and the supernatant was diluted 2-fold with ultrapure water. The diluted supernatant was placed in a filter vial (manufactured by Thomson) and the concentrations of pyrogallol (PG), gallic acid (GA), epigallocatechin (EGC), epigallocatechin gallate (EGCg), epicatechin (EC), epicatechin gallate (ECg), gallocatechin (GC), gallocatechin gallate (GCg), catechin (C), and catechin gallate (Cg) were measured by HPLC. The analytical conditions for HPLC are shown below. The results are shown in Table 1 below.

[0065] [HPLC analysis conditions] Column: COSMOSIL Cholester Column size: 4.6mm ID x 150mm ·Mobile phase: A) Acetonitrile / 20mM phosphate buffer (pH 2.5) = 10 / 90 B) Acetonitrile / 20 mM phosphate buffer (pH 2.5) = 30 / 70 B:0%(0min)→100%(20min)→0%(21min) ·Flow rate: 1mL / min ·Temperature: 30℃ Detector: UV-VIS detector SPD-20AV (Shimadzu Corporation) Detection wavelength: 200nm (except GA), 280nm (GA) ·Injection volume: 10μL.

[0066] <Measurement of lactic acid and acetic acid concentration> The obtained fermented tea composition was centrifuged at 13,000 rpm at 4° C. for 10 minutes, and the supernatant was placed in a filter vial (manufactured by Thomson) and the concentrations of lactic acid and acetic acid were measured by HPLC. The HPLC analysis conditions are shown below. The results are shown in Table 1 below.

[0067] [HPLC analysis conditions] Column: ICSep ICE ORH-801 (Transgenomic) Column size: 6.5mm ID x 300mm Mobile phase: 7.5mM p-toluenesulfonic acid Reaction solution: 7.5 mM p-toluenesulfonic acid + 150 μM EDTA (2Na) + 30 mM Bis Tris ·Flow rate: 0.5mL / min ·Temperature: 55℃ Detector: CDD-10A (Shimadzu Corporation) ·Injection volume: 10μL.

[0068] [Table 1]

[0069] <Confirmation of intestinal regulation effect> NcxKO mice (Ncx / Hox11L.1 gene knockout mice, obtained from Chiba University) were bred and bred to obtain 14 male NcxKO mice of similar age. CLEA Rodent Diet CE-2 (solid, CLEA Japan, Inc.) was used as food. When the mice reached 9-10 weeks of age, they were divided into two groups of 7 mice each to ensure that there was no weight bias: Group 1: Water group (control) Group 2: Fermented tea composition group The mice were divided into groups. The mice were individually housed. After grouping (before administration), feces were collected from each mouse, and then the mice in group 1 were given water and the mice in group 2 were given the fermented tea composition obtained above, with free drinking for four weeks. Food was provided in each cage and the mice were allowed to consume it ad libitum. The food and each administered substance (water or fermented tea composition) were replaced with new ones three times a week, and the mice's body weight was measured every week from the start of administration, and feces was collected after four weeks of breeding from the start of administration.

[0070] 1. Gastrointestinal transit time measurement After 4 weeks of feeding, each mouse was transferred to a cage with a wire mesh floor and orally administered 300μL of 50% barium sulfate suspended in 1% gum arabic solution. After that, the mice in each group were provided with the feed and food in the cage with a wire mesh floor, and were allowed to consume both ad libitum. After administration of barium sulfate, the excreted feces were collected and observed to confirm the presence or absence of coloration due to barium sulfate. The time from administration of barium sulfate to the first appearance of colored feces was measured, and this value was taken as the digestive tract transit time. The contents of the colon and cecum of each mouse were collected the day after administration of barium sulfate.

[0071] The average body weight of mice in each group 4 weeks after the start of administration of water or fermented tea composition, and the average gastrointestinal transit time in each group after 4 weeks are shown in Figures 1 and 2, respectively. As shown in Figure 1, there was no difference in the body weight of mice during the experimental period between the groups, but as shown in Figure 2, the gastrointestinal transit time was significantly shorter in the tea fermented composition group compared to the water group (P value by Student's t-test between the time in the water group and the time in the tea fermented composition group: P<0.05), confirming that the tea fermented composition of the present invention is effective in shortening the gastrointestinal transit time.

[0072] 2. Gut flora analysis-1 (analysis by real-time PCR) DNA was extracted from 20 mg of feces collected before and 4 weeks after administration of water or fermented tea composition using QIAamp DNA Stool Mini kit (QIAGEN) and QIAcube (QIAGEN), and dissolved in 200 μL of water. The extracted DNA was used as template DNA to analyze the bacterial flora in the feces by q-PCR targeting the 16S rRNA gene of the bacterial group. The bacterial groups to be analyzed were (1) Clostridium coccoides group, (2) Clostridium leptum subgroup, and (3) Bacteroides fragilis group, which are the main bacterial groups constituting the intestinal bacterial flora. The PCR reaction conditions for each bacterial group are shown below.

[0073] [PCR conditions] (1)Clostridium coccoides group Primer F (g-Ccoc-F): 5'-aaatgacggtacctgactaa-3' (SEQ ID NO: 1) Primer R (g-Ccoc-R): 5'-ctttgagtttcattcttgcgaa-3' (SEQ ID NO: 2) DNA quantification standard: OB7133 (Clostridium clostridiiforme JCM1291) Template DNA concentration: 100-fold dilution PCR device: QuantStudio 3 real-time PCR system (applied biosystems) PCR reaction mixture composition: PowerUp SYBR Green Mater Mix (ABI): 100 μL Primer F(100pmol / μL):0.18μL Primer R(100pmol / μL):0.18μL Template DNA: 4 μL H 2 O: 5.64 μL Total: 20μL PCR reaction conditions: 50℃-2min 95℃-2min 95℃-15sec / 60℃-1min:45cycles Melting curve analysis (Dissociation stage) (2)Clostridium leptum subgroup Primer F (sg-Clept-F): 5'-gcacaagcagtggagt-3' (SEQ ID NO: 3) Primer R (sg-Clept-R): 5'-cttcctccgttttgtcaa-3' (SEQ ID NO: 4) DNA quantification standard: OB7163 (Clostridium sporosphaeroides NCIMB 10672) Template DNA concentration: 100-fold dilution PCR device, PCR reaction solution composition, and reaction conditions: Same as (1) (3) Bacteroides fragilis group Primer F (g-Bact-F): 5'-atagcctttcgaaagaagat-3' (SEQ ID NO: 5) Primer R (g-Bact-R): 5'-ccagtatcaactgcaatttta-3' (SEQ ID NO: 6) DNA quantification standard: OLB7086 (Bacteroides vulgatus JCM 5826) Template DNA concentration: 100-fold dilution PCR equipment: ABI PRISM 7000 Sequence Detection System PCR reaction mixture composition: 2x SYBR Green PCR Master Mix (QIAGEN): 12.5μL Primer F (100 pmol / λ): 0.225 μL Primer R(100pmol / λ):0.225μL 50mM MgCl 2 : 0.75μL H 2 O: 6.3 μL Template DNA: 5 μL Total: 25μL PCR reaction conditions: 95℃-15min 94℃-15sec / 50℃-30sec / 72℃-30sec:45cycles Melting curve analysis (Dissociation stage).

[0074] The number of bacteria of each bacterial group per 1 g of feces before and after administration of water or fermented tea composition (after 4 weeks of administration) was converted to the copy number per 1 g of feces. 10The detection rate (%) of each bacterial group was calculated by dividing the number of individuals in which each bacterial group was detected by the total number of individuals in each group. The results are shown in Table 2 below. In Table 2, the values ​​of each bacterial count are shown as the average (m) and its standard error (SE) (m±SE) in the mice of each group, and values ​​before and after administration with a P value of less than 0.05 (P<0.05) by Wilcoxon's signed rank test are marked with an asterisk (*) (same as in Table 6 below).

[0075] [Table 2]

[0076] As shown in Table 2, in the water group, there was no significant change in the number of bacteria in each bacterial group before and after administration. In contrast, in the tea fermented composition group, it was confirmed that the number of bacteria in the C. coccoides group, C. leptum subgroup, and B. fragilis group all significantly decreased.

[0077] 3. Gut flora analysis-2 (analysis using next-generation sequencers) (1) Amplification of the 16S rDNA V4 region First, the DNA extracted from each feces in 2 (analysis by real-time PCR) above was used as a template DNA to amplify the 16S rDNA V4 region. The PCR reaction conditions are shown below. The primers used were the 515F (forward primer, SEQ ID NO: 7) PCR primer and the 806rcbc1 to 806rcbc28 (reverse primers, SEQ ID NO: 8 to 35) PCR primers described in Caporaso JG et al., 2012, ISME J., 6(8), p.1621-1624.

[0078] [PCR conditions] Primer F (515F): sequence shown in SEQ ID NO: 7 Primer R (806rcbc1 to 806rcbc28): sequences shown in SEQ ID NOs: 8 to 35, respectively PCR device: Veriti Thermal Cycler (applied biosystems) PCR reaction mixture composition: TaKaRa Ex Taq (RR001, Takara Bio Inc.): 0.125 μL 10xExTaq buffer: 2.5μL Primer F(10μM):0.5μL Primer R(5μM):1μL dNTP: 2 μL dH 2 O: 17.825 μL Template DNA: 1 μL Total: 25μL PCR reaction conditions: 94℃-3min 94℃-45sec / 50℃-60sec / 72℃-90sec:25cycles 72℃-10min 4℃-∞.

[0079] (2) Purification of PCR products Next, each PCR product obtained in (1) above was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the QIAGEN protocol.

[0080] (3) Quantification and concentration adjustment of PCR products Each purified PCR product obtained in (2) above was diluted 10,000-fold with 0.1% Tween 20, and real-time PCR was performed using this as template DNA under the conditions shown below. Illumina Library Quantification DNA Standards 1-6 (Kapa Biosystems) were used as DNA quantification standards. Next, based on the quantitative results of real-time PCR, each real-time PCR product was diluted to 1000ng / mL with EB buffer included in the QIAamp DNA stool mini Kit (QIAGEN).

[0081] [PCR conditions] Primer 1.1: 5'-aatgatacggcgaccaccgagat-3' (SEQ ID NO: 36) Primer 2.1: 5'-caagcagaagacggcatacga-3' (SEQ ID NO: 37) PCR device: QuantStudio 3 real-time PCR system (applied biosystems) PCR reaction mixture composition: 2x SYBR Green PCR Master Mix (QIAGEN): 12.5μL Primer 1.1: 0.3 μL Primer 2.1: 0.3 μL dH 2 O: 7 μL Template DNA: 5 μL Total: 25.1μL PCR reaction conditions: 95℃-15min 94℃-15sec / 60℃-40sec / 72℃-40sec:45cycles 72℃-10min 4℃-∞.

[0082] (4) Check the size of the PCR product Using a microchip electrophoresis device for DNA / RNA analysis (MultiNA, Shimadzu Corporation), the real-time PCR products diluted to 1000 ng / mL in (3) above were electrophoresed to confirm the presence of the target bands. The procedure followed the protocol of Shimadzu Corporation.

[0083] (5)DNA sequence analysis 5 μL of each real-time PCR product diluted to 1000 ng / mL (4 nM) in (3) above was mixed to prepare a 4 nM library. 5 μL of the 4 nM library was mixed with 5 μL of 0.2 N NaOH, and the mixture was left at room temperature for 5 minutes to denature the library. 990 μL of ice-cold HT1 buffer (Illumina) was added to prepare a 20 pM denatured library. 2 μL of 10 nM PhiX Sequencing Control V3 (PhiX; Illumina) was mixed with 3 μL of EB buffer to prepare a 4 nM PhiX. 5 μl of 4 nM PhiX was mixed with 5 μl of 0.2 N NaOH, and the mixture was left at room temperature for 5 minutes to denature PhiX. 990 μL of ice-cold HT1 buffer (Illumina) was added to prepare a 20 pM denatured PhiX.

[0084] Next, 200 μL of the denatured library, 150 μL of the denatured PhiX, and 250 μL of ice-cold HT1 buffer (Illumina) were mixed to obtain the final library. DNA sequence analysis was performed using the final library with Miseq (Illumina). The method of creating the sample sheet required for DNA sequence analysis with Miseq and the method of using the device were in accordance with the manual attached to Miseq.

[0085] (6) Data analysis The data obtained in (5) above was analyzed using the analysis software "BITS (Bitz Co., Ltd.)" to calculate the occupancy rate of each bacterial group relative to the total bacterial groups in each feces. The data analysis method was in accordance with the manual attached to BITS. Among the detected bacterial groups, those in which the maximum number of reads in the total feces was less than 1% (read number %) of the total number of reads in the bacterial domain were excluded. The ratio of the number of reads of Lactobacillales to the total number of reads (occupancy rate, read number %) in each feces before and after administration of water or fermented tea composition (after 4 weeks of administration) is shown in Table 3 below. In Table 3, each value is shown as the mean (m) and its standard error (SE) (m±SE) for each group of mice, and values ​​for which the P value between each pre-administration value and post-administration value by Wilcoxon signed-rank test was less than 0.05 (P<0.05), i.e., values ​​that were found to have a significant difference between pre- and post-administration, are marked with an asterisk (*) (same as in Table 7 below).

[0086] [Table 3]

[0087] As shown in Table 3, in the water group, there was no significant change in the occupancy rate of Lactobacillus order before and after administration.In contrast, in the tea fermentation composition group, it was confirmed that the occupancy rate of Lactobacillus order increased significantly before and after administration.In addition, a similar test was performed except that the tea fermentation composition containing 2 times the amount of Lactobacillus 1 was used, and no significant difference was observed in the occupancy rate of Lactobacillus order between the tea fermentation composition containing 1 times the amount of Lactobacillus 1 and the tea fermentation composition containing 1 times the amount of Lactobacillus 1.Therefore, the increase in the occupancy rate of Lactobacillus order is not the result of detection of the DNA of the lactic acid bacteria contained in the tea fermentation composition of the present invention itself, but suggests that the administration of the tea fermentation composition of the present invention changes the intestinal flora in the direction of improving constipation.

[0088] The data obtained in (5) above was analyzed using the analysis software "BITS (Bitz Co., Ltd.)" to analyze the alpha diversity of bacterial groups in each feces before and after administration of water or fermented tea composition (after 4 weeks of administration). The data analysis method was in accordance with the manual attached to BITS. The number of bacterial species in each feces and the Shannon-Wiener diversity index (H', Shannon diversity index) are shown in Table 4 below. The difference (Δ number of species) obtained by subtracting the number of species in the feces before administration of water or fermented tea composition from the number of species in the feces after administration and the difference in H' (Δ Shannon diversity index) are shown in Figures 3 and 4, respectively. In Table 4, each value is shown as the mean (m) and its standard error (SE) (m±SE) for each group of mice, and an asterisk (*) is indicated for values ​​before and after administration for which the P value in the Wilcoxon signed-rank test between each value before and after administration was less than 0.05 (P<0.05), i.e., values ​​that were found to have a significant difference between before and after administration.

[0089] [Table 4]

[0090] As shown in Table 4 and Figures 3-4, it was confirmed that the number of species and H' value after administration were lower in the tea fermented composition group than before administration, compared to the water group. In particular, as shown in Table 4, there was no significant change in the H' value before and after administration in the water group, whereas the H' value significantly decreased before and after administration in the tea fermented composition group. Furthermore, as shown in Figure 4, the difference in H' in the tea fermented composition group was significantly reduced compared to the difference from before administration after administration in the water group. It has been previously reported that the H' value is significantly larger in patients with constipation than in healthy subjects (e.g., Non-Patent Document 1), and it can be said that the administration of the tea fermented composition of the present invention has changed the intestinal flora in a direction that improves constipation. It is also considered that such a change in the intestinal flora also contributed to the shortening of the digestive time.

[0091] 4. Measurement of putrefactive product concentration in cecal contents First, 100 mg of each cecal content collected the day after the administration of barium sulfate in 1 above was freeze-dried, and the weight after freeze-drying was measured. Next, 1.5 mL of methanol was added to this and mixed to form a suspension, which was then added to Lysing Matrix F (MP Biomedicals), and the suspension was disrupted using FastPrep 24 (MP Biomedicals) at 5.0 m / s for 30 seconds twice. The disrupted material was cooled at -20°C for 1 hour. After 30 minutes had passed since cooling, the material was mixed by inversion. After cooling, the material was centrifuged at 20000g for 20 minutes at 4°C, and 1 mL of the supernatant was filtered through a 0.45 μm filter. The amount of p-cresol and the amount of skatole were measured for each filtrate by LC / MS / MS. The analytical conditions for LC / MS / MS are shown below. The amounts of p-cresol and skatole (concentration, ng / g dry weight) per gram (dry weight) of each cecal content are shown in Figures 5 and 6, respectively.

[0092] [LC / MS / MS analysis conditions (QTRAP4500 LC-MS / MS system)] [LC conditions] Column: Waters BEH C18 1.7μm×10cm Mobile phase: A) water, B) methanol, B:30%(0min)→30%(5min)→99%(10min)→99%(12min)→30%(12.01min) ·Flow rate: 0.3mL / min ·Temperature: 40℃ ·Injection volume: 5μL [MS conditions (APCI probe)] p-Cresol: Q1) 107, Q3) 91.9, CE) -31 Skatole: Q1) 132.2, Q3) 117, DP) 70, CE) 27.

[0093] As shown in Figures 5 and 6, it was confirmed that the concentrations of p-cresol and skatole, which are known to be putrefactive products in the cecal contents, were both lower in the fermented tea composition group compared to the water group. Since p-cresol and skatole are produced by intestinal bacteria, it can be said that the decrease in these substances is due to changes in the intestinal flora.

[0094] 5. Analysis of iNOS gene expression in the colon The colon collected the day after administration of barium sulfate in 1 above was immersed in RNAlater (Ambion) and stored at -20°C until use. The colon was disrupted with TissueLyser II, and RNA was extracted with NucleoSpin RNA Plus (Macherey-Nagel). Next, cDNA reverse transcribed with PrimeScript RT Master Mix (Takara Bio Inc.) was used as template DNA, and q-PCR was performed targeting the iNOS gene and the GAPDH gene as an endogenous control to analyze the expression level of each gene. The PCR reaction conditions are shown below. The primers used were the primers (iNOS gene) described in Boyer L et al., 2011, Lab Invest 91, p.353-362 and the primers (GAPDH gene) described in Eissa N et al., 2016, PLoS One 11, e0156289.

[0095] [PCR conditions] Primer F (iNOS Forward Primer): 5'-cgggcaaacatcacattcagatcccg-3' (SEQ ID NO: 38) Primer R (iNOS Reverse Primer): 5'-tatattgctgtggctcccatgtt-3' (SEQ ID NO: 39) Standard Primer F (GAPDH Forward standard primer): 5'-aggtcggtgtgaacggatttg-3' (SEQ ID NO: 40) Standard Primer R (GAPDH Reverse Standard Primer): 5'-ggggtcgttgatggcaaca-3' (SEQ ID NO: 41) ·Template DNA (cDNA) concentration: 12.5ng / μL PCR device: QuantStudio 3 real-time PCR system (applied biosystems) PCR reaction mixture composition: TB Green Premix Ex Taq (Takara Bio Inc.): 10 μL Primer F (or standard Primer F): 0.4 μL Primer R (or standard Primer R): 0.4 μL Template DNA: 1 μL ROX Reference Dye II: 0.4 μL H 2 O: 7.8 μL Total: 20μL PCR reaction conditions: 95℃-30sec 95℃-5sec / 60℃-30sec:40cycles Melting curve analysis (Dissociation stage).

[0096] The expression level of the iNOS gene in each colon after 4 weeks of administration of water or tea fermentation composition (iNOS gene mRNA amount / GAPDH gene mRNA amount, relative iNOS mRNA level) is shown in FIG. 7. As shown in FIG. 7, it was confirmed that the expression level of the iNOS gene in the colon shows a tendency to decrease in the tea fermentation composition group compared to the water group (P value by Welch's t test between the expression level of the iNOS gene in the water group and the expression level of the iNOS gene in the tea fermentation composition group: P<0.1). It has been reported that administration of NO synthase inhibitor (L-NAME) shortens the gastrointestinal transit time of NcxKO mice, that NO in the colon contributes to intestinal motility disorder in constipated patients, and that the expression level of the iNOS gene in the colon contributes to intestinal motility, and it is considered that the decrease in the expression level of the iNOS gene in the colon contributed to the shortening of the gastrointestinal transit time.

[0097] Example 2 <Comparison of components of fermented tea composition and unfermented tea> The fermented tea composition used was the fermented tea composition obtained in Example 1 (lactic acid bacteria fermented tea). As a control, the above tea extract (heated at 122°C for 10 minutes, pH 6.2, lactic acid bacteria count: 0) was used as unfermented tea. The concentration of each component in the above tea extract was measured in the same manner as in Example 1. The results are shown in Table 5 below. Table 5 also shows the results of Example 1.

[0098] [Table 5]

[0099] As shown in Table 5, in the fermented tea composition of the present invention, lactic acid and acetic acid were produced by fermentation, and the pH decreased. It was also confirmed that pyrogallol (PG) was specifically produced in the fermented tea composition of the present invention. Furthermore, it was confirmed that the fermented tea composition of the present invention had decreased EGCg, ECg, GCg, and Cg concentrations, increased EGC, EC, GC, and C concentrations, and decreased total catechin concentration, compared to unfermented tea.

[0100] <Confirmation of intestinal regulation effect> NcxKO mice and WT mice were bred and kept until 24 male NcxKO mice and 8 male WT mice of similar age were obtained. CLEA Rodent Diet CE-2 (solid, CLEA Japan, Inc.) was used as food. When the mice reached 9-10 weeks of age, they were divided into the following 4 groups of 8 mice each to ensure that there was no weight bias: Group 1: WT mice, water group (control) Group 2: NcxKO mice, water group (control 1) Group 3: NcxKO mice, non-fermented tea group (control 2) Group 4: NcxKO mice, fermented tea composition group The mice were divided into groups of four. After grouping (before administration), feces were collected from each mouse. Then, the mice in groups 1 and 2 were given water, the mice in group 3 were given unfermented tea (the above tea extract, "Meiji Deep Steamed Shizuoka Tea", manufactured by Meiji Co., Ltd., heated at 122°C for 10 minutes), and the mice in group 4 were given the above fermented tea composition, all of which were given ad libitum for four weeks. Feed was placed in each cage and allowed to be consumed ad libitum. The feed and each administered substance (water, unfermented tea, or fermented tea composition) were replaced with new ones three times a week, and the body weight was measured every week from the start of administration. After four weeks of breeding from the start of administration of each administered substance, feces was collected from each mouse, and the cecal contents were also collected from each mouse.

[0101] The average body weight of mice in each group 4 weeks after the start of administration of water, unfermented tea, or fermented tea composition is shown in Figure 8. As shown in Figure 8, there was no difference in the body weight of mice between the groups during the experimental period.

[0102] 1. Gut flora analysis-1 (analysis by real-time PCR) The bacterial flora in the feces was analyzed by q-PCR in the same manner as in Example 1-2, except that feces collected before and after 4 weeks of administration of water, unfermented tea, or the fermented tea composition were used. The number of bacteria (Log 10 The detection rates (%) and the number of cells / g feces are shown in Table 6 below.

[0103] [Table 6]

[0104] As shown in Table 6, there was no significant change in the number of bacteria in each bacterial group before and after administration in the WT mice (group 1), the NcxKO water group (group 2), and the unfermented tea group (group 3). In contrast, it was confirmed that the numbers of bacteria in the C. coccoides group, C. leptum subgroup, and B. fragilis group were significantly reduced in the NcxKO fermented tea composition group (group 4).

[0105] 2. Gut flora analysis-2 (analysis using next-generation sequencers) The occupancy rate of each bacterial group in each stool was calculated in the same manner as in Example 1-3, except that the stool samples were collected before and after 4 weeks of administration of water, unfermented tea, or the fermented tea composition. The ratio of the number of reads of Lactobacillales to the total number of reads (occupancy rate, number of reads %) in each stool sample before and after (after 4 weeks of administration) of water, unfermented tea, or the fermented tea composition is shown in Table 7 below.

[0106] [Table 7]

[0107] As shown in Table 7, in the WT mice (group 1), the NcxKO mice water group (group 2) and the non-fermented tea group (group 3), there was no significant change in the Lactobacillus occupancy rate before and after administration. In contrast, in the NcxKO mice fermented tea composition group (group 4), it was confirmed that the Lactobacillus occupancy rate increased significantly before and after administration.

[0108] 3. Measurement of putrefactive product concentration in cecal contents The amount of skatole was measured in the same manner as in 4 of Example 1, except that the cecal contents collected after 4 weeks of administration of water, unfermented tea, or fermented tea composition were used. The amount of skatole (concentration, ng / g dry weight) per 1 g (dry weight) of each cecal content is shown in Figure 9.

[0109] As shown in Figure 9, it was confirmed that the concentration of skatole in the cecal contents of the NcxKO mice fermented tea composition group (group 4) was lower than that of the NcxKO mice water group (group 2) and unfermented tea group (group 3). [Industrial Applicability]

[0110] As described above, according to the present invention, it is possible to provide a novel fermented tea composition for intestinal regulation, which has intestinal regulating effects such as regulating the intestinal flora and shortening the digestive tract transit time, and a method for producing the same. The fermented tea composition for intestinal regulation of the present invention is particularly useful as, for example, a composition for regulating the intestinal flora, a composition for shortening the digestive tract transit time, a composition for reducing the expression level of iNOS gene in the intestine, a composition for preventing or ameliorating constipation, or a food or beverage composition. [Accession number]

[0111] 1. (1) Identification: Lactobacillus pentosus OLL203969 (2) Accession number: NITE BP-01986 (3) Original acceptance date: January 5, 2015 (4) Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depository Center 2. (1) Identification: Lactobacillus pentosus OLL203982 (2) Accession number: NITE BP-01987 (3) Original acceptance date: January 5, 2015 (4) Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depository Center 3. (1) Identification: Lactobacillus pentosus OLL203984 (2) Accession number: NITE BP-01988 (3) Original acceptance date: January 5, 2015 (4) Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depository Center [Sequence List Free Text]

[0112] SEQ ID NO:1 <223> g-Ccoc-F SEQ ID NO:2 <223> g-Ccoc-R SEQ ID NO:3 <223> sg-Clept-F SEQ ID NO:4 <223> sg-Clept-R SEQ ID NO:5 <223> g-Bact-F SEQ ID NO:6 <223> g-Bact-R SEQ ID NO:7 <223> 515F SEQ ID NO:8 <223> 806rcbc1 SEQ ID NO:9 <223> 806rcbc2 SEQ ID NO:10 <223> 806rcbc3 SEQ ID NO:11 <223> 806rcbc4 SEQ ID NO:12 <223> 806rcbc5 SEQ ID NO:13 <223> 806rcbc6 SEQ ID NO:14 <223> 806rcbc7 SEQ ID NO:15 <223> 806rcbc8 SEQ ID NO:16 <223> 806rcbc9 SEQ ID NO:17 <223> 806rcbc10 SEQ ID NO:18 <223> 806rcbc11 SEQ ID NO:19 <223> 806rcbc12 SEQ ID NO:20 <223> 806rcbc13 SEQ ID NO:21 <223> 806rcbc14 SEQ ID NO:22 <223> 806rcbc15 SEQ ID NO:23 <223> 806rcbc16 SEQ ID NO:24 <223> 806rcbc17 SEQ ID NO:25 <223> 806rcbc18 SEQ ID NO:26 <223> 806rcbc19 SEQ ID NO:27 <223> 806rcbc20 SEQ ID NO:28 <223> 806rcbc21 SEQ ID NO:29 <223> 806rcbc22 SEQ ID NO:30 <223> 806rcbc23 SEQ ID NO:31 <223> 806rcbc24 SEQ ID NO:32 <223> 806rcbc25 SEQ ID NO:33 <223> 806rcbc26 SEQ ID NO:34 <223> 806rcbc27 SEQ ID NO:35 <223> 806rcbc28 SEQ ID NO:36 <223> Primer 1.1 SEQ ID NO:37 <223> Primer 2.1 SEQ ID NO:38 <223> iNOS Forward Primer SEQ ID NO:39 <223> iNOS Reverse Primer SEQ ID NO:40 <223> GAPDH Forward standard primer SEQ ID NO:41 <223> GAPDH Reverse standard primer

Claims

1. A tea fermentation product containing, as an active ingredient, at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus identified by accession number NITE BP-01986, Lactobacillus pentosus identified by accession number NITE BP-01987, and Lactobacillus pentosus identified by accession number NITE BP-01988, The lactic acid bacteria are pyrogallol-producing lactic acid bacteria, The fermented tea product is a lactic acid bacteria fermentation product of a tea extract, and further contains the tea extract and at least one selected from the group consisting of the lactic acid bacteria and processed products thereof, A composition for shortening gastrointestinal transit time. A fermented tea composition for intestinal regulation.

2. The fermented tea composition for intestinal regulation according to claim 1, characterized in that the fermented tea product contains pyrogallol.

3. The fermented tea composition for intestinal regulation according to claim 1 or 2, characterized in that it is a composition for regulating the intestinal flora.

4. The fermented tea composition for intestinal regulation according to any one of claims 1 to 3, which is a composition for preventing or improving constipation.

5. The fermented tea composition for intestinal regulation according to any one of claims 1 to 4, which is a food or drink composition.

6. A method for producing a fermented tea composition for intestinal regulation, A step of preparing a fermentation mix containing a tea extract and at least one lactic acid bacterium selected from the group consisting of Lactobacillus pentosus identified by accession number NITE BP-01986, Lactobacillus pentosus identified by accession number NITE BP-01987, and Lactobacillus pentosus identified by accession number NITE BP-01988; fermenting the tea extract with the lactic acid bacteria in the fermentation mix to obtain a tea fermented product; Including, The lactic acid bacteria are pyrogallol-producing lactic acid bacteria, and The fermented tea composition for intestinal regulation is a composition for shortening the digestive tract transit time. A method for producing a fermented tea composition for intestinal regulation.

7. The method for producing a fermented tea composition for intestinal regulation according to claim 6, characterized in that the fermented tea product contains pyrogallol.

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