Food for improving intraintestinal environment

Abstract

A food for improving intestinal environment by promoting proliferation of intestinal bifidobacteria is described herein. The food for improving intestinal environment can contain a combination of (1) an enteric composition containing an amino acid or a salt thereof and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide.

Claims

1. A food for improving an intestinal environment in a subject comprising: (1) an enteric composition comprising an amino acid or a salt thereof in an amount of 0.1 to 50 g/60 kg body weight of the subject, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide, wherein the indigestible polysaccharide is in an amount of 0.1 to 50 g/60 kg body weight of the subject; wherein a synergistic effect exceeding an additive effect in the proliferation of Bifidobacteria is observed after combining (1) and (2).

2. The food according to claim 1, wherein the indigestible polysaccharide is an oligosaccharide.

3. The food according to claim 1, wherein the amino acid is selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, histidine, lysine, proline, valine, and combinations thereof.

4. The food according to claim 1, wherein the amino acid is selected from the group consisting of glutamic acid, lysine, and a combination thereof.

5. The food according to claim 1, wherein the form of the enteric composition is selected from the group consisting of a granule, a granulated substance, a tablet, a hard capsule, and a soft capsule.

6. A method for improving an intestinal environment in a human comprising ingestion by the human of an effective amount of a food comprising: (1) an enteric composition comprising an amino acid or a salt thereof in an amount of 0.1 to 50 g/60 kg body weight of the human, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide, wherein the indigestible polysaccharide is in an amount of 0.1 to 50 g/60 kg body weight of the human; wherein a synergistic effect exceeding an additive effect in the proliferation of Bifidobacteria is observed after combining (1) and (2).

7. A method of producing a food for improving the intestinal environment in a subject comprising combining (1) an enteric composition comprising an amino acid or a salt thereof in an amount of 0.1 to 50 g/60 kg body weight of the subject, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide, wherein the indigestible polysaccharide is in an amount of 0.1 to 50 g/60 kg body weight of the subject; wherein a synergistic effect exceeding an additive effect in the proliferation of Bifidobacteria is observed after combining (1) and (2).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1-1 shows the results of Experimental Example 1-1.

(2) FIG. 1-2 shows the results of Experimental Example 1-2.

(3) FIG. 2 shows the results of Experimental Example 2.

(4) FIG. 3 shows the results of relative amounts of cecal bifidobacteria in Experimental Example 3.

(5) FIG. 4 shows the results of cecal lysine concentration in Experimental Example 3.

DETAILED DESCRIPTION

(6) The food for improving intestinal environment as described herein is composed of a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide.

(7) The food is a combined-use food of (1) and (2) in which (1) and (2) are used in combination.

(8) In the food as described herein, (1) and (2) may be simultaneously formulated and contained in the same preparation (food), or (1) and (2) may be formulated separately and ingested simultaneously or at different times by the same route or different routes. That is, the food as described herein includes a food containing (1) and (2) in one preparation, and a food combining (1) and (2) formulated separately.

(9) The food as described herein is a concept that broadly encompasses foods that can be taken orally, excluding pharmaceuticals, and includes not only so-called “food” but also includes drinks, health supplements, foods with health claims, such as foods for specified health uses or foods with functional claims, supplements, and the like.

(10) The phrase “improvement of intestinal environment” can refer to increasing the relative bifidobacteria present in the lower gastrointestinal tract. To relatively increase can mean to promote the predominance of bifidobacteria in the intestinal bacterial flora. The predominance of bifidobacteria in the intestinal bacterial flora can be confirmed by measuring the amount of bifidobacteria in feces by quantitative PCR or the like.

(11) The “lower gastrointestinal tract” can mean the ileum, cecum, colon, and rectum.

(12) Enteric Composition Containing an Amino Acid or a Salt Thereof

(13) Examples of the amino acid include alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine; and alanine, glutamine, glutamic acid, isoleucine, histidine, lysine, proline, and valine are particular examples. These amino acids can be in an L form. One or more of these amino acids can be used in combination.

(14) As the amino acid, L-alanine, L-glutamine, L-glutamic acid, L-isoleucine, L-histidine, L-lysine, L-proline, L-valine, or a combination of these, such as a combination of L-glutamic acid and L-lysine is a particular example, and L-glutamic acid, L-lysine, or a combination of L-glutamic acid and L-lysine is another particular example.

(15) The amino acid may be in the form of a salt. Examples include salts acceptable as medicament or food. Examples include alkali metal salts such as sodium salt, potassium salt and the like; alkaline earth metal salts such as calcium salt, magnesium salt, barium salt, and the like; aluminum salt; salts with organic bases such as ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, triethanolamine, and the like; salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; and salts with organic acids such as formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; and sodium salt, potassium salt, or hydrochloride are particular examples.

(16) The enteric composition is a composition that does not disintegrate in the stomach or the upper part of the small intestine where the pH is low, but disintegrates in the lower part of the small intestine or large intestine where the pH is high, and the amino acid or a salt thereof can be effective.

(17) The enteric composition can disintegrate at pH 4 or more, pH 4-pH 9, or pH 5-pH 9.

(18) The disintegration property can be measured, for example, according to the rules for enteric preparations in the disintegration test method, the Japanese Pharmacopoeia 17th edition.

(19) Examples of the form of the enteric composition include granules including fine granules, a granulated substance, tablet, hard capsule, and soft capsule.

(20) The enteric composition can be produced by a method known in the field of food preparation or pharmaceutical preparation.

(21) For example, methods of production can include mixing, granulating and/or tableting an amino acid or a salt thereof together with a carrier, such as excipient, binder, disintegrant, lubricant, protector, to give a granule or tablet, then applying an enteric coating to the granule or tablet to give an enteric granule or enteric tablet; filling granules or tablets containing an amino acid or a salt thereof in a hard capsule applied with an enteric coating to give an enteric capsule; and encapsulating a suspension of an amino acid or a salt thereof in a carrier, such as an oil component, in a soft capsule composed of an enteric substrate to give an enteric capsule. In addition, the above-described enteric granules may be encapsulated in a capsule, such as a cellulose capsule, to give a capsule. Also, a molten mixture of oil and fat (protector) and an amino acid or a salt thereof may be solidified by cooling to give an enteric granulated substance.

(22) Commercially available products can also be used.

(23) Examples of the enteric coating base include cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, cellulose acetate phthalate and the like; acrylic acid polymers such as methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, and the like; and naturally occurring substances such as shellac and the like. A coating additive such as a plasticizer may also be used during coating.

(24) The carrier, coating base, and coating additive are used in amounts conventionally employed in the technical field of preparation formulation.

(25) (2) Indigestible Polysaccharide or Composition Containing Indigestible Polysaccharide

(26) The indigestible polysaccharide is a polysaccharide that is not easily digested or absorbed in the stomach or small intestine, and that reaches the large intestine to be utilized by bifidobacteria.

(27) The degree of polymerization of the indigestible polysaccharides is not less than 2 or not less than 3. The upper limit of the degree of polymerization of indigestible polysaccharides is not particularly limited and can be, for example, not more than 100, not more than 60, not more than 10, not more than 6, or not more than 5.

(28) The degree of polymerization of the indigestible polysaccharides can be 2-100, 2-60, 2-10, or 2-6.

(29) Examples of the indigestible polysaccharides include oligosaccharides, water-soluble dietary fiber, and the like.

(30) Indigestible polysaccharides, such as oligosaccharides or water-soluble dietary fiber, can be used as they are, or can be in a composition containing indigestible polysaccharides, such as oligosaccharides or water-soluble dietary fiber.

(31) The oligosaccharide can have 2 to 10 monosaccharides bonded thereto.

(32) Examples of the oligosaccharide include coffee bean manno-oligosaccharide, lactosucrose oligosaccharide, galacto-oligosaccharide, fructo-oligosaccharide, soybean oligosaccharide, xylo-oligosaccharide, and isomalto-oligosaccharide; and fructo-oligosaccharide or galacto-oligosaccharide are particular examples. One or more kinds of oligosaccharides can be used in combination.

(33) The oligosaccharide can be produced by a known method, and commercially available products can also be used.

(34) Examples of the fructo-oligosaccharide include fructo-oligosaccharides with polymerization degree of 3-5 in which 1 to 3 fructoses are bonded to sucrose. Specifically, for example, FOS: Wako Pure Chemical Industries, Ltd., #064-02385 is a particular example.

(35) Examples of the galacto-oligosaccharide include galacto-oligosaccharide with polymerization degree of 2-6 in which galactose is bonded to lactose. Specifically, for example, GOS: Wako Pure Chemical Industries, Ltd., #076-05945 is a particular example.

(36) Water-soluble dietary fiber is a dietary fiber that is not easily digested or absorbed in the stomach or small intestine, and can reach the large intestine to be degraded by intestinal bacteria and utilized by bifidobacteria.

(37) Examples of the water-soluble dietary fiber include inulin.

(38) The water-soluble dietary fiber such as inulin can be produced by a known method, and commercially available products can also be used.

(39) Inulin generally has a structure in which 2-60 molecules of fructose are polymerized by a β(2-1) bond and a glucose molecule is bonded to the terminal.

(40) Inulin is degraded by intestinal bacteria in the large intestine (partial hydrolysis) to fructo-oligosaccharide. Thus, an effect similar to the effect of fructo-oligosaccharide was confirmed in the Experimental Examples 1-1, 1-2, and is expected to be present in the gastrointestinal tract.

(41) The form of the composition containing indigestible polysaccharides such as an oligosaccharide or water-soluble dietary fiber is not particularly limited and may be, for example, a powder, granule (including fine granules), tablet, hard capsule, soft capsule, liquid (e.g., solution, suspension, milky lotion), drink, jelly, pudding, yogurt, candy, chewing gum, or the like. These can be produced by a known method. For example, indigestible polysaccharides, such as oligosaccharides or water-soluble dietary fiber, can be mixed with carriers, such as an excipient, binder, disintegrant, lubricant, or solvent, to produce a powder, granule, tablet, capsule, liquid, and the like by a method known in the field of food preparation or pharmaceutical preparation. In addition, they can also be produced by adding and mixing indigestible polysaccharides such as oligosaccharide or water-soluble dietary fiber to and with food and drink, such as water or a soft drink. Commercially available products, such as fructo-oligosaccharide “Meiorigo P granule” manufactured by Meiji Food Materia Co., Ltd., can also be used.

(42) As described above, the food as described herein includes a food containing the above-mentioned (1) and (2) in one preparation, and a food combining the above-mentioned (1) and (2) formulated separately.

(43) As the food combining (1) and (2) formulated separately, for example, a food is described combining (1) in the form of an enteric granule, enteric granulated substance, enteric tablet, enteric hard capsule, or enteric soft capsule containing amino acid or a salt thereof, and (2) in the form of a drink containing an indigestible polysaccharide, such as oligosaccharide or water-soluble dietary fiber.

(44) (1) and (2) may be ingested simultaneously or at different times by the same route or different routes. Specifically, for example, an enteric capsule such as a hard capsule or soft capsule containing an amino acid or a salt thereof, enteric granule, enteric granulated substance, and the like may be ingested together with drink containing an indigestible polysaccharide such as an oligosaccharide or water-soluble dietary fiber. The ingestion amount of an amino acid or a salt thereof in the food for improving intestinal environment can be generally 0.1-50 g, 0.2-20 g, or 0.5-10 g, based on amino acid, per day for an adult (body weight 60 kg).

(45) The ingestion amount of an indigestible polysaccharide such as an oligosaccharide or water-soluble dietary fiber in the food for improving intestinal environment can be generally 0.1-50 g, 0.2-20 g, or 0.5-10 g, per day for an adult (body weight 60 kg).

(46) The food can be safely given to humans and animals other than human, such as mammals and birds such as domestic animals, poultry, experimental animals, and the like. When providing the food to animals other than human, it may be added to the animal's feed.

(47) Further embodiments include a commercial package containing a food containing a combination of (1) an enteric composition containing an amino acid or a salt thereof and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide, and written matter describing and explaining uses of the food for improving intestinal environment; a food with an indication that it is useful for improving intestinal environment, which contains a combination of (1) an enteric composition containing amino acid or a salt thereof and (2) indigestible polysaccharide or a composition containing indigestible polysaccharide; a food for enhancing the intestinal environment improving effect containing a combination of (1) an enteric composition containing an amino acid or a salt thereof and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide; and a food for enhancing the intestinal environment improving effect of an indigestible polysaccharide or a composition containing an indigestible polysaccharide, which food is an enteric composition containing an amino acid or a salt thereof. The “enteric composition containing an amino acid or a salt thereof”, “an indigestible polysaccharide or a composition containing indigestible polysaccharide”, “intestinal environment improvement”, “ingestion amount of amino acid or a salt thereof” and “ingestion amount of indigestible polysaccharides” are the same as exemplifications and definitions indicated for the above-described food for improving intestinal environment.

EXAMPLES

(48) The present invention is explained in more detail by referring to the following non-limiting Experimental Examples, Examples, Comparative Examples, and Reference Examples.

(49) Abbreviations mean the following:

(50) Ala: L-alanine

(51) Arg: L-arginine

(52) Asn: L-asparagine

(53) Cys: L-cysteine

(54) Gln: L-glutamine

(55) Glu: L-glutamic acid

(56) Gly: glycine

(57) His: L-histidine

(58) Ile: L-isoleucine

(59) Leu: L-leucine

(60) Lys: L-lysine

(61) Met: L-methionine

(62) Phe: L-phenylalanine

(63) Pro: L-proline

(64) Ser: L-serine

(65) Thr: L-threonine

(66) Val: L-valine

(67) FOS: fructo-oligosaccharide

(68) GOS: galacto-oligosaccharide

(69) PBS: phosphate buffered saline

(70) The PBS solutions for the Examples and Comparative Examples shown in Table 1-1 were prepared and used as media for a feces culture. PBS alone was used as a control. Fructo-oligosaccharide (FOS) manufactured by Wako Pure Chemical Industries, Ltd. (#064-02385) was used.

(71) Thereafter, 10 ml of each solution was dispensed into glass vials and the glass vials were sterilized by autoclave, made anaerobic, and sealed with a butyl rubber stopper. The feces suspension was prepared by collecting about 10 g of feces from 3 healthy individuals (one man and two women) in their 20 s to 40 s and suspending same in 80 ml of anaerobic PBS. The feces suspension was added to each vial by 0.5 ml, placed in an incubator at 37° C., and anaerobically cultured for 4 days with tumble blending once per day. After completion of the culture, an intestinal bacterial pellet was obtained from the entire amount of the culture medium by centrifugation (8,000×g, 5 min). DNA was extracted from this pellet using ISOFECAL for BeadsBeat (manufactured by NIPPON GENE CO., LTD.), and quantitative PCR was performed using GeneAce SYBR qPCR Mix α (manufactured by NIPPON GENE CO., LTD.). Quantitative PCR was performed under the conditions of 45 cycles of 95° C. for 30 sec and 60° C. for 60 sec after 95° C. for 10 min. Primers for quantitative PCR used for detection bifidobacteria are as follows.

(72) TABLE-US-00001 (SEQ ID NO: 1) g-Bifid-F: 5′-CTCCTGGAAACGGGTGG-3′ (SEQ ID NO: 2) g-Bifid-R: 5′-GGTGTTCTTCCCGATATCTACA-3′

(73) TABLE-US-00002 TABLE 1-1 medium for feces culture Example 1-1 0.1% Ala and 0.5% FOS-containing PBS solution Example 1-2 0.5% Ala and 0.5% FOS-containing PBS solution Example 1-3 1.0% Ala and 0.5% FOS-containing PBS solution Example 2-1 0.1% Arg and 0.5% FOS-containing PBS solution Example 2-2 0.5% Arg and 0.5% FOS-containing PBS solution Example 2-3 1.0% Arg and 0.5% FOS-containing PBS solution Example 3-1 0.1% Gln and 0.5% FOS-containing PBS solution Example 3-2 0.5% Gln and 0.5% FOS-containing PBS solution Example 3-3 1.0% Gln and 0.5% FOS-containing PBS solution Example 4-1 0.1% Glu and 0.5% FOS-containing PBS solution Example 4-2 0.5% Glu and 0.5% FOS-containing PBS solution Example 4-3 1.0% Glu and 0.5% FOS-containing PBS solution Example 5-1 0.1% Gly and 0.5% FOS-containing PBS solution Example 5-2 0.5% Gly and 0.5% FOS-containing PBS solution Example 5-3 1.0% Gly and 0.5% FOS-containing PBS solution Example 6-1 0.1% Ile and 0.5% FOS-containing PBS solution Example 6-2 0.5% Ile and 0.5% FOS-containing PBS solution Example 6-3 1.0% Ile and 0.5% FOS-containing PBS solution Example 7-1 0.1% Lys and 0.5% FOS-containing PBS solution Example 7-2 0.5% Lys and 0.5% FOS-containing PBS solution Example 7-3 1.0% Lys and 0.5% FOS-containing PBS solution Example 8-1 0.1% Ser and 0.5% FOS-containing PBS solution Example 8-2 0.5% Ser and 0.5% FOS-containing PBS solution Example 8-3 1.0% Ser and 0.5% FOS-containing PBS solution Example 9-1 0.1% Val and 0.5% FOS-containing PBS solution Example 9-2 0.5% Val and 0.5% FOS-containing PBS solution Example 9-3 1.0% Val and 0.5% FOS-containing PBS solution Example 10-1 0.1% Glu, 0.1% Lys and 0.5% FOS-containing PBS solution Example 10-2 0.5% Glu, 0.5% Lys and 0.5% FOS-containing PBS solution Example 10-3 1.0% Glu, 1.0% Lys and 0.5% FOS-containing PBS solution Comparative 1.0% Ala-containing PBS solution Example 1 Comparative 1.0% Arg-containing PBS solution Example 2 Comparative 1.0% Gln-containing PBS solution Example 3 Comparative 1.0% Glu-containing PBS solution Example 4 Comparative 1.0% Gly-containing PBS solution Example 5 Comparative 1.0% Ile-containing PBS solution Example 6 Comparative 1.0% Lys-containing PBS solution Example 7 Comparative 1.0% Ser-containing PBS solution Example 8 Comparative 1.0% Val-containing PBS solution Example 9 Comparative 1.0% Glu and 1.0% Lys-containing Example 10 PBS solution Comparative 0.5% FOS-containing PBS solution Example 11 Reference PBS alone (control) Example 1

(74) Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

(75) The results are shown in FIG. 1-1. The data shows a mean of z-score±standard error (n=3).

(76) As a result, proliferation of bifidobacteria was not observed when any amino acid was added alone. However, when Ala, Gln, Glu, Ile, Lys, and Val were respectively added together with 0.5% FOS, greater proliferation of bifidobacteria than that with FOS alone was observed. In addition, when Glu and Lys were mixed and added together with 0.5% FOS, greater proliferation of bifidobacteria than that with FOS alone was similarly observed.

(77) Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and an oligosaccharide.

Experimental Example 1-2: Addition Test of Amino Acid and Fructo-Oligosaccharide to Human Feces Suspension

(78) The PBS solutions for the Examples and Comparative Examples shown in Table 1-2 were prepared and used as media for a feces culture. PBS alone was used as a control. Feces culture and detection of bifidobacteria were performed according to methods similar to those in Experimental Example 1-1.

(79) TABLE-US-00003 TABLE 1-2 medium for feces culture Example 11-1 0.1% Asn and 0.5% FOS-containing PBS solution Example 11-2 0.5% Asn and 0.5% FOS-containing PBS solution Example 11-3 1.0% Asn and 0.5% FOS-containing PBS solution Example 12-1 0.1% Cys and 0.5% FOS-containing PBS solution Example 12-2 0.5% Cys and 0.5% FOS-containing PBS solution Example 12-3 1.0% Cys and 0.5% FOS-containing PBS solution Example 13-1 0.1% His and 0.5% FOS-containing PBS solution Example 13-2 0.5% His and 0.5% FOS-containing PBS solution Example 13-3 1.0% His and 0.5% FOS-containing PBS solution Example 14-1 0.1% Leu and 0.5% FOS-containing PBS solution Example 14-2 0.5% Leu and 0.5% FOS-containing PBS solution Example 14-3 1.0% Leu and 0.5% FOS-containing PBS solution Example 15-1 0.1% Met and 0.5% FOS-containing PBS solution Example 15-2 0.5% Met and 0.5% FOS-containing PBS solution Example 15-3 1.0% Met and 0.5% FOS-containing PBS solution Example 16-1 0.1% Phe and 0.5% FOS-containing PBS solution Example 16-2 0.5% Phe and 0.5% FOS-containing PBS solution Example 16-3 1.0% Phe and 0.5% FOS-containing PBS solution Example 17-1 0.1% Pro and 0.5% FOS-containing PBS solution Example 17-2 0.5% Pro and 0.5% FOS-containing PBS solution Example 17-3 1.0% Pro and 0.5% FOS-containing PBS solution Example 18-1 0.1% Thr and 0.5% FOS-containing PBS solution Example 18-2 0.5% Thr and 0.5% FOS-containing PBS solution Example 18-3 1.0% Thr and 0.5% FOS-containing PBS solution Comparative 0.5% FOS-containing PBS solution Example 11 Comparative 1.0% Asn-containing PBS solution Example 12 Comparative 1.0% Cys-containing PBS solution Example 13 Comparative 1.0% His-containing PBS solution Example 14 Comparative 1.0% Leu-containing PBS solution Example 15 Comparative 1.0% Met-containing PBS solution Example 16 Comparative 1.0% Phe-containing PBS solution Example 17 Comparative 1.0% Pro-containing PBS solution Example 18 Comparative 1.0% Thr-containing PBS solution Example 19 Reference PBS alone (control) Example 1

(80) Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

(81) The results are shown in FIG. 1-2. The data shows a mean of z-score standard error (n=3).

(82) As a result, proliferation of bifidobacteria was not observed when any amino acid was added alone. However, when His and Pro were respectively added together with 0.5% FOS, greater proliferation of bifidobacteria than that with FOS alone was observed.

(83) Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and an oligosaccharide.

Experimental Example 2: Addition Test of Amino Acid and Galacto-Oligosaccharide to Human Feces Suspension

(84) The PBS solutions for the Examples and Comparative Examples shown in Table 2 were prepared and used as media for feces culture. Feces culture and detection of bifidobacteria were performed according to methods similar to those in Experimental Example 1-1. Galacto-oligosaccharide (GOS) manufactured by Wako Pure Chemical Industries, Ltd. (#076-05945) was used.

(85) TABLE-US-00004 TABLE 2 medium for feces culture Example 19-1 0.1% Glu and 0.5% GOS-containing PBS solution Example 19-2 0.5% Glu and 0.5% GOS-containing PBS solution Example 19-3 1.0% Glu and 0.5% GOS-containing PBS solution Example 20-1 0.1% Lys and 0.5% GOS-containing PBS solution Example 20-2 0.5% Lys and 0.5% GOS-containing PBS solution Example 20-3 1.0% Lys and 0.5% GOS-containing PBS solution Example 21-1 0.1% Glu, 0.1% Lys and 0.5% GOS-containing PBS solution Example 21-2 0.5% Glu, 0.5% Lys and 0.5% GOS-containing PBS solution Example 21-3 1.0% Glu, 1.0% Lys and 0.5% GOS-containing PBS solution Comparative 0.1% Glu-containing PBS solution Example 20-1 Comparative 0.5% Glu-containing PBS solution Example 20-2 Comparative 1.0% Glu-containing PBS solution Example 20-3 Comparative 0.1% Lys-containing PBS solution Example 21-1 Comparative 0.5% Lys-containing PBS solution Example 21-2 Comparative 1.0% Lys-containing PBS solution Example 21-3 Comparative 0.1% Glu and 0.1% Lys-containing Example 22-1 PBS solution Comparative 0.5% Glu and 0.5% Lys-containing Example 22-2 PBS solution Comparative 1.0% Glu and 1.0% Lys-containing Example 22-3 PBS solution Comparative 0.5% GOS-containing PBS solution Example 23 Reference PBS alone (control) Example 2

(86) Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

(87) The results are shown in FIG. 2. The data shows a mean of z-score±standard error (n=3).

(88) As a result, proliferation of bifidobacteria was not observed when amino acid was added alone at any concentration. However, when Glu and Lys were respectively added together with 0.5% GOS, greater proliferation of bifidobacteria than that with GOS alone was observed. In addition, when Glu and Lys were mixed and added together with 0.5% GOS, greater proliferation of bifidobacteria than that with GOS alone was similarly observed.

(89) Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and an oligosaccharide irrespective of the type thereof.

(90) Experimental Example 3: Administration test of enteric Lys and FOS to pigs

(91) Six 2 to 3 months-old male pigs (HI-COOP SPF pig LWD) were purchased from ZEN-NOH, divided into 2 test groups each containing 3 pigs such that the average body weight was uniform, and kept for 4 weeks at 3 pigs/pen. In each test group, 400 g/head of pig MIRAI CEX (ZEN-NOH) was fed twice per day. In each test group, the test substance was administered by the dosage and method shown in Table 3. A lysine preparation “AjiPro-L” for dairy cows manufactured by Ajinomoto Co., Inc. (lysine hydrochloride content is 40% or more) was used as Enteric Lys, and fructo-oligosaccharide “Mayoligo P granule” manufactured by Meiji Food Materia Co., Ltd. (FOS content is 95% or more) was used as FOS. After 4 weeks from the start of administration, the cecum was collected, and bifidobacteria in the cecal contents were examined by quantitative PCR in the same manner as in Experimental Examples 1-1, 1-2 and 2. The concentration of Lys in the cecum contents was measured using 6470 Triple Quad LC/MS/MS system manufactured by Agilent and Intrada Amino Acid column manufactured by Imtakt.

(92) TABLE-US-00005 TABLE 3 dosage and administration method of test substance Example 22 FOS: Meiorigo P granule (10 g) was mixed with each meal Enteric Lys: Oral administration of 2 cellulose capsules after each meal, each capsule encapsulating 6 g of AjiPro-L Comparative FOS: Meiorigo P granule (10 g) was mixed with each meal Example 24 placebo: Oral administration of 2 empty cellulose capsules after each meal

(93) The results obtained by quantitative PCR are shown as relative amounts when Comparative Example 24 (placebo) is 1. The results of the relative amount of bifidobacteria in the cecum are shown in FIG. 3 (mean±standard deviation, n=3). As a result, pig intestinal bifidobacteria proliferation is about 3 times higher on average when the combination of enteric Lys and FOS was ingested than by the ingestion of FOS alone.

(94) In addition, the results of the concentration of Lys in the cecum at that time are shown in FIG. 4 (mean±standard deviation, n=3). As a result, it was confirmed that the concentration of Lys in the cecum increased in the enteric Lys ingestion group.

(95) Thus, a synergistic effect with fructo-oligosaccharide was observed by increasing the concentration of Lys in the lower gastrointestinal tract by the ingestion of enteric Lys.