JP2024152695A - Composition and method for growing bifidobacteria, and composition and method for increasing lactic acid and/or pyruvic acid - Google Patents

Abstract

The present invention provides a technique for effectively growing bifidobacteria and a technique for effectively increasing lactic acid and/or pyruvic acid in the intestine.
[Solution] A composition for growing bifidobacteria, containing oligosaccharides and proteins as active ingredients. According to the present invention, the number of bifidobacteria can be effectively increased. Therefore, useful effects known for bifidobacteria (intestinal regulation effects such as improvement of constipation, immunostimulatory effects such as prevention of infectious diseases and prevention/amelioration of allergic diseases, etc.) can be expected.
[Selected Figure] Figure 1

Description

The present invention relates to a composition and method for growing bifidobacteria using oligosaccharides and proteins, and a composition and method for increasing lactic acid and/or pyruvic acid in the intestine.

Bifidobacteria (bifidobacteria) are normally present in the intestines of humans and animals, and are known to play useful roles such as acidifying the intestinal environment with their metabolic product, lactic acid, and preventing the growth of so-called bad bacteria.

In addition, lactic acid and pyruvic acid produced by the metabolism of intestinal bacteria, including bifidobacteria, have been reported in recent years to act directly on macrophages in the small intestine and increase resistance to pathogenic bacteria (Non-Patent Document 1), making them useful substances that are expected to have immune-activating effects.

Therefore, research and development has been conducted on substances that increase the proliferation of bifidobacteria in the intestines or substances that increase lactic acid or pyruvic acid in the intestines. For example, Patent Document 1 discloses an agent for increasing the proliferation of bifidobacteria in the intestinal environment, the active ingredient of which is an oligosaccharide with a purity of 90% by weight or more of 1-kestose.

Patent No. 4669235

Osaka University and Japan Agency for Medical Research and Development, Top > News > Press Release > Elucidation of the mechanism by which lactate and pyruvate produced by intestinal bacteria activate the immune system, Posted on January 24, 2019, Last updated on January 24, 2019, [Retrieved May 6, 2022], Internet <URL: https://www.amed.go.jp/news/release_20190124-01.html>

As shown in Patent Document 1, 1-kestose was known to have the effect of increasing bifidobacteria in the intestines, but no substance was known that could be used in combination with it to more effectively increase bifidobacteria.

On the other hand, protein is one of the three major nutrients, along with carbohydrates and lipids, and is an essential food component for maintaining life. Traditionally, athletes and others tended to actively consume protein to build their bodies, but in recent years, people who do not regularly engage in intense exercise, including the elderly, have been actively consuming protein to maintain and improve their health or for beauty purposes. However, it has been reported that a high protein intake leads to a decrease in the number of bifidobacteria in the intestine and the proliferation of so-called bad bacteria, which deteriorates the intestinal environment (Chunlong Mu, et al. Temporal microbiota changes of high-protein diet intake in a rat model. Anaerobe 2017;47:218-225.), which has been a problem.

The present invention has been made to solve these problems, and aims to provide a technology that can effectively grow bifidobacteria and maintain a good intestinal environment even when a large amount of protein is ingested. It also aims to provide a technology that can effectively increase lactic acid and/or pyruvic acid in the intestine.

As a result of intensive research, the inventors have found that simultaneous intake of oligosaccharides and protein can significantly increase bifidobacteria in the intestines, and can significantly increase lactic acid and pyruvic acid in the intestines, even at intake levels that would not show a noticeable effect when taken alone. Based on this knowledge, the inventors have completed the following inventions.

(1) The composition for growing bifidobacteria according to the present invention contains oligosaccharides and proteins as active ingredients.

(2) The composition for increasing intestinal lactic acid and/or pyruvic acid according to the present invention contains oligosaccharides and proteins as active ingredients.

(3) In the present invention, the oligosaccharide may be one or more selected from the group consisting of 1-kestose, lactulose, lactosucrose, isomaltooligosaccharides, and galactooligosaccharides.

(4) In the present invention, the oligosaccharide may be 1-kestose.

(5) The method for growing bifidobacteria according to the present invention (sometimes simply referred to as the "growing method" in the present invention) includes a step of having a human or animal ingest oligosaccharides and proteins. This method may be one excluding medical procedures.

(6) The method of the present invention for increasing intestinal lactate and/or pyruvic acid (sometimes simply referred to as the "method of increasing" in the present invention) includes a step of having a human or animal ingest oligosaccharides and proteins. This method may be exclusive of medical procedures.

(7) In the present invention, the intake of oligosaccharides may be 0.02 g/kg body weight or more per day, and the intake of protein may be 0.1 g/kg body weight or more per day.

According to the present invention, the number of bifidobacteria can be effectively increased. Therefore, it is possible to expect useful effects known for bifidobacteria (intestinal regulation effects such as improving constipation, immunostimulatory effects such as preventing infectious diseases and preventing/improving allergic diseases, etc.).

According to the present invention, the number of bifidobacteria can be effectively increased by ingesting oligosaccharides together with protein, and this is expected to suppress the deterioration of the intestinal environment caused by ingesting large amounts of protein and maintain a good intestinal environment.

According to the present invention, it is possible to effectively increase lactic acid and pyruvic acid in the intestine. Therefore, it is possible to expect useful effects known to be produced by lactic acid and pyruvic acid in the intestine (for example, the effect of inhibiting the growth of harmful bacteria by lowering the pH level in the intestine, the effect of improving constipation, the immune activation effect described above, etc.).

FIG. 1 is a box plot showing the occupancy rate of bifidobacteria in the cecal contents of rats fed a diet high in 1-kestose and/or casein. FIG. 1 is a box plot showing the occupancy of the genus Bifidobacterium in the cecal contents of rats fed a diet high in 1-kestose and/or casein. 1 is a bar graph showing the relative concentrations of lactate and pyruvate in the cecal contents of rats fed diets high in 1-kestose and/or casein. FIG. 1 is a box plot showing the occupancy of Bifidobacteriaceae in the cecal contents of rats fed a diet high in 1-kestose and/or whey. FIG. 1 is a box plot showing the occupancy of the genus Bifidobacterium in the cecal contents of rats fed a diet high in 1-kestose and/or whey. 1 is a bar graph showing the relative concentrations of lactate and pyruvate in the cecal contents of rats fed diets high in 1-kestose and/or whey. FIG. 1 is a box plot showing the occupancy rate of bifidobacteria in the cecal contents of rats fed a diet high in 1-kestose and/or soy protein. FIG. 1 is a box plot showing the occupancy of the genus Bifidobacterium in the cecal contents of rats fed a diet high in 1-kestose and/or soy protein.

The present invention will be described in detail below. In the present invention, the "composition for growing bifidobacteria" and the "composition for increasing lactic acid and/or pyruvic acid in the intestine" may be collectively referred to as "the composition" or may refer to either one of these compositions.

The present invention uses oligosaccharides and proteins as active ingredients, which have the effect of promoting the growth of bifidobacteria, a type of intestinal bacteria, and increasing their numbers.

Bifidobacteria are bacteria belonging to the family Bifidobacteriaceae (Watanabe, Koichi, Current Status and Trends of Taxonomy of Bifidobacteria, Intestinal Microbiology Journal, 2016, Vol. 30, pp. 129-139). Examples of bifidobacteria include the genera Bifidobacterium, Gardnerella, Alloscardovia, Aeriscardovia, Parascardovia, Scardovia, Bombiscardovia, Galliscardovia, Neoscardovia, and Pseudoscardovia. Among these, the type genus of bifidobacteria is the genus Bifidobacterium, and examples of the genus Bifidobacterium include Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium pseudocatenulatum, Bifidobacterium gallicum ... Examples of Bifidobacterium include Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium pseudolongum, Bifidobacterium gallinarum, Bifidobacterium pullorum, Bifidobacterium boum, Bifidobacterium thermophilum, Bifidobacterium magnum, Bifidobacterium merycicum, and Bifidobacterium minimum.

The present invention uses oligosaccharides and proteins as active ingredients, and the active ingredients have the effect of increasing lactic acid and/or pyruvic acid in the intestine. Lactic acid is a type of carboxylic acid represented by the chemical formula C ₃ H ₆ O _3. Pyruvic acid is a type of carboxylic acid represented by the chemical formula C ₃ H ₄ O _3. In vivo, lactic acid and pyruvic acid are produced by metabolizing and decomposing carbohydrates through glycolysis (anaerobic metabolism). It is known that lactic acid and pyruvic acid in the intestine are mainly produced by intestinal bacteria (Non-Patent Document 1). Therefore, the mechanism of action of the present composition is considered to be that it acts on intestinal bacteria that produce lactic acid and pyruvic acid, increasing the number of bacteria, or improving the production activity of lactic acid and pyruvic acid, thereby increasing lactic acid and pyruvic acid in the intestine.

Oligosaccharides are sugars in which a relatively small number of monosaccharides, the smallest unit, are linked together, and are also called oligosaccharides. The number of polymerized monosaccharides in an oligosaccharide can be, for example, 2 or more, 3 or more, 30 or less, 25 or less, 20 or less, 15 or less, a dozen or so or 10 or less. In the present invention, oligosaccharides are preferably those which are hardly digested or absorbed in the stomach or small intestine and reach the large intestine as is (so-called resistant oligosaccharides), or those which can be digested and absorbed in the stomach or small intestine but have an undigested portion that reaches the large intestine (oligosaccharides intermediate between resistant and easy digestibility). Specific examples of oligosaccharides include trehalose, lactose, 1-kestose, lactulose, lactosucrose, maltosyltrehalose, isomaltooligosaccharides, galactooligosaccharides, fructooligosaccharides, gentiooligosaccharides, nigerooligosaccharides, and xylooligosaccharides.

1-kestose is a trisaccharide oligosaccharide consisting of one glucose molecule and two fructose molecules. 1-kestose can be produced by performing an enzymatic reaction using sucrose as a substrate with an enzyme as disclosed in JP-A-58-201980. Specifically, β-fructofuranosidase is first added to a sucrose solution, and the solution is left to stand at 37°C to 50°C for about 20 hours to perform an enzymatic reaction, thereby obtaining a 1-kestose-containing reaction solution. This 1-kestose-containing reaction solution is subjected to a chromatographic separation method as disclosed in JP-A-2000-232878 to separate and purify 1-kestose from other sugars (glucose, fructose, sucrose, and oligosaccharides of 4 or more sugars), thereby obtaining a high-purity 1-kestose solution. Next, this high-purity 1-kestose solution is concentrated and then crystallized using a crystallization method such as that disclosed in Japanese Patent Publication No. 6-70075, thereby obtaining 1-kestose as crystals.

In addition, since 1-kestose is contained in commercially available fructooligosaccharides, it may be used as is, or 1-kestose may be separated and purified from fructooligosaccharides by the above-mentioned method. That is, as the 1-kestose of the present invention, a 1-kestose-containing mixture such as an oligosaccharide containing 1-kestose may be used. When a 1-kestose-containing mixture is used, the purity of 1-kestose is preferably 70% or more, and more preferably 75% or more, 80% or more, 85% or more, or 90% or more can be mentioned as examples.

In the present invention, the "purity" of a particular oligosaccharide such as 1-kestose refers to the mass percentage of the oligosaccharide when the total mass of the sugar is taken as 100.

Lactulose is a disaccharide in which galactose is β-linked to the hydroxyl group at the 4-position of fructose (4-O-β-D-Galactopyranosyl-β-D-fructofuranose), and is also called lactulose. Lactulose can generally be produced industrially by an isomerization method using lactose as the starting material, and a commercially available product produced in this manner can be used in the present invention. It is also contained in small amounts in milk obtained by sterilizing raw milk. The lactulose of the present invention may be a lactulose-containing mixture such as an oligosaccharide containing lactulose. In such a mixture, the purity of lactulose is preferably 70% or more, and more preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more can be given as examples.

Lactosucrose is a trisaccharide consisting of one molecule each of galactose, glucose, and fructose bound together, and is also called galactosylsucrose (4G-β-D-galactosylsucrose) or lactosylsucrose. Lactosucrose can be enzymatically synthesized from lactose and sucrose by the action of levansucrase, or it can be industrially produced by the action of β-fructofuranosidase on the same raw materials through a sugar transfer reaction. In the present invention, a commercially available product produced in this manner can be used. Commercially available products may contain monosaccharides, disaccharides, other trisaccharides (1-kestose), and tetrasaccharides that are generated by enzyme reactions or side reactions, and such lactosucrose-containing mixtures may be used in the present invention. In such mixtures, the purity of lactosucrose can be, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, and the like.

Isomaltooligosaccharides are oligosaccharides formed by the polymerization of glucose molecules and have α-1,6 bonds. They are widely contained in traditional fermented foods in nature, although in small amounts, and are industrially produced by microbial enzyme reactions using starch as a raw material. In the present invention, commercially available products produced in this manner can be used. Commercially available products may be mixtures of isomaltooligosaccharides and other sugars (glucose, maltose, etc.). They may also be mixtures of isomaltooligosaccharides with various degrees of polymerization (isomaltose, isomaltotriose, panose, isomaltotetraose, etc.). In the present invention, such isomaltooligosaccharide-containing mixtures may be used. In such mixtures, the purity of isomaltooligosaccharides may be, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, etc.

Galactooligosaccharides are oligosaccharides formed by binding one or more galactoses to the non-reducing terminal galactose of lactose. They are naturally contained in breast milk and cow's milk, and industrially produced by treating lactose with β-galactosidase derived from microorganisms. In the present invention, commercially available products produced in this manner can be used. Commercially available products may be mixtures of isomers of disaccharides to hexasaccharides or octasaccharides in which multiple galactoses are extended at the glucose terminal. They may also be mixtures of galactooligosaccharides and other sugars (galactose, glucose, lactose, etc.). In the present invention, such galactooligosaccharide-containing mixtures may be used. In such mixtures, the purity of the galactooligosaccharides may be, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, etc.

Proteins are substances formed by linear polymerization of amino acids (usually about 100 or more). The protein, which is the active ingredient of the present composition, is a protein that can be ingested by human or animal living bodies. In other words, any size, type, amino acid sequence, amino acid composition, and organism of origin may be used as long as it is edible. Proteins may also be artificially synthesized or derived from organisms such as animals and plants. For example, edible proteins are generally derived from animals and plants, and their types, amino acid sequences, and amino acid compositions vary widely. However, in the present invention, as shown in the examples described below, it has been found that the same effect of increasing bifidobacteria and lactic acid and pyruvic acid can be obtained even if various different proteins such as milk-derived casein, milk-derived whey protein, and soybean-derived protein are used.

Examples of proteins that have traditionally been considered edible include albumin, collagen, gluten, collagen, gelatin, mucin, casein, and whey protein. In terms of the organism from which they are derived, examples include vegetable proteins such as those derived from soybeans, wheat, and beans, and animal proteins such as those derived from eggs, milk, animal muscles, animal skin, and animal tendons.

In addition, in the present invention, food and drink that contains a relatively large amount of protein may be used as the protein.

The active ingredient is used by being ingested by humans or animals. That is, the present invention also provides a method for growing bifidobacteria and a method for increasing lactic acid and/or pyruvic acid in the intestine, which includes a step of ingesting oligosaccharides and proteins to humans or animals.

The method of having a human or animal ingest the active ingredient may be any administration method that allows the active ingredient to reach the intestinal lumen. Specific examples include an embodiment in which a solid, semi-solid, or liquid active ingredient is orally ingested, or an embodiment in which the active ingredient is added to an enteral nutritional agent and administered by enteral nutrition via a tube inserted into the digestive tract, such as the stomach or small intestine.

Oligosaccharides and protein are used in a manner that allows them to be ingested simultaneously, but "simultaneously" does not mean "at the same time" in the strict sense of the word, and there may be a certain interval between the ingestion of the two. When oligosaccharides and protein are ingested separately, it is preferable to ingest both within an interval of about 24 hours, although this depends on the rate at which the subject digests, absorbs, or metabolizes food and drink.

The composition may take the form of a medicine, food additive, feed additive, health food such as supplements, functional food, food for specified health uses, nutritional supplements, special purpose foods (food for artificially fed infants or for sick people, etc.), infant food, confectionery, beverages, processed foods, and other ordinary food and drink forms, as well as feed. When used in the form of a medicine, food additive, feed additive, or supplement, the dosage form may be, for example, a solid or liquid dosage form such as powder, tablet, sugar-coated agent, capsule, granule, dry syrup, liquid, syrup, drop, or drink. When used in the form of a food or drink, the active ingredient can be added during the normal manufacturing process.

In the present invention, the intake amount (dosage) of oligosaccharides can be, for example, 0.0216 g/kg body weight or more per day. Regarding this intake amount, it has been reported that 1-kestose does not cause the proliferation of intestinal bifidobacteria (Watanabe A, et al. Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats. Foods. 2019;9(1):4.), but according to the present invention (co-administration of oligosaccharides and protein), it has a significantly high bifidobacteria proliferation effect, so that intake amount is considered to be effective. That is, the intake amount (dosage amount) of oligosaccharides per day can be exemplified as 0.02 g/kg body weight or more, 0.03 g/kg body weight or more, 0.04 g/kg body weight or more, 0.05 g/kg body weight or more, 0.06 g/kg body weight or more, 0.07 g/kg body weight or more, 0.08 g/kg body weight or more, 0.09 g/kg body weight or more, 0.1 g/kg body weight or more, 0.11 g/kg body weight or more, 0.12 g/kg body weight or more, 0.13 g/kg body weight or more, 0.14 g/kg body weight or more, 0.15 g/kg body weight or more, 0.16 g/kg body weight or more, 0.17 g/kg body weight or more, 0.18 g/kg body weight or more, 0.19 g/kg body weight or more, or 0.2 g/kg body weight or more. The intake amount of the active ingredient is not limited to once a day, and may be divided and taken several times.

On the other hand, the protein intake (dosage) is, for example, 0.1 g/kg body weight or more, 0.2 g/kg body weight or more, 0.3 g/kg body weight or more, 0.4 g/kg body weight or more, 0.5 g/kg body weight or more, 0.6 g/kg body weight or more, 0.7 g/kg body weight or more, 0.8 g/kg body weight or more, 0.9 g/kg body weight or more, 1.0 g/kg body weight or more, 1.2 g/kg body weight or more, 1.3 g/kg body weight or more, 1.4 g/kg body weight or more, 1.5 g/kg body weight or more, 1.6 g/kg body weight or more, 1.7 g/kg body weight or more, 1.8 g/kg body weight or more, 1.9 g/kg body weight or more, body weight or more, 2.0 g/kg body weight or more, 2.1 g/kg body weight or more, 2.2 g/kg body weight or more, 2.3 g/kg body weight or more, 2.4 g/kg body weight or more, 2.5 g/kg body weight or more, 2.6 g/kg body weight or more, 2.7 g/kg body weight or more, 2.8 g/kg body weight or more, 2.9 g/kg body weight or more, 3.0 g/kg body weight or more, 3.1 g/kg body weight or more, 3.2 g/kg body weight or more, 3.3 g/kg body weight or more, 3.4 g/kg body weight or more, 3.5 g/kg body weight or more, 3.6 g/kg body weight or more, 3.7 g/kg body weight or more and 3.8 g/kg body weight or more. The amount of intake of the active ingredient is not limited to once a day, and may be taken in multiple divided doses.

In the present invention, "growing intestinal bacteria" means increasing the number of the bacteria in the intestinal tract.

For example, since the number of bifidobacteria in the intestine is thought to correlate with the number of the bacteria in feces or cecal contents (hereinafter referred to as "feces, etc."), it is possible to confirm whether the number of the bacteria in the intestine has increased by measuring the number of the bacteria in the feces, etc. Specifically, for example, feces, etc. before and after ingestion of the composition, or feces, etc. from an individual who has ingested the composition and feces, etc. from an individual who has not ingested the composition are used as samples, and the 16S rRNA gene (16S rDNA) copy number is measured by performing a real-time PCR method using primers specific to bifidobacteria. Since the 16S rDNA copy number and the number of bifidobacteria are correlated, this copy number can be used as an indicator of the number of bacteria. Therefore, if the 16S rDNA copy number of bifidobacteria is measured and the copy number in the sample after ingestion is greater than that before ingestion, or if the copy number in the sample from an individual who has taken the composition is greater than that of an individual who has not taken the composition, it can be determined that the number of bifidobacteria has increased (promoted proliferation) by the composition.

Primers specific to the 16S rDNA of bifidobacteria can be designed based on known base sequences. For example, they can be designed based on the partial sequence (SEQ ID NO: 1) of the 16S rDNA of the genus Bifidobacterium shown in the Examples below.

As shown in the examples described later, feces before and after ingestion of the active ingredient, or feces from individuals who have ingested the active ingredient and feces from individuals who have not, are used as samples, and a comprehensive analysis of bacterial 16S rDNA in the samples is performed using a next-generation sequencer to calculate the abundance ratio of sequence data with high homology to the partial sequence (SEQ ID NO: 1) of 16S rDNA of bifidobacteria. It has been reported that the homology of 16S rRNA gene sequences in bifidobacteria (Bifidobacteriaceae) is 90% or more (Watanabe, Koichi, Current Status and Trends of Taxonomy of Bifidobacteria, Journal of Intestinal Microbiology, 2016, Vol. 30, pp. 129-139). It can be said that the abundance ratio is correlated with the 16S rDNA copy number of bifidobacteria, i.e., the number of bifidobacteria. Therefore, if the abundance ratio of the sequence data in the sample after ingestion is greater than that before ingestion, or if the abundance ratio of the sequence data in the sample from an individual who has ingested the product is greater than that of an individual who has not ingested the product, it can be determined that the number of bifidobacteria has increased according to the present invention.

The amount of lactic acid and pyruvic acid in the intestine is thought to be correlated with the amount of lactic acid and pyruvic acid in feces or cecal contents (hereinafter referred to as "feces, etc."), so by measuring the amount of lactic acid and pyruvic acid in feces, etc., it is possible to confirm whether these organic acids have increased in the intestine. Specifically, for example, feces, etc. before and after ingestion of an active ingredient, or feces, etc. from an individual who has ingested the active ingredient and feces, etc. from an individual who has not ingested the active ingredient are used as samples to measure the content of these organic acids using a gas chromatograph mass spectrometer. As a result, if the amount of lactic acid and pyruvic acid in the sample after ingestion is greater than before ingestion, or if the amount of lactic acid and pyruvic acid in the sample from an individual who has ingested the active ingredient is greater than that of an individual who has not ingested the active ingredient, it can be determined that lactic acid and pyruvic acid in the intestine has increased according to the present invention.

The present invention will be described below based on each example. The technical scope of the present invention is not limited to the characteristics shown in these examples.

<Evaluation Sample>
(1) Oligosaccharides The following five types of oligosaccharides were used:
[1-1] 1-kestose As 1-kestose, "a composition containing 1-kestose with a purity of 98% by mass or more (Butsusan Food Science Co., Ltd.)" (powder) was used.

[1-2] Lactulose Lactulose was used as "Milk oligosaccharide lactulose" (containing 97% or more lactulose, powder product) (Nichiga).

[1-3] Lactosucrose Lactosucrose used was "LS-55P" (containing 55.0 to 60.0% lactosucrose, powder product) (Ensuiko Sugar Refining Co., Ltd.).

[1-4] Isomalto-oligosaccharides Isomalto-oligosaccharides were used as "Isomalto 900P" (powdered product containing 85% or more isomalto-oligosaccharides) (Showa Sangyo). The sugar composition of this product is 6% monosaccharide (glucose), 38% disaccharide (isomaltose, etc.), 29% trisaccharide (isomaltotriose, panose), and 27% tetrasaccharide (isomaltotetraose, etc.) (Kimura Kazumasa et al., Milk Science Vol. 67, No. 2, 2018, pp. 88-101).

[1-5] Galactooligosaccharides Galactooligosaccharides were used as "Oligomate 55NP" (powdered product containing 55% or more galactooligosaccharides in the solid content) (Yakult Pharmaceutical Co., Ltd.). The sugar composition of this product is 31% monosaccharides (galactose 9%, glucose 22%), 22% disaccharides (lactose and others), 33% trisaccharides (4'-galactosyllactose), and 8% galactooligosaccharides of tetrasaccharides or more (Kimura Kazumasa et al., Milk Science Vol.67, No. 2, 2018, pp.88-101).

(2) Protein The following three types of protein were used:
[2-1] Casein Product name: SureProtein (registered trademark) LACTIC CASEIN 720 (nzmp)
Product Specifications: Acid casein made from skim milk. Appearance: Cream color, granular (particle size 30 mesh). Density: 0.67 g/mL. Nutritional Information (per 100 g product) Energy (kJ): 1520. Protein (N x 6.38) (g): 86.9. Moisture (g): 11.5. Fat (g): 1.2. Carbohydrate (g): 0.1. Ash (g): 1.8.
[2-2] Whey protein Product name: SureStart (registered trademark) WHEY PROTEIN ISOLATE 895 (nzmp)
Product specifications: Protein (N x 6.38) per dry weight (% m/m): 95.0 or more. Ash content (% m/m): 4.0 or less. Moisture (% m/m): 5.5 or less. Lipid (% m/m): 1.0 or less. Lactose (% m/m): 1.0 or less. pH (5%, 20°C): 6.5 or higher.
[2-3] Soy protein Product name: SUPRO (registered trademark) 661 Isolated Soy Protein (Solae)
Product Specifications: Nutritional Information (per 100g product): Calories: 381kcal. Protein (N x 6.25) (g): 87.5. Moisture (g): 4.3. Ash (g): 4.4. Fat (g): 3.1. Carbohydrates (g): 1g or less.

The amino acid compositions of casein and soy protein are shown in Table 1.

Example 1 1-Combined Use of Kestose and Casein (1) Rat Rearing 28 male Sprague-Dawley (SD) rats (Japan SLC) were divided into 4 groups of 7 rats each and reared for 2 weeks with free access to the feed and water shown in Table 2. Rearing conditions were a temperature of 23±1°C, with a light period of 12 hours (8:00-20:00) and a dark period of 12 hours (20:00-8:00).

The composition of the feed is shown below.
Composition of standard feed (unit: mass %): Corn starch 39.7486, milk casein 20, pregelatinized corn starch 13.2, granulated sugar 10, refined soybean oil 7, cellulose powder 5, mineral mix 3.5, vitamin mix 1, L-cystine 0.3, choline bitartrate 0.25, tert-butylhydroquinone 0.0014.
<Composition of high protein feed> A feed in which casein has been added to standard feed so that the final protein content is 50% (w/w).

In other words, the "control group" was a group that did not ingest 1-kestose and had a normal protein intake, the "kestose group" was a group that ingested 1-kestose and had a normal protein intake, the "protein group" was a group that did not ingest 1-kestose and had a high protein intake, and the "kestose 2% + protein group" was a group that ingested 1-kestose and had a high protein intake. Note that because rats drink more water when fed a high-protein diet, the "kestose 2% + protein group" had a greater intake of 1-kestose than the kestose group.

During the rearing period, the intake of food and the intake of water were recorded every day, and the intake of protein and 1-kestose (the actual intake of each component) was calculated based on the records. It has also been reported that the intake of a drug in rats can be converted to the intake in an adult human by the following formula 1 (paragraph [0065] of JP 2014-526521 A, Shannon Reagan-Shaw et al., The FASEB Journal, Vol. 22, March 2007, pp. 659-661). Therefore, the daily intake of 1-kestose and protein in rats was converted to that in an adult human by the formula 1. The calculation results are shown on the right side of Table 1.
Formula 1: Daily intake of 1-kestose or protein in an adult human (g/kg body weight) = Daily intake of 1-kestose or protein in a rat (g/kg body weight) x 6/37

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 1 (1) were dissected to remove the cecum, and the cecal contents were collected and frozen. Total DNA was extracted from the cecal contents according to the method described in Shunsuke Takahashi et al., PLosONE, Vol. 9, No. 8, e105592, August 2014: Reference 1. Specifically, 100 mg of the cecal contents melted on ice was suspended in an aqueous solution containing 4 M guanidine thiocyanate, 100 mM Tris-HCl (pH 9.0), and 40 mM EDTA, and the suspension was obtained by pulverizing with zirconia beads using FastPrep FP100A (MP Biomedicals). DNA was extracted from the suspension using Magtration System 12GC (Precision System Science) and GC series MagDEA DNA 200 (Precision System Science). The DNA concentration was measured using a spectrophotometer ND-1000 (NanDrop Technologies) and adjusted to 10 ng/μL, which was used as the total DNA of the cecal contents.

A comprehensive analysis of the types and abundance ratios of bacteria contained in the cecal contents was performed according to the method described in Reference 1. That is, first, the total DNA of the cecal contents was used as a template, and a polymerase chain reaction (PCR) was performed using the universal primers of SEQ ID NOs: 2 and 3 below by the Dual-index method (Hisada Takayoshi et al., Arch Microbiol, Vol. 197, No. 7, pp. 919-34, June 2015) to amplify the V3-V4 region of the bacterial 16S rDNA.
Forward primer (341f): CCTACGGGAGGCAGCAG (SEQ ID NO: 2)
Reverse primer (R806): GGACTACHVGGGTWTCTAAT (SEQ ID NO: 3)

Then, the base sequence of the PCR amplified product was deciphered by the paired-end method using a next-generation sequencer MiSeq (Illumina). The obtained base sequence data was excluded if it was of low quality or derived from a chimeric sequence. A database search was performed for the determined base sequence, and a taxonomic group detected with an identity of 97% or more was identified as a single bacterial species (genus). One of the sequences (partial sequence of 16S rDNA) identified as Bifidobacteria is shown in SEQ ID NO:1.

[SEQ ID NO: 1]
TGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGCTTCGGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTC CATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGAGCGAACAGGAT

The occupancy rate was calculated as a percentage of the number of reads of each bacterial species (genus, family) in the total number of reads. Quality checks were performed according to the methods described in Reference 1, chimeric sequence checks in Robert C. Edgar et al., BIOINFORMATICS, Vol. 27, No. 16, pp. 2194-2200, June 2011, and database searches in Hisada Takayoshi et al., Arch Microbiol, Vol. 197, No. 7, pp. 919-34, June 2015. The occupancy rates of Bifidobacteriaceae and Bifidobacterium in this comprehensive analysis (median values in each group (N = 7)) are shown in Figures 1 and 2, respectively.

As shown in Figure 1, the occupancy rate of bifidobacteria was 0.1% in the control group and 0.3% in the kestose group, which was slightly higher than the control group. In the protein group, it was 0.0%, which was lower than the control group. On the other hand, the occupancy rate in the kestose 2% + protein group was 31.2%, which was significantly higher than the control group. That is, the occupancy rate of bifidobacteria was significantly higher in rats that simultaneously ingested 1-kestose and a feed containing a high proportion of casein (high-casein feed). Furthermore, the degree of increase was far greater than the combined effect of increasing bifidobacteria when 1-kestose and high-casein feed were ingested alone. Furthermore, as shown in Figure 2, the occupancy rate values of the Bifidobacterium genus were all the same as the occupancy values of bifidobacteria in each group. These results demonstrate that simultaneous intake of 1-kestose and protein can effectively increase the number of bifidobacteria in the intestines.

(3) Quantitative Determination of Lactic Acid and Pyruvic Acid The rats in each group in Example 1(1) were dissected to remove the cecum, and the concentrations of lactic acid and pyruvic acid in the cecal contents (Absorbance Unit; au) were measured using a gas chromatograph mass spectrometer (GC-MS/MS). The average of the measured values was then calculated for each group, and the relative values were calculated when the average value of the control group was set to 1, and were plotted in a bar graph. The results are shown in FIG. 3.

As shown in Figure 3, the lactate concentration was lower in the kestose and protein groups than in the control group, whereas the kestose 2% + protein group was significantly higher than the control group. The pyruvate concentration was similar to that of the control group in the kestose group and slightly higher in the protein group than in the control group, whereas the kestose 2% + protein group was significantly higher than the control group. That is, the cecal lactate and pyruvate concentrations were significantly higher in rats that simultaneously ingested 1-kestose and a high-casein diet. Furthermore, the degree of increase was far greater than the combined increase in lactate and pyruvate when 1-kestose and a high-casein diet were each ingested alone. These results demonstrate that the amount of lactate and pyruvate in the intestine can be significantly increased by simultaneously ingesting 1-kestose and protein.

Example 2 Combined Use of 1-kestose and Whey Protein (1) Rat Rearing Rats were reared according to the method described in Example 1 (1). However, in addition to the "kestose 2% + protein group," a "kestose 1% + protein group" was also set up as a group that ingested 1-kestose and had a large protein intake. In addition, whey protein was blended in place of casein in the high protein feed. The calculated results of the intake of feed and water, as well as 1-kestose and protein, given to each group are shown in Table 3.

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 2(1) were dissected to remove the cecum, and meta-16S analysis was performed by the method described in Example 1(2) to determine the occupancy rate of bifidobacteria and Bifidobacterium (median value in each group (N=7)). The results are shown in Figures 4 and 5.

As shown in FIG. 4, the occupancy rate of bifidobacteria was 0.1% in the control group, 6.5% in the kestose group, and 0.4% in the protein group, both of which were slightly higher than the control group. In contrast, the occupancy rates of the 1% kestose + protein group and the 2% kestose + protein group were 23.2% and 46.2%, respectively, which were significantly higher than the control group. That is, the occupancy rate of bifidobacteria was significantly higher in rats that simultaneously ingested 1-kestose and a feed containing a high proportion of whey protein (high-whey feed). Furthermore, the degree of increase was far greater than the sum of the bifidobacteria-increasing effects when 1-kestose and high-whey feed were ingested alone. Furthermore, as shown in FIG. 5, the occupancy rate values of the genus Bifidobacterium were all the same as the occupancy values of bifidobacteria in each group. These results demonstrate that simultaneous intake of 1-kestose and protein significantly increases the number of bifidobacteria in the intestine, regardless of the type of protein.

(3) Quantitative Determination of Lactic Acid and Pyruvate The rats in each group in Example 2(1) were dissected to remove the cecum, and the concentrations (au) of lactic acid and pyruvic acid in the cecal contents were measured using GC-MS/MS. The average of the measured values was then calculated for each group, and the relative values were calculated when the average value of the control group was set to 1, and were plotted in a bar graph. The results are shown in FIG.

As shown in Figure 6, the lactate concentration was lower in the kestose and protein groups than in the control group, whereas it was significantly higher in the 1% kestose + protein and 2% kestose + protein groups than in the control group. The pyruvate concentration was also equal to or lower in the kestose and protein groups than in the control group, whereas it was significantly higher in the 1% kestose + protein and 2% kestose + protein groups than in the control group. In other words, the cecal lactate and pyruvate concentrations were significantly higher in rats that simultaneously ingested 1-kestose and a high-whey diet. These results demonstrate that the amount of lactate and pyruvate in the intestine can be significantly increased by simultaneously ingesting 1-kestose and protein, regardless of the type of protein.

Example 3 1-Combination Use of Kestose and Soy Protein (1) Rat Rearing Rats were reared according to the method described in Example 1 (1), except that soy protein was added to the high protein feed instead of casein.

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 3(1) were dissected to remove the cecum, and meta-16S analysis was performed by the method described in Example 1(2) to determine the occupancy rate of bifidobacteria and Bifidobacterium (median value in each group (N=7)). The results are shown in Figures 7 and 8.

As shown in Figure 7, the bifidobacteria occupancy rate was 0.1% in the control group, 6.5% in the kestose group, and 0.4% in the protein group, both of which were slightly higher than the control group. In contrast, the rate in the 2% kestose + protein group was 46.4%, significantly higher than the control group. In other words, the bifidobacteria occupancy rate was significantly higher in rats that simultaneously ingested 1-kestose and a feed containing a high proportion of soy protein (high soy protein feed). Furthermore, the degree of increase was far greater than the combined effect of increasing bifidobacteria when 1-kestose and high soy protein feed were ingested alone.

As shown in Figure 8, the occupancy rate of the genus Bifidobacterium also showed a similar trend, being 0.1% in the control group, 0.2% in the kestose group, and 0% in the protein group, while it was 12.3% in the 2% kestose + protein group, and was significantly higher only in the 2% kestose + protein group. In other words, the occupancy rate of the genus Bifidobacterium was significantly higher in rats that simultaneously ingested 1-kestose and a diet high in soy protein.

These results demonstrate that simultaneous intake of 1-kestose and protein, regardless of the type of protein, can significantly increase the number of bifidobacteria in the intestine.

Example 4: Combined Use of Other Oligosaccharides and Casein (1) Rat Breeding Rats were bred according to the method described in Example 1 (1), except that lactulose, lactosucrose, isomaltooligosaccharide, or galactooligosaccharide was used instead of 1-kestose as the oligosaccharide.

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 4(1) were dissected to remove the cecum, and meta-16S analysis was performed by the method described in Example 1(2) to determine the occupancy rate of bifidobacteria and Bifidobacterium (median value in each group (N=7)).

As a result, the occupancy rate of bifidobacteria in the various oligosaccharides and protein groups was slightly higher than that of the control group. In contrast, the occupancy rate of the 2% various oligosaccharides + protein group was significantly higher than that of the control group. That is, the occupancy rate of bifidobacteria was significantly higher in rats that simultaneously ingested various oligosaccharides and a casein-rich feed. The degree of increase was far greater than the sum of the bifidobacteria-increasing effects when the various oligosaccharides and the casein-rich feed were ingested alone. The occupancy rate of the genus Bifidobacterium was also similar to the occupancy rate of bifidobacteria in each group. These results revealed that the number of bifidobacteria in the intestine can be significantly increased by simultaneously ingesting various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein.

(3) Quantitative determination of lactic acid and pyruvic acid The rats in each group in Example 4(1) were dissected to remove the cecum, and the concentrations (au) of lactic acid and pyruvic acid in the cecal contents were measured using GC-MS/MS. The average of the measured values was then calculated for each group, and the relative values were calculated when the average value of the control group was set to 1, and the results were plotted in a bar graph.

As a result, the lactate and pyruvic acid concentrations in the various oligosaccharides and protein groups were equal to or lower than those in the control group, whereas the 2% oligosaccharides + protein group was significantly higher than the control group. In other words, the cecal lactate and pyruvic acid concentrations were significantly higher in rats that had simultaneously ingested various oligosaccharides and a high casein content diet. These results demonstrated that the amount of lactate and pyruvic acid in the intestine can be significantly increased by simultaneously ingesting various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein.

Example 5: Combined Use of Other Oligosaccharides and Whey Protein (1) Raising Rats Rats were bred according to the method described in Example 1 (1). However, the oligosaccharide used was lactulose, lactosucrose, isomaltooligosaccharide or galactooligosaccharide instead of 1-kestose. In addition, whey protein was added instead of casein to the high protein feed.

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 5(1) were dissected to remove the cecum, and meta-16S analysis was performed by the method described in Example 1(2) to determine the occupancy rate of bifidobacteria and Bifidobacterium (median value in each group (N=7)).

As a result, the occupancy rate of bifidobacteria in the various oligosaccharides group and the protein group was slightly higher than that of the control group. In contrast, the occupancy rate of the 2% various oligosaccharides + protein group was significantly higher than that of the control group. That is, the occupancy rate of bifidobacteria was significantly higher in rats that simultaneously ingested various oligosaccharides and whey-rich feed. The degree of increase was far greater than the sum of the bifidobacteria-increasing effects when various oligosaccharides and whey-rich feed were ingested alone. The occupancy rate of the genus Bifidobacterium was also similar to the occupancy rate of bifidobacteria in each group. These results revealed that the simultaneous intake of various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein can significantly increase the number of bifidobacteria in the intestine, regardless of the type of protein.

(3) Quantitative Determination of Lactic Acid and Pyruvate The rats in each group in Example 5(1) were dissected to remove the cecum, and the concentrations (au) of lactic acid and pyruvic acid in the cecal contents were measured using GC-MS/MS. The average of the measured values was then calculated for each group, and the relative values were calculated when the average value of the control group was set to 1, and were plotted in a bar graph.

As a result, the lactate and pyruvic acid concentrations in the various oligosaccharides and protein groups were equal to or lower than those in the control group, whereas the 2% oligosaccharides + protein group was significantly higher than the control group. In other words, the cecal lactate and pyruvic acid concentrations were significantly higher in rats that simultaneously ingested various oligosaccharides and a high whey content diet. These results demonstrated that the amount of lactate and pyruvic acid in the intestine can be significantly increased by simultaneously ingesting various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein, regardless of the type of protein.

Example 6: Combination of other oligosaccharides with soybean protein (1) Rat breeding Rats were bred according to the method described in Example 1 (1). However, the oligosaccharide used was lactulose, lactosucrose, isomaltooligosaccharide, or galactooligosaccharide instead of 1-kestose. Also, soybean protein was added to the high protein feed instead of casein.

(2) Confirmation of the number of bifidobacteria by meta-16S analysis The rats in each group in this Example 6(1) were dissected to remove the cecum, and meta-16S analysis was performed by the method described in Example 1(2) to determine the occupancy rate of bifidobacteria and Bifidobacterium (median value in each group (N=7)).

As a result, the occupancy rate of bifidobacteria in the various oligosaccharides group and the protein group was slightly higher than that of the control group. In contrast, the occupancy rate of the 2% various oligosaccharides + protein group was significantly higher than that of the control group. That is, the occupancy rate of bifidobacteria was significantly higher in rats that simultaneously ingested various oligosaccharides and whey-rich feed. The degree of increase was far greater than the sum of the bifidobacteria-increasing effects when various oligosaccharides and soy protein-rich feed were ingested alone. The occupancy rate of the genus Bifidobacterium was also similar to the occupancy rate of bifidobacteria in each group. These results revealed that the simultaneous intake of various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein can significantly increase the number of bifidobacteria in the intestine, regardless of the type of protein.

(3) Quantitative Determination of Lactic Acid and Pyruvate The rats in each group in Example 6(1) were dissected to remove the cecum, and the concentrations (au) of lactic acid and pyruvic acid in the cecal contents were measured using GC-MS/MS. The average of the measured values was then calculated for each group, and the relative values were calculated when the average value of the control group was set to 1, and the results were plotted in a bar graph.

As a result, the lactate and pyruvic acid concentrations in the various oligosaccharides and protein groups were equal to or lower than those in the control group, whereas the 2% oligosaccharides + protein group was significantly higher than the control group. In other words, the cecal lactate and pyruvic acid concentrations were significantly higher in rats that had simultaneously ingested various oligosaccharides and a high soy protein content diet. These results demonstrated that the amount of lactate and pyruvic acid in the intestine can be significantly increased by simultaneously ingesting various oligosaccharides (lactulose, lactosucrose, isomaltooligosaccharides, or galactooligosaccharides) and protein, regardless of the type of protein.

Claims (8)

A composition for the growth of bifidobacteria, containing oligosaccharides and proteins as active ingredients. A composition for increasing lactic acid and/or pyruvic acid in the intestines, containing oligosaccharides and proteins as active ingredients. The composition according to claim 1 or 2, wherein the oligosaccharide is one or more oligosaccharides selected from the group consisting of 1-kestose, lactulose, lactosucrose, isomaltooligosaccharides, and galactooligosaccharides. The composition according to claim 1 or 2, wherein the oligosaccharide is 1-kestose. A method for growing bifidobacteria (excluding medical procedures) that includes a step of administering oligosaccharides and proteins to humans or animals. A method (excluding medical procedures) for increasing lactate and/or pyruvate in the intestine, comprising the step of administering oligosaccharides and proteins to a human or animal. The method according to claim 5 or 6, wherein the oligosaccharide is one or more oligosaccharides selected from the group consisting of 1-kestose, lactulose, lactosucrose, isomaltooligosaccharides and galactooligosaccharides. The method according to claim 5 or claim 6, wherein the oligosaccharide is 1-kestose.