JP2022048629A - Composition for improving antibiotic-induced intestinal dysbiosis - Google Patents

Abstract

To provide a composition for improving antibiotic-induced intestinal dysbiosis.SOLUTION: A composition for improving antibiotic-induced intestinal dysbiosis contains barley with reduced expression levels and/or activity of starch synthase II.SELECTED DRAWING: Figure 1

Description

The present invention relates to a composition for improving antibiotic-induced intestinal disbiosis, as well as foods and drinks and pharmaceuticals containing the composition.

Enterobacteriaceae in the intestines of organisms such as mammals such as humans, monkeys, mice, rats, dogs, cats, pigs and rabbits, and insects such as flies, mosquitoes, abs, bees, ants, cockroaches and butterflies. Many bacteria called are inhabited. Gut microbiota is generally classified into good bacteria, bad bacteria, and opportunistic bacteria, and the presence of these bacteria in a well-balanced manner at a fixed ratio forms and maintains the intestinal flora, that is, the intestinal flora. There is.

Research on the intestinal flora is being actively conducted, and the disorder of the intestinal flora is generally called intestinal disbiosis, and it may be closely related to the disease and health of animals such as humans. It is being revealed. In recent years, it has become a problem that the use of antibiotics (administration, administration, etc.) causes this intestinal disbiosis. Specifically, for example, the use of antibiotics kills not only pathogens but also other beneficial gut bacteria, resulting in reduced diversity of gut bacteria that form the gut flora. In addition, the production of short-chain fatty acids, which is produced by the intestinal bacteria and is involved in the suppression of inflammation and the suppression of the rise in blood glucose, is caused.

Previous attempts to improve intestinal disbiosis include, for example, a composition containing eggshell membrane-containing powder as an active ingredient (Patent Document 1), a certain amount of β-glucan, resistant starch, and the like. Examples thereof include a composition containing fructan (Patent Document 2).

Japanese Unexamined Patent Publication No. 2018-052854 Japanese Unexamined Patent Publication No. 2018-05526

However, in the above-mentioned Patent Documents 1 and 2, a colitis model mouse and a healthy human are used as specimens, respectively. Therefore, in the above-mentioned Patent Documents 1 and 2, no study has been made on the improvement of intestinal disbiosis in the sample after the use of the antibiotic.

An object of the present invention is to provide a composition for improving antibiotic-induced intestinal disbiosis.

[1]
A composition for improving antibiotic-induced intestinal disbiosis, which comprises barley in which the expression level and / or activity of starch synthase II is suppressed.
[2]
Antibiotics-induced, containing barley containing 2% to 10% by weight of resistant starch, 5% to 15% by weight of fructan and 2% to 15% by weight of β-glucan per crushed barley husk. A composition for improving sex intestinal disbiosis.
[3]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is to increase and / or decrease the number of intestinal bacteria.
[4]
Improving intestinal disbiosis is to increase the number of gut bacteria belonging to one or more families and / or genera selected from the genus Lachnospiraceae, Ruminococcaceae and Bacteroides. [1] Or the composition according to [2].
[5]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is to reduce the number of intestinal bacteria belonging to the family Enterobacter.
[6]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is to increase the diversity of intestinal bacteria.
[7]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is to increase the alpha diversity of intestinal bacteria.
[8]
An increase in intestinal dysbiosis indicates an increase in the alpha diversity of one or more gut microbiota selected from the group consisting of Observed species, Phylogenetic Diversity color tree, Shannon diversity index and Chao1 index. The composition according to [1] or [2].
[9]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis promotes the production of short-chain fatty acids in the intestine.
[10]
The improvement of intestinal disbiosis is to promote the production of one or more intestinal short chain fatty acids selected from the group consisting of acetic acid, propionic acid and butyric acid, according to [1] or [2]. Composition.
[11]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is to lower the pH in the intestine.
[12]
The composition according to [1] or [2], wherein the improvement of intestinal disbiosis is performed within 14 days from the day when the composition is provided.
[13]
A food or drink containing the composition according to any one of [1] to [12].
[14]
A pharmaceutical product containing the composition according to any one of [1] to [12].

According to the present invention, antibiotic-induced intestinal disbiosis can be improved.

It is a figure which shows the measurement result of the DNA amount in feces obtained by Example 1. FIG. It is a figure which shows the analysis result of the diversity of the intestinal flora obtained by Example 2. FIG. It is a figure which shows the analysis result of the abundance ratio of the intestinal bacterium obtained by Example 3. FIG. It is a figure which shows the measurement result of the short chain fatty acid in the intestine obtained by Example 4. FIG. It is a figure which shows the measurement result of the pH in the intestine obtained by Example 5.

<Composition for ameliorating antibiotic-induced intestinal disbiosis of the present invention>
The composition for improving the antibiotic-induced intestinal disbiosis of the present invention (hereinafter, may be abbreviated as "the composition of the present invention") is an expression level of starch synthase II and / or It contains barley with suppressed activity.
Alternatively, the composition of the present invention contains 2% by mass to 10% by mass of resistant starch, 5% by mass to 15% by mass of fructan, and 2% by mass of β-glucan per crushed barley shell grain. It contains barley containing up to 15% by mass.
In still another form, the composition of the present invention is barley in which the expression level and / or activity of starch synthase II is suppressed, and resistant starch is added in an amount of 2% by mass or more per crushed barley shell grain. It contains barley containing 10% by mass, 5% by mass to 15% by mass of fructan, and 2% to 15% by mass of β-glucan.
Among the above-mentioned compositions, the composition of the present invention includes (I) barley in which the expression level and / or activity of starch synthase II is suppressed, and (II) resistant starch per crushed barley shell grain. 2-10% by mass, fructan 5-15% by mass and β-glucan 2-15% by mass, and (III) starch synthase II expression level or activity-suppressed barley. It contains 2% by mass to 10% by mass of resistant starch, 5% by mass to 15% by mass of fructan, and 2% by mass to 15% by mass of β-glucan per crushed barley husk grain. Is preferable, and (III) is more preferable.

[Barley according to the present invention]
The barley according to the present invention includes (i) barley in which the expression level and / or activity of starch synthase II is suppressed (hereinafter, may be abbreviated as barley A according to the present invention), and (ii) the barley shell. Barley containing 2% by mass to 10% by mass of resistant starch, 5% by mass to 15% by mass of fructan, and 2% to 15% by mass of β-glucan per crushed grain (hereinafter referred to as barley B according to the present invention). (May be abbreviated) and (iii) Barley in which the expression level and / or activity of starch synthase II is suppressed, and the resistant starch per crushed barley shell grain is 2% by mass to 10% by mass. Barley containing 5% by mass to 15% by mass of fructan and 2% by mass to 15% by mass of β-glucan (hereinafter, may be abbreviated as barley C according to the present invention) is preferable, and (iii) is more preferable.
The above (i), (ii) and (iii) will be described in detail below.

(I) Barley A according to the present invention (barley in which the expression level and / or activity of starch synthase II is suppressed).
The barley A according to the present invention is barley disclosed in Japanese Patent Laid-Open No. 2004-512427, Japanese Patent Application Laid-Open No. 2013-500022, Japanese Patent Application Laid-Open No. 2018-050526 and Japanese Patent Application Laid-Open No. 2020-0282227.

The barley A according to the present invention is, for example, a wild-type starch synthase II gene by introducing a mutation or an exogenous nucleic acid molecule in the endogenous starch synthase II gene that downwardly controls the expression of the starch synthase II gene. A method of introducing a mutation that reduces the expression of the starch synthase II gene in comparison with the wild-type starch synthase II polypeptide, and the expression of a starch synthase II polypeptide having a lower synthetic activity as compared with the wild-type starch synthase II polypeptide in barley. It can be obtained by a method of introducing a mutation or the like.
In addition, Burley Max (registered trademark), which is a breed-improved barley, has an suppressed expression level and / or activity of starch synthase II. Therefore, Barley Max (registered trademark), which is a breed-improved barley, is an example of barley A according to the present invention.

The "starch synthase II" in barley A according to the present invention means the starch synthase II gene and catalyzes the biosynthesis of starch. Specifically, it catalyzes the formation of α-1,4 bonds with glucose residues at the non-reducing ends of amylose and amylopectin, and the sequence information thereof is described in JP-A-2004-512427. , Japanese Patent Publication No. 2013-500022, etc. As isoforms of the starch synthase II gene, starch synthase IIa gene, starch synthase IIb gene, starch synthase IIc gene and the like are known. , Starch synthase IIa gene is preferred.

The "expression level of starch synthase II" in barley A according to the present invention specifically means the amount of mRNA and / or the amount of protein of the starch synthase II gene. Further, the "amount" is represented by any of an absolute value such as volume and mass or a relative value such as a value calculated from an ionic strength, an absorbance, a fluorescence intensity, a turbidity, a peak area value and the like. Is.

The "activity of starch synthase II" in barley A according to the present invention specifically means the enzymatic activity of the starch synthase II gene, and more specifically, the catalytic action of the starch synthase II gene. Means the strength of. The "activity and intensity" are expressed by values (titer, unit, number of units, etc.) usually used in the enzyme activity measurement method used in this field. Further, the "measurement of the enzyme activity of the starch synthase II gene" may be performed by any method usually performed in this field, and specifically, for example, a spectrophotometer, a radioactive isotope, or the like is used. And / or a method of measuring the amount of the substrate and / or the amount of the reaction product. The "measurement of the enzyme activity of the starch synthase II gene" may be measured using a commercially available kit.

"Suppressed" in barley A according to the present invention means that the expression level and / or the enzyme activity value of the starch synthase II gene is unmodified, that is, the starch synthase II gene in wild-type barley shell grains. It means that the expression level and / or the enzyme activity is decreased by at least 40%, preferably 60%, more preferably 75%, further preferably 90%, and particularly preferably 95%.
Here, the above-mentioned "barley shell grain" means a barley grain having a husk after cultivation and harvesting or a state in which the husk after refining is removed. In the present invention, from the viewpoint of processability, it is preferable that the barley grains are in a state where the outer skin after purification is removed. Further, the "barley shell grain" has the same meaning in the barley B according to the present invention described later.

(Ii) Barley B according to the present invention (2% by mass to 10% by mass of resistant starch, 5% by mass to 15% by mass of fructan, and 2% by mass to 15% by mass of β-glucan per crushed barley shell grain). % Including barley)
The barley B according to the present invention can be obtained by breeding methods such as a separate breeding method, a cross breeding method, a radiation breeding method, and a protoplast culture method, which are usually performed in this field.
In addition, Burley Max (registered trademark), which is a variety-improved barley, contains 2% by mass to 10% by mass of resistant starch, 5% by mass to 15% by mass of fructan, and β-glucan per crushed barley shell grain. It contains 2% by mass to 15% by mass. Therefore, Barley Max (registered trademark), which is a breed-improved barley, is an example of barley B according to the present invention.

The "crushed barley shell grain" in the barley B according to the present invention means the above-mentioned solid substance in which the barley shell grain is crushed, and preferably the barley in a state where the hull after milling is removed and the bran is attached. It is whole grain flour made by crushing shell grains. The "crushing" may be performed manually using a mortar, kitchen knife, cutter knife, scissors, etc. However, if a large amount of material is to be processed in a short time, a mill, a hammer type crusher, a mixer, etc. It may be performed using a device such as a blender.

The "resistant starch" in barley B according to the present invention means a fraction of starch contained in grains and the like that is indigestible to digestive enzymes.
Resistant starch is classified into resistant starch 1 (Resistant Starch 1: RS1) to resistant starch (Resistant Starch 4: RS4) according to the difference in the mechanism of digestion resistance. RS1 is one that cannot be physically contacted with digestive enzymes such as starch of beans and unground whole grains. RS2 is a starch such as raw potato, immature banana, high amylose corn and the like. RS3 is aged starch (starch once gelatinized (pregelatinized) recrystallized (β-formulated)). RS4 is modified starch (chemically modified starch such as crosslinked starch). In the present invention, the resistant starch may be any of the above RS1 to RS4, but it can be easily ingested as food and is in the intestine (particularly, the descending colon, the S-shaped (S-shaped) colon and the rectum). RS1 which is easy to reach is preferable, and resistant starch contained in Burley Max (registered trademark), which is a variety-improved barley, is more preferable.
In the barley B according to the present invention, the content of resistant starch is usually 2% by mass to 10% by mass, preferably 3% by mass to 8% by mass, and more preferably 4% by mass to 6% by mass. be.

The "fructose" in barley B according to the present invention means a fructose polysaccharide containing fructooligosaccharide and the like. Fructose includes homopolysaccharides in which only fructose is polymerized, as well as polysaccharides in which fructose is polymerized in sucrose. The "polysaccharide" means an oligomer formed by polymerizing three or more sugars.
Fructans are mainly present in microorganisms and plants (for example, leaves and stems of plants of the family Allium), and levan (D-fructofranose is β2), which is a bacterially secreted polysaccharide produced from sucrose by the action of bacteria. → 6 bonds), inulin (D-fructan flanose β2 → 1 bond), levan, garlic, onion, etc. present in roots, rhizomes, grains, etc. of plants such as Kiku, Yuri, Ayame, and Orchid Fructan (containing both β2 → 6 bond and β2 → 1 bond; see Patent No. 3111378) contained in the bulb (scale) of a plant of the genus Allium is known. The fructan may be any of the above-mentioned levan, inulin and fructan derived from plants of the genus Allium, but linear and / or branched inulin is preferable. Further, fructan also includes a hydrolyzate of fructan extracted from a microorganism or a plant, and examples of the method of hydrolysis include a method of limitingly decomposing fructan with a hydrolase such as inulinase. Further, the fructan used in the present invention may be either a synthetic product or a natural product, and in the case of a natural product, those derived from microorganisms, plants and the like are used.
In the barley B according to the present invention, the content of fructan is usually 5% by mass to 15% by mass, preferably 7% by mass to 13% by mass, and more preferably 9% by mass to 12% by mass.

The "β-glucan" in barley B according to the present invention means a polysaccharide in which glucose is bound to 1-3 and 1-4, that is, (1-3) and (1-4) -β-D-glucan. It is also a type of dietary fiber. Further, in the barley B according to the present invention, β-glucan may have a mass average molecular weight of tens of thousands to millions.
In the barley B according to the present invention, the content of β-glucan is usually 2% by mass to 15% by mass, preferably 4% by mass to 10% by mass, and more preferably 6% by mass to 8% by mass. be.

(Iii) Barley C according to the present invention (barley in which the expression level and / or activity of starch synthase II is suppressed, and resistant starch per crushed barley shell grain is 2% by mass to 10% by mass, Barley containing 5% by mass to 15% by mass of fructan and 2% to 15% by mass of β-glucan)
The barley C according to the present invention has the functions, characteristics and compositions of the barleys A and B according to the present invention described above, respectively, as described above.
The barley C according to the present invention can be obtained by the method described in the above-mentioned sections of barley A and B according to the present invention.
Burley Max (registered trademark), which is a variety-improved barley, is a barley in which the expression level and / or activity of starch synthase II is suppressed, and the amount of resistant starch per crushed barley shell grain is 2% by mass. It contains% to 10% by mass, 5% by mass to 15% by mass of fructan, and 2% to 15% by mass of β-glucan. Therefore, Barley Max (registered trademark), which is a breed-improved barley, is an example of barley C according to the present invention.

[Improvement of antibiotic-induced intestinal disbiosis according to the present invention]
"Improvement of antibiotic-induced intestinal dysbiosis" in the composition of the present invention means improvement of dysbiosis (disturbance, abnormality, etc.) of the intestinal flora caused by administration of antibiotics. ..
Here, the above-mentioned "antibiotic-induced" means that it is caused by the use of an antibiotic described later, and has the same meaning as the expression such as "antibiotic-induced". The above-mentioned "improvement" means recovery from disbiosis of the intestinal flora caused by the use of antibiotics, in other words, recovery from disbiosis of the intestinal flora after use of antibiotics. .. Specifically, recovery to a state less than the same level as the intestinal flora before the use of antibiotics, recovery to the same level as the intestinal flora before the use of antibiotics, and before the use of antibiotics. It also includes the meaning of recovery / promotion to a state better than the state of the intestinal flora. Further, the above-mentioned "intestine" means a small intestine and / or a large intestine, preferably a large intestine, and more preferably a descending colon, an S-shaped (S-shaped) colon and / or a rectum.

The "antibiotic" in the composition of the present invention is a substance that can inhibit the growth of other microorganisms, and may be any generally used substance, and is a natural product produced by the microorganism. However, it may be artificially synthesized. Specifically, for example, β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as streptomycin, polypeptide antibiotics such as colistin, glycopeptide antibiotics such as vancomycin, and penicillin antibiotics such as penicillin. , Cephem antibiotics such as cefazoline, carbapenem antibiotics such as melopenem, monobactam antibiotics such as azthreonum, tetracycline antibiotics such as tetracycline, macrolide antibiotics such as erythromycin, and lincomycin antibiotics such as lincomycin. Examples thereof include β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as streptomycin, polypeptide antibiotics such as colistin, and glycopeptide antibiotics such as vancomycin. Specific examples of the route of use of the antibiotic include non-local, oral, ocular, nasal, transdermal, intravenous, intramuscular and intradermal injection, depending on the dosage form of the antibiotic. Oral is mentioned.

Specific examples of the "improvement of antibiotic-induced intestinal disbiosis" in the composition of the present invention include (i) increase and / or decrease in the number of intestinal bacteria, and (ii) intestine. Increasing bacterial diversity, (iii) promoting the production of short-chain fatty acids in the intestine, (iv) lowering the intestinal pH, and the like.
The above (i), (ii), (iii) and (iv) will be described in detail below.

(I) Increase and / or decrease in the number of enterobacteriaceae One of the improvements in antibiotic-induced enterobacteriaceae in the composition of the present invention is "increase and / or decrease in the number of enterobacteriaceae". "" Means an enterobacteriaceae belonging to one or more families and / or genera selected from the group consisting of the family Lachnospiraceae, the family Luminococcaceae and the genus Bacteroides, which form the intestinal flora. An increase in the number, type and / or proportion and / or a decrease in the number, type and / or proportion of enterobacteriaceae belonging to the family Enterobacteriaceae. Specifically, according to the composition of the present invention, Lachnospiraceae, Ruminococcaceae.
) The number, type and / or proportion of gut microbiota belonging to one or more families or genera selected from the group consisting of the family and the genus Bacteroides can be increased. Further, according to the composition of the present invention, it is possible to reduce the number, type and / or proportion of enterobacteriaceae belonging to the family Enterobacteriaceae.
Here, the above-mentioned "intestinal bacterium belonging to the family Lachnospiraceae" may be any intestinal bacterium belonging to the family Lachnospiraceae, and specifically, for example, the genus Blautia, It is an intestinal bacterium belonging to the genus Rosebria, the genus Coprococcus, and more specifically, it is an intestinal bacterium such as Blautia obium, Rosebulia inulinivorans, and Coprococcus catus. The above-mentioned "intestinal bacterium belonging to the Ruminococcaceae family" may be any intestinal bacterium belonging to the Ruminococcaceae family, and specifically, for example, Faecalibacterium. It is an intestinal bacterium belonging to the genus, the genus Subdoligranulum, the genus Ruminococcus, etc., and more specifically, the genus Faecalibacterium prausnitzii, the bacterium Ruminococcus in the genus Ruminococcus, and the like. The above-mentioned "intestinal bacterium belonging to the genus Bacteroides" may be any intestinal bacterium belonging to the genus Bacteroides. It is the intestinal bacteria of. Further, the above-mentioned "intestinal bacterium belonging to the family Enterobacteriaceae" may be any intestinal bacterium belonging to the family Enterobacteriaceae, and specifically, for example, the genus Eschericia. , Shigella, Salmonella, and the like, and more specifically, Shigella coli, Shigella sonnei, Salmonella, and the like.

(Ii) Increasing Gut Bacterial Diversity The term "increased gut microbiota", which is one of the improvements in antibiotic-induced intestinal disbiosis in the composition of the present invention, is defined as "increased gut microbiota diversity". It is an increase in the types of gut microbiota that form the flora, and more specifically, an increase in the alpha (α) diversity of the gut microbiota that forms the gut microbiota. According to the composition of the present invention, Observed species, Phylogenetic Diversity whole tree (PD walle tree), which are indicators of alpha (α) diversity of intestinal bacteria, Shannon diversity index and Che 1 increase the value of Shannon tree. can.
Here, the above-mentioned "alpha (α) diversity" means the diversity of species in a certain environment (for example, in the intestine). The above-mentioned "Observed species" means the number of confirmed species, and is an index of the abundance of species. The higher this value, the more abundant species are present. The above-mentioned "Phylogenetic Diversity color tree" means the total distance of phylogenetic trees and is an index of phylogenetic diversity. The higher this value, the more species of organisms are present. The above-mentioned "Shannon diversity index" is an index of equality (Evenness) between species calculated from the ratio of each organism species. The higher this value, the more evenly each species is present. Further, "Chao1 index" is an index of abundance of organisms estimated based on the arrangement of an organism species (singleton) confirmed only once and an organism species (doubleton) confirmed only twice. The higher this number, the more species of various classifications exist.

(Iii) Promotion of production of short-chain fatty acids in the intestine "Promotion of production of short-chain fatty acids in the intestine", which is one of the improvements in antibiotic-induced intestinal disbiosis in the composition of the present invention. Is an increase in the amount of short-chain fatty acids in the intestine, which is thought to be involved in suppressing inflammation in the intestine and suppressing the rise in blood glucose. According to the composition of the present invention, the production of short-chain fatty acids in the intestine can be promoted.
Here, the above-mentioned "short-chain fatty acid in the intestine" means a fatty acid having 6 or less carbon atoms produced by intestinal bacteria, and specifically, for example, acetic acid, propionic acid, isobutyric acid, butyric acid, iso. Examples thereof include valeric acid, valeric acid, succinic acid, formic acid, lactic acid, butyric acid, and acetic acid, propionic acid and butyric acid are preferable, acetic acid and butyric acid are more preferable, and butyric acid is particularly preferable.

(Iv) Decrease in intestinal pH One of the improvements in antibiotic-induced intestinal disbiosis in the composition of the present invention, "decrease in intestinal pH" is the value of intestinal pH. It is a decrease in. According to the composition of the present invention, the pH in the intestine can be lowered from alkaline to acidic. Here, the above-mentioned "pH" is a Potential of Hydrogen, and is also referred to as a hydrogen ion index, a hydrogen ion concentration index, or a hydrogen index.

Since the composition of the present invention contains barley with abundant eating experience, even if it is continuously ingested for a long period of time, there is little concern that it will have a harmful effect on the subject who needs it, and it is safe. Is guaranteed.
Here, examples of the above-mentioned "subject" include mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, and rabbits, with humans, mice, or rats being preferred, and humans being more preferred.

In addition, according to the composition of the present invention, antibiotic-induced intestinal disbiosis can be improved at an early stage. Specifically, according to the composition of the present invention, improvement of antibiotic-induced intestinal disbiosis is achieved within 14 days from the date on which the composition of the present invention is provided. More specifically, improvement of antibiotic-induced intestinal disbiosis on the 3rd, 5th, 7th, 10th or 14th day from the day when the composition of the present invention was provided. Is achieved.
The above-mentioned "third day from the day when the composition of the present invention is provided" is the number of days calculated with the day when the composition of the present invention is provided as the 0th day, and is "the composition of the present invention". "5th day from the day when the product was served", "7th day from the day when the composition of the present invention was served", "10th day from the day when the composition of the present invention was served" and "the present invention". The same applies to "14th day from the day when the composition is provided".

The amount of the composition of the present invention to be provided may be any amount as long as it achieves a desired effect, that is, an improvement in antibiotic-induced intestinal disbiosis, and is provided in the form of the composition. It can be changed as appropriate according to the species, symptoms, age, gender, etc. of the subject. Specifically, as the amount of the composition of the present invention to be provided, for example, the barley according to the present invention contained in the composition of the present invention is usually 1 g to 20 g per day for an adult (weight: 60 kg). It is preferably 3 g to 17 g, more preferably 5 g to 15 g, and even more preferably 7 g to 13 g.

[Food and drink of the present invention]
The food and drink of the present invention is characterized by containing the composition of the present invention.

The food and drink of the present invention can be used as a food and drink for improving antibiotic-induced intestinal disbiosis, and the form thereof is specifically, for example, rice (for example, rice cake, lunch box). Rice, porridge), confectionery (eg ice, potato chips), bakeries (eg bread, pie, cakes, cookies, biscuits, crackers), noodles (eg udon, buckwheat, ramen), frozen or refrigerated Distributed processed foods, baby foods, baby foods, pet foods, animal feeds, beverages (eg fruit juice beverages, soft beverages, alcoholic beverages, tea, sports beverages), fermented foods such as medicinal liquors, seasonings (eg mirin, Foods such as vinegar, soy sauce, miso, sauces, sports foods, health foods, functional foods, nutritional supplements, foods with health claims (eg foods for specified health use, foods with nutritional claims, foods with functional claims), foods for special use (eg foods with functional claims) , Infant foods, pregnant women's foods, sick foods), and rice, snack foods, bakeries, noodles, nutritional supplements and health functional foods are preferable. In the food and drink of the present invention, when the form is a dietary supplement or a health functional food, supplements in the form of tablets, capsules and the like, granola-like cereals, granola-like snake bar cereal bars and the like can be mentioned.
Here, the above-mentioned "health functional food" is subject to legal restrictions in each country from the viewpoint of health when manufacturing and / or selling foods and drinks for the purpose of improving intestinal disbiosis. It is a food or drink that may occur. Such foods and drinks can be labeled with the possibility of reducing the risk of illness, working on health (maintenance / promotion), safety, etc., for example, the product body of foods and drinks, containers, packaging, instructions, etc. It can be displayed on attached documents and advertisements.

The food and drink of the present invention can be produced with reference to known production techniques usually performed in this field. At that time, if necessary, fungi such as vitamins, minerals, sugars, flavors, fruit juices, additives, stabilizers, lactic acid bacteria, and bifidobacteria may be added. Further, the food or drink of the present invention can be manufactured according to the manufacturing technique for a pharmaceutical product described later, and in that case, a pharmaceutically acceptable carrier, additive or the like can also be used.

Mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, and rabbits are mentioned as targets for ingesting the food and drink of the present invention, and humans, mice, and rats are preferable, and humans are more preferable.

The amount of the food or drink of the present invention may be any amount as long as it can obtain a desired effect of improving intestinal dysbiosis, depending on the form of the food or drink, the species of the solid to be ingested, symptoms, age, gender, etc. It can be changed as appropriate. As the intake of the food and drink of the present invention, specifically, for example, the barley according to the present invention contained in the food and drink of the present invention is usually 1 g to 20 g, preferably 1 g to 20 g, per day for an adult (weight: 60 kg). It is 3 g to 17 g, more preferably 5 g to 15 g, still more preferably 7 g to 13 g.

[Pharmaceutical product of the present invention]
The pharmaceutical product of the present invention is characterized by containing the composition of the present invention.

The pharmaceutical product of the present invention is a drug for improving an antibiotic-induced intestinal disbiosis (improving drug), and a disease that can be caused by an antibiotic-induced intestinal disbiosis (for example, obesity, diabetes, arteries). It can also be used as a therapeutic agent for sclerosis, inflammatory bowel disease, rheumatoid arthritis, colon cancer, Parkinson's disease), and its form is specifically, for example, tablets, capsules, granules, powders, syrups. , Oral preparations such as dry syrups, liquids and suspensions, enteric preparations such as inhalants, transdermal preparations and suppositories, parenteral preparations such as drip and injections, tablets, capsules and granules. And powders are preferred.
The liquid and suspension may be dissolved or suspended in water or an appropriate medium immediately before administration, and the tablets and granules may be coated on the surface thereof, if necessary. .. Further, the pharmaceutical product of the present invention may contain a pharmaceutically acceptable carrier and / or other medicinal ingredient, if necessary. Specific examples of the pharmaceutically acceptable carrier include excipients, binders, diluents, additives, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, and dispersants. , Suspension agents, preservatives and the like, and examples of the other medicinal ingredients include, for example, vitamins, minerals and crude drugs.

The pharmaceutical product of the present invention is produced by blending the composition of the present invention with the above-mentioned pharmaceutically acceptable carrier and / or other medicinal ingredient, and referring to a known manufacturing technique usually performed in this field. be able to.

Targets for using (administering, taking, etc.) the pharmaceutical product of the present invention include mammals such as humans, monkeys, mice, rats, dogs, cats, pigs, and rabbits, and humans, mice, and rats are preferable, and humans are preferred. More preferred.

The amount of the drug of the present invention used (administered, taken, etc.) may be any amount as long as it can obtain the desired effect of improving intestinal disbiosis, and the form of the drug, the species of the solid to be administered, the symptoms, and the age may be used. , Gender, etc. may be changed as appropriate.

Hereinafter, the present invention will be described in more detail with reference to Examples, but this does not limit the scope of the present invention.

[(1) Preparation of feed]
The feed was based on AIN-93G, which is a standard purified feed published by the National Institute of Health and Nutrition (AIN), and adjusted as shown in Table 1 to obtain a control feed and a barley feed, respectively. Specifically, 5% of cellulose was added to the AIN-93G as a control feed, and 30.4% of barley flour was added to the barley feed, and the total dietary fiber content was adjusted to 5%. In the barley feed, the amounts of cornstarch and milk casein added were adjusted so that the amounts of starch and protein were similar to those of the control feed.
The cellulose and AIN-93G were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. and Oriental Yeast Co., Ltd., respectively. Further, as the barley flour, Barley Max (registered trademark) (The Healthy Grain Proprietary Limited), which is a breed-improved barley, was used (an example of barley A, B and C according to the present invention).

[(2) Preparation of sample]
Six-week-old C57BL / 6J male mice that had been acclimated for one week were classified into four groups: Cont group, Ab group, Ab-CL group, and Ab-BM group. Then, tap water is added to the Cont group, and the antibiotics ampicillin (1 mg / mL), streptomycin (1 mg / mL), colistin (1 mg / mL) and vancomycin are given to the Ab group, Ab-CL group and Ab-BM group. Water containing (1 mg / mL) was allowed to drink freely for 7 days each. In addition, during this period, all four groups were fed CE-2, which is a feed for breeding and breeding. After that, the Cont group and Ab group were dissected, and the contents of the cecum and feces were collected, respectively. For the Ab-CL group and Ab-BM group, continue breeding with free drinking tap water, the Ab-CL group is fed with the control feed adjusted in (1) above, and the Ab-BM group is fed with the above (1). ) Was fed for 14 days each. The day when feeding was started was set as the 0th day, and 14 days after that, dissection was performed, and the contents of the cecum and feces were collected, respectively. The collected samples were stored frozen until use.
All of the above antibiotics were purchased from Fujifilm Wako Pure Chemical Industries, Ltd.

[(3) Analysis of intestinal bacterial flora]
As an analysis of the intestinal bacterial flora in the sample prepared in (2) above, we requested Bacterial Giken Co., Ltd. to analyze the bacterial 16S rRNA in feces.
Specifically, first, each stool sample was freeze-dried using VD-250R Freeze Dryer (Tietech Co., Ltd.), and then pulverized using Shake Master Neo (Biomedical Science Co., Ltd.). DNA was extracted from each of the pulverized samples using the MPure Bacterial DNA Execution Kit (MP Biomedical). Then, using Synergy H1 (BioTek Instrument) and QuantiFluor dsDNA System (Promega), the concentration of the extracted DNA solution was measured, respectively. Then, a library was prepared from the extracted DNA by 2Step tailed PCR using a base sequence containing a base sequence commonly conserved in 16S rRNA of various intestinal bacteria as a primer. Next, sequencing was performed using MiSeq (Illumina Co., Ltd.) under the condition of 2 × 300 base pairs. From the obtained sequence, only the sequence in which the start of reading the sequence exactly matched the primer used above was extracted using the fastq barcode stiltter of Fastx toolkit, and then the primer sequence of the sequence was removed. Then, using sickle tools, sequences having a quality value of less than 20 were removed, and sequences having a length of 150 bases or less and their pair sequences were discarded. Arrays that passed quality filtering were merged with the pair-end merge script FLASH. The conditions for merging were a fragment length of 420 bases after merging, a read fragment length of 280 bases, and a minimum overlap length of 10 bases. Chimeric sequences were confirmed for sequences that had passed all filtering using the uchime algorithm of usarch. For the database, 97% OTU of Greengene attached to the pipeline for bacterial flora analysis was used to extract all sequences that were not determined to be chimeric sequences and used for analysis. OTU creation and system estimation were performed using Qime's workflow script with no reference and all parameters under default conditions. The alpha (α) diversity analysis was performed using a Qime script.
The primers and the like used in the above 2Step tailed PCR can also be designed with reference to the Journal of Gut Bacteriology (19: 47-52, 2005) and the like.

[(4) Measurement of short-chain fatty acids in the intestine]
As shown in Table 2 below, short-chain fatty acids in the intestine were measured using a high performance liquid chromatograph Prominence (Shimadzu Corporation). Specifically, 0.1 g of the cecal content derived from the sample prepared in (2) above was suspended in Milli-Q water, and then centrifuged (15,350 × g, 10 minutes). Then, the obtained supernatant was filtered through a 0.2 μm filter (Merck Millipore) and used as a measurement sample. The identification of short-chain fatty acids acetic acid, propionic acid and butyric acid was performed by comparing the retention times of the measurement sample and the standard solution, and the quantification was performed by comparing the peak area values.

[Example 1: Measurement of DNA amount in feces]
When the amount of DNA extracted from feces was measured from the above (3), the results as shown in FIG. 1 were obtained.
Specifically, in the Ab group, a decrease in the amount of DNA was confirmed after administration of the antibiotic as compared with the Cont group. In the Ab-CL group, after administration of the antibiotic, recovery of the DNA amount was confirmed as compared with the Ab group, but the value was about the same as that in the Cont group. On the other hand, in the Ab-BM group, surprisingly, after administration of the antibiotic, a significant recovery / increase in the amount of DNA was confirmed as compared with the Cont group, the Ab group and the Ab-CL group.
From the results of Example 1, since the amount of DNA extracted from feces is proportional to the number of intestinal bacteria, ingestion of barley feed improves (recovers) the decrease in the number of intestinal bacteria caused by the administration of antibiotics.・ Increased).

[Example 2: Analysis of diversity of intestinal bacterial flora]
From the above (3), the alpha (α) diversity of the intestinal flora (Observed species, PD walle tree, Shannon diversity index and Chao1 index) was analyzed, and the results shown in FIG. 2 were obtained.
Specifically, in the Ab group, after administration of the antibiotic, a decrease in the above four index values indicating alpha (α) diversity was confirmed as compared with the Cont group. In the Ab-CL group, after administration of the antibiotic, a further decrease in the above four index values showing alpha (α) diversity was confirmed as compared with the Cont group and the Ab group, respectively. On the other hand, in the Ab-BM group, surprisingly, after administration of the antibiotic, recovery / increase of the above four index values showing alpha (α) diversity was confirmed as compared with the Ab-CL group.
From the results of Example 2, the decrease (recovery / increase) in the diversity of the intestinal flora (intestinal flora) caused by the administration of the antibiotic was improved (recovered / increased) by the intake of the barley feed.

[Example 3: Analysis of abundance ratio of intestinal bacteria]
When the abundance ratio of various intestinal bacteria was analyzed from the above (3), the results as shown in FIG. 3 were obtained.
Specifically, in the Ab group, after administration of antibiotics, the abundance ratio of intestinal bacteria belonging to the Lachnospiraceae family, Ruminococcaceae family and Bacteroides genus was reduced as compared with the Cont group. Each was confirmed. In the Ab-CL group, after administration of antibiotics, a further decrease in the abundance ratio of intestinal bacteria belonging to the Lachnospiraceae family and the Ruminococcaceae family was confirmed, respectively, as compared with the Cont group and the Ab group. However, it was confirmed that the abundance ratio of intestinal bacteria belonging to the genus Bacteroides was restored and increased. On the other hand, in the Ab-BM group, the abundance ratio of intestinal bacteria belonging to the Lachnospiraceae family, Ruminococcaceae family and Bacteroides genus is higher than that in the Cont group, Ab group and Ab-CL group. Significant recovery and increase were confirmed respectively.
In addition, in the Ab group and Ab-CL group, an increase in the abundance ratio of enterobacteriaceae belonging to the family Enterobacteriaceae was confirmed after administration of antibiotics, respectively, as compared with the Cont group, but the Ab-BM group. It was confirmed that this increase was suppressed even after administration of antibiotics.
From the results of Example 3, since the intestinal bacteria belonging to the family Lachnospiraceae and the family Ruminococcaceae contain bacteria known as butyric acid-producing bacteria, by ingesting barley feed, The decrease (recovery / increase) in the number of butyric acid-producing bacteria caused by the administration of antibiotics was improved. In addition, since pathogens such as Escherichia, Shigella, and Salmonella belong to the family Enterobacteriaceae, it is caused by administration of antibiotics by ingestion of barley feed. The increase in the number of these pathogens was improved (recovery / suppression).

[Example 4: Measurement of short-chain fatty acids in the intestine]
From the above (4), when the short-chain fatty acids acetic acid, butyric acid and propionic acid were measured, the results as shown in FIG. 4 were obtained.
Specifically, in the Ab group, a decrease in the amounts of acetic acid, butyric acid, propionic acid and total short-chain fatty acids was confirmed after administration of the antibiotic as compared with the Cont group. In the Ab-CL group, after administration of the antibiotic, the amount of butyric acid was similar to that in the Ab group, but recovery / increase in the amounts of acetic acid, propionic acid and total short-chain fatty acid was confirmed. On the other hand, in the Ab-BM group, a significant recovery / increase in the amounts of acetic acid, butyric acid, propionic acid and total short-chain fatty acids was confirmed as compared with the Ab group and the Ab-CL group.
From the results of Example 4, the decrease (recovery / increase) in the amount of short-chain fatty acids caused by the administration of antibiotics was improved (recovered / increased) by the intake of barley feed.

[Example 5: Measurement of intestinal pH]
From the above (2), when the pH of the cecal content was measured with a pH meter, the results shown in FIG. 5 were obtained.
Specifically, in the Ab-CL group, an increase in pH was confirmed after administration of the antibiotic as compared with the Cont group, but in the Ab-BM group, even after the administration of the antibiotic, Suppression of this increase was confirmed.
From the results of Example 5, ingestion of barley feed improved (recovered / suppressed) the increase in intestinal pH caused by the administration of antibiotics.

The present invention can be utilized in the production industry of compositions, foods and drinks and pharmaceuticals for improving antibiotic-induced intestinal disbiosis.

Claims (14)

A composition for improving antibiotic-induced intestinal disbiosis, which comprises barley in which the expression level and / or activity of starch synthase II is suppressed. Antibiotics-induced, containing barley containing 2% to 10% by weight of resistant starch, 5% to 15% by weight of fructan and 2% to 15% by weight of β-glucan per crushed barley husk. A composition for improving sex intestinal disbiosis. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis is to increase and / or decrease the number of intestinal bacteria. An improvement in intestinal disbiosis is to increase the number of gut bacteria belonging to one or more families and / or genera selected from the group consisting of Lachnospiraceae, Ruminococcus and Bacteroides. Or the composition according to 2. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis is to reduce the number of intestinal bacteria belonging to the family Enterobacter. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis is to increase the diversity of intestinal bacteria. The composition according to claim 1 or 2, wherein the improvement of the intestinal disbiosis is to increase the alpha diversity of the intestinal bacteria. An increase in intestinal dysbiosis indicates an increase in the alpha diversity of one or more gut microbiota selected from the group consisting of Observed species, Phylogenetic Diversity color tree, Shannon diversity index and Chao1 index. The composition according to claim 1 or 2. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis promotes the production of short-chain fatty acids in the intestine. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis promotes the production of one or more short-chain fatty acids in the intestine selected from the group consisting of acetic acid, propionic acid and butyric acid. .. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis is to lower the pH in the intestine. The composition according to claim 1 or 2, wherein the improvement of intestinal disbiosis is performed within 14 days from the date when the composition is provided. A food or drink containing the composition according to any one of claims 1 to 12. A pharmaceutical product containing the composition according to any one of claims 1 to 12.

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