CN118614617A - Composition for promoting secretion of appetite stimulating hormone - Google Patents

Abstract

The present invention provides compositions for promoting the secretion of orexin. The present invention addresses the problem of providing a novel food material effective for promoting the secretion of an orexin. The composition for stimulating appetite, which comprises a bacterium belonging to the genus Streptococcus (Streptococcus), and is used for any one selected from the group consisting of regulating autonomic nerves, stimulating growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, increasing gastric acid secretion, increasing pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting myocardial cells.

Description

Composition for promoting secretion of appetite stimulating hormone

Technical Field

The present invention relates to a composition for promoting the secretion of appetite stimulating hormone.

Background Art

Ghrelin is a peptide hormone mainly secreted in the stomach, and its effects of promoting appetite, regulating gastrointestinal motility, promoting growth hormone, regulating energy metabolism, promoting cardiac function, and anti-diabetic effects have been reported. These are mediated by autonomic nervous system regulation such as suppressing sympathetic nerves/hyperactivity of parasympathetic nerves through ghrelin, and ghrelin is very important for maintaining life homeostasis.

Regarding appetite stimulants, Patent Documents 1 and 2 record hormone secretion promoters with skim milk or skim milk powder as active ingredients, and the hormones referred to in these are recorded as appetite stimulants or growth hormones. In addition, Patent Document 3 records a preparation for promoting the secretion of appetite stimulants, which contains: whey protein hydrolyzate and fermented milk protein as proteins; medium-chain fatty acids, milk lecithin and fish oil as lipids; and palatinose (registered trademark) as a carbohydrate. Patent Document 4 records a preparation for improving loss of appetite, which contains Lactobacillus gasseri OLL2716 (FERM BP-6999) (recently reclassified as Lactobacillus paragasseri) as an active ingredient, and records that the improvement of loss of appetite is brought about by the secretion promotion effect of appetite stimulants. Furthermore, Patent Document 5 describes peptides having a specific sequence of 4 to 12 amino acids in length derived from a purified rice endosperm protein subtilisin digest, and describes that these peptides are effective for treating anorexia caused by decreased secretion of ghrelin. In addition, Non-Patent Document 1 describes the beneficial effect of ghrelin in suppressing the overly tense sympathetic nerve activity after heart disease regarding the cardioprotective effect of ghrelin.

On the other hand, thermophilic streptococcus (Streptococcus thermophilus, S.thermophilus) is a kind of lactic acid bacteria (such as aforementioned patent document 3, patent document 6) that is sometimes used as the starter of fermented milk. In addition, about thermophilic streptococcus, patent document 7 records vagus nerve activator, which contains lactic acid bacteria body and/or lactic acid bacteria processed material with vagus nerve activation as effective ingredient, and as the example of lactic acid bacteria, thermophilic streptococcus is enumerated. Patent document 8 records the auxiliary agent composition for alleviating the side effect of chemotherapeutic agent, and it comprises the thermophilic streptococcus ST4 strain of effective amount. And then, about the lactic acid bacteria of Streptococcus, patent document 9 records the pharmaceutical composition or the diet product with regulating autonomic nerve effect with specific compound as effective ingredient, as effective ingredient, can be the culture supernatant of the microorganism with the ability of producing formula (I) compound or its processed product or the microorganism with the ability of producing compound, in addition, such microorganism can be lactic acid bacteria, and as the example of lactic acid bacteria, the lactic acid bacteria belonging to Streptococcus can be enumerated.

Prior art literature

Patent Literature

Patent Document 1: Japanese Patent Application Laid-Open No. 2015-205829

Patent Document 2: Japanese Patent Application Laid-Open No. 2015-205830

Patent Document 3: Japanese Patent No. 6657084 (International Publication No. WO2015/199192)

Patent Document 4: Japanese Patent No. 6813974 (Japanese Patent Application Laid-Open No. 2018-8901)

Patent Document 5: International Publication No. WO2020/218450

Patent Document 6: International Publication No. WO2022/191093

Patent Document 7: Japanese Patent Application Publication No. 2012-17282

Patent Document 8: Japanese Patent Application Publication No. 2021-20896

Patent Document 9: International Publication No. WO2008/120713

Non-patent literature

Non-patent literature 1: Expert Rev Endocrinol Metab, 4:283-289, 2014. https://doi.org/10.1586/eem.09.7

Summary of the invention

Problem that the invention aims to solve

A new food material effective in promoting the secretion of appetite stimulating hormones is desired.

Solutions for solving problems

The present invention provides the following inventions.

[1] A composition for promoting the secretion of anorectic hormone, comprising bacteria belonging to the genus Streptococcus.

[2] The use according to 1, wherein the bacteria belonging to the genus Streptococcus have any species selected from the following: a polynucleotide comprising a sequence of sequence number 5 or its ortholog, a polynucleotide comprising a sequence of sequence number 7 or its ortholog, and a polynucleotide comprising a sequence of sequence number 9 or its ortholog.

[3] A composition for use in any one of the following: regulating the autonomic nervous system, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, promoting gastric acid secretion, promoting pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting cardiomyocytes, wherein the composition contains any one of the bacteria belonging to the genus Streptococcus.

[4] The composition according to 3, which is based on promoting the secretion of appetite-stimulating hormone.

[5] The composition according to any one of 1 to 4, wherein the bacteria belonging to the genus Streptococcus belongs to Streptococcus thermophilus (S. thermophilus).

[6] The composition according to any one of 1 to 5, wherein the bacteria belonging to the genus Streptococcus is any species selected from the group consisting of Streptococcus thermophilus OLS3289 (NCIMB 8510T), Streptococcus thermophilus 1131, Streptococcus thermophilus OLS3294 (Deposit No.: NITE BP-00077), and Streptococcus thermophilus OLS3615 (Deposit No.: NITE BP-01696).

[7] The composition according to any one of 1 to 6, which is used for administering bacteria belonging to the genus Streptococcus at a dose of 1.3×10 ¹⁰ bacteria/day or more.

[8] The composition according to any one of 1 to 7, which is in the form of fermented milk that does not contain bacteria belonging to Lactobacillus paragaseri.

[11] A composition containing bacteria belonging to the genus Streptococcus, and a method for promoting the secretion of appetite stimulating hormone. Use of bacteria belonging to the genus Streptococcus in the manufacture of a composition for promoting the secretion of appetite stimulating hormone. A method or non-therapeutic method for promoting the secretion of appetite stimulating hormone, comprising the step of administering a composition containing bacteria belonging to the genus Streptococcus to a subject. Use or non-therapeutic use of a composition containing bacteria belonging to the genus Streptococcus for promoting the secretion of appetite stimulating hormone.

[12] A composition containing bacteria belonging to the genus Streptococcus, and a method for promoting the secretion of an appetite stimulating hormone, the composition comprising any one selected from the following: a polynucleotide containing a sequence of SEQ ID NO: 5 or an ortholog thereof, a polynucleotide containing a sequence of SEQ ID NO: 7 or an ortholog thereof, and a polynucleotide containing a sequence of SEQ ID NO: 9 or an ortholog thereof. Use of bacteria belonging to the genus Streptococcus having any one selected from the group consisting of a polynucleotide containing a sequence of SEQ ID NO: 5 or an ortholog thereof, a polynucleotide containing a sequence of SEQ ID NO: 7 or an ortholog thereof, and a polynucleotide containing a sequence of SEQ ID NO: 9 or an ortholog thereof in the manufacture of a composition for promoting the secretion of an appetite stimulating hormone. A method or non-therapeutic method for promoting the secretion of an appetite stimulating hormone, comprising the step of administering to a subject a composition containing bacteria belonging to the genus Streptococcus having any one selected from the group consisting of a polynucleotide containing a sequence of SEQ ID NO: 5 or an ortholog thereof, a polynucleotide containing a sequence of SEQ ID NO: 7 or an ortholog thereof, and a polynucleotide containing a sequence of SEQ ID NO: 9 or an ortholog thereof. The use or non-therapeutic use of a composition for promoting the secretion of appetite stimulating hormone, wherein the composition comprises a bacterium belonging to the genus Streptococcus having any species selected from a polynucleotide comprising a sequence of sequence number 5 or a direct homolog thereof, a polynucleotide comprising a sequence of sequence number 7 or a direct homolog thereof, and a polynucleotide comprising a sequence of sequence number 9 or a direct homolog thereof.

[13] A composition containing bacteria belonging to the genus Streptococcus, which is used for any method selected from the group consisting of regulating the autonomic nervous system, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, causing hypersecretion of gastric acid, causing hypersecretion of pancreatic exocrine, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting myocardial cells. Use of bacteria belonging to the genus Streptococcus in the manufacture of a composition, wherein the composition is used for any method selected from the group consisting of regulating the autonomic nervous system, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, causing hypersecretion of gastric acid, causing hypersecretion of pancreatic exocrine, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting myocardial cells. A method or non-therapeutic method for any one of the group consisting of regulating the autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, hypersecretion of gastric acid, hypersecretion of pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting cardiomyocytes, comprising the step of administering a composition containing bacteria belonging to the genus Streptococcus to a subject. A composition containing bacteria belonging to the genus Streptococcus for use or non-therapeutic use of any one of the group consisting of regulating the autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, hypersecretion of gastric acid, hypersecretion of pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting cardiomyocytes.

[14] The composition, application in manufacture, method or non-therapeutic method, or application or non-therapeutic application according to item 13, wherein the composition is selected from any one of the group consisting of regulating the autonomic nervous system, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, increasing gastric acid secretion, increasing pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells based on promoting the secretion of appetite-stimulating hormone.

[15] The composition, use in manufacturing, method or non-therapeutic method, or use or non-therapeutic use according to any one of 11 to 14, wherein the bacteria belonging to the genus Streptococcus are bacteria belonging to Streptococcus thermophilus (S. thermophilus).

[16] The composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to any one of 11 to 15, wherein the bacterium belonging to the genus Streptococcus is any species selected from the group consisting of Streptococcus thermophilus OLS3289 (NCIMB8510T), Streptococcus thermophilus 1131, Streptococcus thermophilus OLS3294 (Deposit Number: NITE BP-00077), and Streptococcus thermophilus OLS3615 (Deposit Number: NITE BP-01696).

[17] The composition, use in manufacture, method or non-therapeutic method, or use or non-therapeutic use according to any one of 11 to 16, wherein the composition is used to administer bacteria belonging to the genus Streptococcus at a dose of 1.3×10 ¹⁰ bacteria/day or more.

[18] The composition, use in production, method or non-therapeutic method, or use or non-therapeutic use according to any one of 11 to 17, wherein the composition is in the form of fermented milk that does not contain bacteria belonging to Lactobacillus paragaseri.

Effects of the Invention

The secretion of appetite stimulating hormone can be promoted through highly safe ingredients.

By providing materials derived from highly safe food ingredients or materials with confirmed safety as preparations for promoting the secretion of appetite stimulants, it is possible to prevent or improve poor physical conditions caused by imbalance of the autonomic nervous system, and to help healthy people maintain/improve their health and work more efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Streptococcus thermophilus significantly increased the expression of ghrelin mRNA. Mean ± SE n = 3, *p < 0.05, relative to the control (based on Dunnett's test).

FIG. 2 : Sequences of rRNA genes of strains used in the Examples.

Figure 3: Sequence of sequence numbers 5 to 14.

DETAILED DESCRIPTION

The present invention is described in detail below.

The present invention relates to a composition containing specific bacteria. More specifically, it relates to a composition containing specific bacteria as an effective ingredient for promoting the secretion of appetite stimulating hormone. It should be noted that, in the present invention, when any species is mentioned, it means that the number and type are arbitrary.

[Active ingredients]

(Bacteria belonging to the genus Streptococcus)

The composition of the present invention comprises bacteria belonging to the genus Streptococcus as an active ingredient. The bacteria belonging to the genus Streptococcus are preferably bacteria belonging to Streptococcus thermophilus (or, sometimes referred to as Streptococcus salivarius subsp. thermophilus). In the composition, as long as the bacteria belong to Streptococcus thermophilus, it is not limited to a specific strain, and various bacteria belonging to Streptococcus thermophilus can be used as an active ingredient.

As examples of strains belonging to Streptococcus thermophilus, OLS3289 (NCIMB8510 ^T ), 1131, OLS3294 (Accession No.: NITE BP-00077), OLS3615 (Accession No.: NITE BP-01696), and strains taxonomically equivalent thereto can be used.

The strain that is taxonomically equivalent to a certain strain (hereinafter referred to as strain S) refers to any of the following species, for example.

A strain that belongs to the same genus as strain S and has a sequence homology of 90% or more, preferably 95% or more, more preferably 98% or more, further preferably 98.5% or more, further preferably higher than 98.7%, further preferably 99% or more, further preferably 100% in all or a characteristic part of its 16S rRNA gene sequence (V1 region, V2 region, or all or part of V1 region and V2 region, or a part including V1 region and V2 region, etc.) with the sequence of strain S;

· Strains with the same mycological properties as strain S.

It should be noted that, regarding the criteria for determining the differences and similarities between species based on 16S rRNA gene sequences, those skilled in the art may refer to Stackebrandt E, Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006; 33: 152-155.

(Streptococcus thermophilus NCIMB8510 ^T )

Streptococcus thermophilus NCIMB 8510 ^T can be purchased from NCIMB Research Institute (The National Collections of Industrial, Food and Marine Bacteria, Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK).

NCIMB8510 ^T is a type strain of Streptococcus thermophilus. A type strain is a strain that has the ability to derive a lineage from the type that serves as the basis for naming.

The sequence of the V1 to V3 region of the 16S rRNA gene of NCIMB8510 ^T (corresponding to positions 27 to 520 in the full-length sequence of the rRNA gene) is shown in FIG2 and is shown as sequence number 2 in the sequence listing.

(Streptococcus thermophilus 1131)

Thermophilus Streptococcus 1131 can be isolated from Meiji Bulgarian Yogurt LB81 (Meiji Co., Ltd.). For information about Meiji Bulgarian Yogurt LB81, please refer to "Yogurt without stabilizers_Yogurt without preservatives_Safe and healthy sugar-free yogurt_Low-fat yogurt_Meiji Yogurt Official Website (meijibulgaria.cn), URL https://www.meijibulgaria.cn/brand.html" and the like.

The mycological properties of 1131 are shown below.

Morphology: Streptococcus, does not use glucose to produce gas, Gram-positive, catalase-negative, lactic acid optical rotation L-type, grows at 45°C, can assimilate glucose, lactose, fructose, etc. to produce acid, does not form spores, and is non-motile.

A portion of the 16S rRNA gene sequence of 1131 is shown in FIG2 , and is shown as SEQ ID NO: 1 in the sequence listing.

(Streptococcus thermophilus OLS3294)

Streptococcus thermophilus OLS3294 (deposit number: NITE BP-00077) is deposited with the National Institute for Product Evaluation and Research Patent Microorganism Depository (NITE-NPMD, National Institute for Technology Evaluation, Patent Microorganism Depository) under the deposit number NITE BP-00077 (Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) (depositor: Meiji Co., Ltd., domestic deposit date: February 10, 2005, receipt date of request for conversion to international deposit based on the Budapest Treaty: March 2, 2022).

The mycological properties of OLS3294 are shown below.

Gram-positive, facultative anaerobic cocci

Sugar assimilation: arabinose-, galactose-, glucose+, salicin-, sucrose+, cellobiose-, starch-, maltose-, mannitol-, mannose-, lactose+, raffinose-, ribose-

Motility-, presence or absence of spores-, gas production-, catalase-, nitrate reduction-, indole production-, hydrogen sulfide production-, optically active L-form of lactic acid, development at 15°C-

The sequence of the V1 to V3 regions of the 16S rRNA gene of OLS3294 (corresponding to positions 27 to 520 in the full-length sequence of the rRNA gene) is shown in FIG2 , and is shown as SEQ ID NO: 3 in the sequence listing.

(Streptococcus thermophilus OLS3615)

Streptococcus thermophilus OLS3615 (deposit number: NITE BP-01696) is deposited with the National Institute for Product Evaluation and Research Patent Microorganism Depository (NITE-NPMD, National Institute for Technology Evaluation, Patent Microorganism Depository) under the deposit number NITE BP-01696 (Room 122, 2-5-8, Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) (depositor: Meiji Co., Ltd., deposit date based on the Budapest Treaty: August 23, 2013).

The mycological properties of OLS3615 are shown below.

Colony characteristics on culture medium (M17 agar, BD): round, white (translucent), smooth, hemispherical

Bacterial morphology: cocci, Gram staining: positive, lactic acid fermentation form: homolactic fermentation, aerobic development: +

A portion of the 16S rRNA gene sequence of OLS3615 is shown in FIG2 , and is shown as SEQ ID NO: 4 in the sequence listing.

The target effect of the composition containing any of the above 4 strains as an active ingredient is particularly excellent, so it is preferred. According to the research of the present inventors, one of the common characteristics of these 4 strains is that they have polynucleotides or their orthologs with sequences recorded as sequence numbers 5, 7 and 9 in the sequence table. The amino acid sequences of the proteins encoded by these polynucleotides (acyl CoA thioesterase, site-specific DNA methyltransferase and histidine phosphatase family protein) are recorded in sequence as sequence numbers 6, 8, and 10 in the sequence table. Therefore, in a preferred embodiment, the composition may contain the following bacteria belonging to thermophilic streptococci as an active ingredient, and the bacteria have polynucleotides or their orthologs containing sequences of sequence numbers 5, 7 and 9 in the sequence table, more preferably more than 2, and more preferably all. In addition, in another preferred embodiment, the composition may contain the following bacteria belonging to thermophilic streptococci as an active ingredient, and the bacteria have proteins or their orthologs containing sequences of sequence numbers 6, 8 and 10 in the sequence table, more preferably more than 2, and more preferably all. In another preferred embodiment, the composition may contain any one, more preferably two or more, and even more preferably all of the proteins comprising the sequences of SEQ ID NOs. 6, 8, and 10 in the sequence listing or their orthologs as active ingredients.

In the present invention, orthologs refer to the corresponding relationship between genes derived from a common ancestral gene as species diverge, or a group of genes in such a corresponding relationship. Orthologs are grouped in the same orthology group (orthgroup) in Orthofinder (http://www.stevekellylab.com/software/orthofinder).

The polynucleotides comprising the sequence of SEQ ID NO: 5, 7 or 9 and their orthologs are specifically as follows.

(A) a polynucleotide comprising the sequence of SEQ ID NO: 5, 7 or 9;

(B) a polynucleotide comprising a sequence having high sequence homology to the sequence of SEQ ID NO: 5, 7 or 9, and encoding a protein having the same activity as the protein comprising the corresponding sequence of SEQ ID NO: 6, 8 or 10;

(C) A polynucleotide comprising a sequence in which one or more nucleotides are replaced, deleted, inserted and/or added in the sequence of sequence number 5, 7 or 9, and encoding a protein having the same activity as the protein comprising the corresponding sequence of sequence number 6, 8 or 10.

Orthologs of the polynucleotide comprising the sequence of SEQ ID NO: 7 include the following.

(A) a polynucleotide comprising the sequence of SEQ ID NO: 11 or 13;

(B) a polynucleotide comprising a sequence having high sequence homology to the sequence of SEQ ID NO: 11 or 13, and encoding a protein having the same activity as the protein comprising the corresponding sequence of SEQ ID NO: 12 or 14;

(C) A polynucleotide comprising a sequence in which one or more nucleotides are replaced, deleted, inserted and/or added to the sequence of SEQ ID NO: 11 or 13, and encoding a protein having the same activity as the protein comprising the corresponding sequence of SEQ ID NO: 12 or 14.

The polynucleotides containing the sequences recorded in SEQ ID NOs: 5, 7, 9, 11, and 13 correspond to the proteins containing the sequences recorded in SEQ ID NOs: 6, 8, 10, 12, and 14, respectively.

In the present invention, high sequence homology means a homology value of 70% or more, more preferably 80% or more, further preferably 90% or more, further preferably 95% or more, further preferably 98% or more.

Regarding the present invention, when being called homology (homology), except the situation that has special record, it refers to the percentage of the number of nucleotides consistent between two sequences when two sequences are arranged in the best mode. About the search and analysis of sequence homology, it can be carried out by those skilled in the art by known algorithms or programs, such as GENETIX (registered trademark) ver.14 (GENETIX Co., Ltd.), BLASTN, BLASTP, BLASTX, ClustalW. The parameters when using the program can be appropriately set by those skilled in the art, and the default parameters of each program can be used in addition. The specific methods of these analytical methods are also known to those skilled in the art.

It should be noted that, in the composition, as long as the function as the target is not impaired, lactic acid bacteria other than bacteria belonging to the genus Streptococcus may also be contained. In addition, the composition may contain microorganisms such as yeast other than the lactic acid bacteria as the target. In a preferred embodiment, the composition contains one or more lactic acid bacteria, but does not contain microorganisms other than lactic acid bacteria, such as yeast. In addition, in another preferred embodiment, the composition does not contain any one of Lactobacillus gasseri and Lactobacillus paragasseri or does not contain these two. In addition, in another preferred embodiment, the composition does not contain lactic acid bacteria other than bacteria belonging to the genus Streptococcus. In addition, in another preferred embodiment, the composition does not contain ingredients that promote the secretion of appetite stimulating hormones other than bacteria belonging to the genus Streptococcus.

The lactic acid bacteria (effective bacteria) as an active ingredient contained in the composition can be produced by culturing. There are no particular limitations on the culturing conditions as long as the target effect is achieved.

In the composition of the present invention, the effective bacteria can be contained in any state as long as they can exert the target effect. For example, in addition to the effective bacteria itself, the effective bacteria may also be a culture of the effective bacteria (including the effective bacteria and the culture supernatant). As long as the bacteria can exert the target effect, they can be in a living state (live bacteria) or in a dead state (dead bacteria).

The effective bacteria may be contained in the composition in the form of dead bacteria. The dead bacteria can be obtained by sterilizing the effective bacteria. The sterilization treatment is not particularly limited as long as it can exert the target effect, and heating, sterilization lamp (UV), ozone, drugs, high osmotic pressure, etc. can be used.

The dead bacteria are preferably heat-treated dead bacteria obtained by heat-treating live bacteria. The heat treatment used to obtain the heat-treated dead bacteria is not particularly limited as long as the target effect is achieved, and is implemented by a temperature and time sufficient to kill the effective bacteria used. Such conditions also depend on the effective bacteria used, and the heat treatment temperature is, for example, above 55°C, preferably above 60°C, more preferably above 65°C, and further preferably above 70°C. It can be set to above 80°C or above 90°C. The upper limit of the heat treatment temperature can be appropriately set, for example, below 121°C, below 100°C, below 90°C, or below 80°C. In addition, according to the heat treatment temperature, the heat treatment time can be set to more than 1 minute, can be set to more than 3 minutes, can be set to more than 10 minutes, more than 15 minutes, more than 30 minutes, or more than 45 minutes. The upper limit of the heat treatment time can be, for example, below 120 minutes, can be below 100 minutes, can be below 90 minutes, or can be below 80 minutes.

The effective bacteria to be included in the composition, whether live or dead, can be prepared in the form of a dry product, a suspension, a paste, a gel, or the like.

[use]

In one embodiment, the composition can be used to promote the secretion of ghrelin. Ghrelin is a peptide hormone that is mainly secreted in the stomach.

It is known that appetite stimulants have the effects of regulating the autonomic nerves (non-patent document 1), promoting growth hormone secretion (patent documents 1 and 2), improving loss of appetite (patent document 4), promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, promoting gastric acid secretion, promoting pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting myocardial cells (patent document 3). Therefore, in one embodiment, the composition can be used for any one selected from the group consisting of regulating the autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, promoting gastric acid secretion, promoting pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility, and protecting myocardial cells.

Whether a certain component promotes the secretion of ghrelin can be confirmed by various methods by those skilled in the art. For example, the evaluation can be performed as follows: the component is added to MKN45 cells (a human gastric cancer cell line) and other human gastric cells, and after a suitable period of time, the expression level of the ghrelin gene in the cells is measured to evaluate. The expression level of the ghrelin gene can be specifically measured as the expression level of mRNA. It is preferably corrected by the expression level of a gene expressed in a constitutive manner, such as β-actin.

[Composition]

(Food composition, etc.)

The composition of the present invention can be set as a food composition or a pharmaceutical composition. Except for the cases specifically recorded, food and medicine include not only food and medicine for humans, but also food and medicine for animals other than humans. Except for the cases specifically recorded, food includes general food, functional food, nutritional composition, and also includes therapeutic food (food that meets the purpose of treatment. Food cooked based on a menu prepared by a doctor, a dietitian, etc. according to the recipe.), diet therapy food, formula food, nursing food, and treatment support food. Except for the cases specifically recorded, food includes not only solids, but also liquid foods, such as beverages, drinking agents, liquid food and soup. Functional food refers to food that can give a specified functionality to an organism, including, for example, specific health foods (including special protection [specific health foods] with additional conditions), functional labeled foods, health functional foods including nutritional functional foods, special purpose foods, nutritional supplements, health supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquid preparations and other various dosage forms of food), beauty foods (for example, weight loss foods) and other all health foods. In addition, the "functional food" in the present invention includes health foods to which health claims based on food standards of the Codex (FAO/WHO Joint Food Standards Commission) are applied.

(Target)

In one embodiment, the composition is suitable for being taken by an object that needs or wishes to promote the secretion of appetite stimulating hormone. In addition, in one embodiment, the composition is suitable for being taken by an object that needs or wishes to be selected from the group consisting of regulating the autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, hypersecretion of gastric acid, hypersecretion of pancreatic exocrine, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells. The object can be healthy (for example, the stomach is healthy, does not suffer from a disease to be treated, does not receive a drug for treatment, such as a chemotherapeutic agent).

The above-mentioned subjects include adults (over 15 years old), middle-aged and elderly people, elderly people (over 65 years old (according to the definition of the World Health Organization (WHO))), people in the late stage of illness, pregnant women, parturients, infants and children. In addition, the active ingredient of the composition of the present invention is a fungus that has been eaten for a long time, so it is suitable for long-term intake, and is particularly suitable for subjects who want to promote the secretion of appetite stimulants in daily life.

(Giving method)

The composition of the present invention may be administered parenterally, for example, via a tube (gastrostomy fistula, enteral fistula), nasally, or orally, and is preferably administered orally.

(Content and dosage of active ingredients)

The content of effective bacteria in the composition of the present invention can be any amount that can exert the target effect. The content of effective bacteria in the composition can be appropriately set in consideration of various factors such as the age, weight, and symptoms of the ingestion object and the administration object. One unit of the composition can be set to the amount ingested/administered per day or per time (it can be administered/ingested once a day, or it can be administered multiple times a day, for example, 3 times).

In one embodiment, the effective bacterial content per unit of the composition can be, for example, 1×10 ⁵ or more, 1×10 ⁶ or more, 1×10 ⁷ or more, 1×10 ⁸ or more, 5×10 ⁸ or more, 5.7×10 ⁸ or more, 6×10 ⁸ or more, 7×10 ⁸ or more, 8×10 ⁸ or more, 9×10 ⁸ or more, 1×10 ⁹ or more, 5×10 ⁹ or more, preferably 1×10 ¹⁰ or more, more preferably 1×10 ¹¹ or more, and further preferably 2×10 ¹¹ or more. The upper limit can be appropriately set, and the lower limit in any case can be, for example, 1×10 ¹⁴ or less, 1×10 ¹³ or less, or 1×10 ¹² or less.

In one embodiment, the intake amount of effective bacteria can be set to 1×10 ⁵ cells/day or more, can be set to 1×10 ⁶ cells/day or more, can be set to 1×10 ⁷ cells/day or more, can be set to 1×10 ⁸ cells/day or more, can be set to 5×10 ⁸ cells/day or more, 5.7×10 ⁸ cells/day or more, 6×10 ⁸ cells/day or more, 7×10 ⁸ cells/day or more, 8×10 ⁸ cells/day or more, 9×10 ⁸ cells/day or more, 1×10 ⁹ cells/day or more, 5×10 ⁹ cells/day or more, preferably 1×10 ¹⁰ cells/day or more, more preferably 1.3×10 ¹⁰ cells/day or more, further preferably 2×10 ¹⁰ cells/day or more, 1×10 ¹¹ cells/day or more, 2×10 ¹¹ cells/day or more. The upper limit value can be appropriately set, and the lower limit value in any case can be, for example, 1×10 ¹⁴ pieces/day or less, 1×10 ¹³ pieces/day or less, or 1×10 ¹² pieces/day or less.

It should be noted that commercially available yogurt may contain about 5×10 ⁶ thermophilic Streptococci/g, and it has been calculated that 5 to 5.6×10 ⁸ thermophilic Streptococci are contained in one product.

In Test Example 2 of Patent Document 3, the amount of Streptococcus thermophilus in the appetite stimulating hormone secretion preparation powder containing fermented milk protein as an active ingredient administered to mice was calculated to be 6.6×10 ¹⁰ cells/16 g, and the amount of Streptococcus thermophilus ingested by mice was calculated to be 1.24×10 ¹⁰ cells/day.

In addition, when the content of effective bacteria is set as above, in any case, 1 unit can be set to, for example, 0.5 g or more, 1 g or more, 5 g or more, 10 g or more, 20 g or more, or 30 g or more. When the composition takes the form of a food such as fermented milk, 1 unit can be set to an amount that is easy to take as a food at one time, for example, 50 g or more, 60 g or more, 70 g or more, 80 g or more, 90 g or more, or 100 g or more. The upper limit can be set appropriately, and the lower limit can be set to 500 g or less in any case, 400 g or less, 300 g or less, 200 g or less, 150 g or less, or 125 g or less.

The composition has effective bacteria with rich dietary experience as an effective ingredient. Therefore, the composition of the present invention can be taken repeatedly or for a long time, for example, it can be taken/administered continuously for more than 3 days, preferably more than 1 week, more preferably more than 15 days, further preferably more than 3 weeks, more than 4 weeks, or more than 1 month.

(Other ingredients, additives)

The composition of the present invention can include other effective ingredients and nutrients that are acceptable as food or medicine. Examples of such ingredients are amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid and nicotinic acid), minerals (e.g., copper, zinc, iron, cobalt, manganese), antibiotics, dietary fiber, protein, lipids, etc.

In addition, the composition may further include additives acceptable as food or medicine. Examples of such additives include inactive carriers (solid, liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH regulators, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, water, other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevioside, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

(Dosage form and shape)

The food composition of the present invention can be made into any form such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule. In addition, the food composition of the present invention can be made into any form such as dairy products, supplements, cakes, beverages, beverages, flavorings, processed foods, side dishes, soups, etc. More specifically, the composition of the present invention can be made into fermented milk, lactic acid bacteria beverage, milk beverage, milk beverage, refreshing beverage, ice cream, tablets, cheese, bread, biscuits (biscuits), crackers (cracker), pizza crust, formula milk powder, liquid food, patient food, nutritious food, frozen food, processed food, etc., and can be made into forms such as granules, powders, pastes, concentrated solutions for mixing in beverages, foods and taking in. Fermented milk refers to the fermented milk and lactic acid bacteria beverage defined in Japan's "provincial order on the composition specifications of milk and dairy products (hereinafter referred to as "provincial order such as milk".)". Fermented milk in the Ministry of Milk and Others Ordinance refers to a paste or liquid substance obtained by fermenting milk or milk containing equal to or more non-fat milk solids with lactic acid bacteria or yeast, or a frozen product thereof.

The pharmaceutical composition of the present invention can be prepared into any dosage form suitable for oral administration, such as solid preparations such as tablets, granules, powders, pills, capsules, liquid preparations, suspensions, liquid preparations such as syrups, gels, aerosols, etc.

(other)

In the manufacture of the composition of the present invention, the compounding stage of effective bacteria can be appropriately selected. As long as the characteristics of the effective bacteria are not significantly damaged, the compounding stage is not particularly limited. For example, by mixing milk, dairy products and water, homogenizing as needed, sterilizing, inoculating effective bacteria and fermenting them, it is possible to manufacture a composition in the form of fermented milk containing a sufficient amount of effective bacteria. The milk and dairy products as raw materials can be appropriately selected according to the effective bacteria used in the fermentation. In a preferred embodiment, the milk and dairy products as raw materials include those of animal origin. In addition, the milk and dairy products as raw materials may not contain vegetable milk, such as soy milk, almond milk, oat milk, cocoa milk, rice milk, and hemp seed milk.

The composition may be labeled with the intended use (purpose), and may also be labeled with the content recommended for ingestion by a specific subject. The labeling may be direct or indirect, and examples of direct labeling include recording on tangible objects such as the product itself, packaging, containers, labels, and signs, and examples of indirect labeling include advertising/promotional activities based on places or means such as web pages, stores, brochures, exhibitions, media seminars, books, newspapers, magazines, television, radio, mail, email, audio, etc.

Examples of uses and functions indicated on the composition as a food include promoting the secretion of appetite stimulating hormone, regulating the autonomic nerves, promoting the secretion of growth hormone, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, increasing gastric acid secretion, increasing pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells.

[Example]

[Example 1]

MKN45 cells were cultured in 24-well plates with RPMI-1640 medium without antibiotics for 24 hours.

The strains of the thermophilic Streptococcus species shown in the table below were subjected to activation culture and main culture in an M17 medium containing 1% lactose for 20 hours.

The cultured bacteria were washed with PBS and suspended in a culture medium at OD660 = 5, and live bacteria were added to the plate at a final concentration of 1%. After incubation for 4 hours and 6 hours, the cells were washed 3 times with PBS, 200 μL of the reagent T/E solution of the Maxwell RSCsimplyRNA Cells Kit (Promega) and 200 μL of the lysis buffer were added, and the cells were recovered. After that, RNA was extracted and cDNA was converted, and the expression level of the appetite stimulating hormone gene was measured by qPCR (using the insertion method of SYBR Green). It was standardized with β-actin and analyzed by the ΔΔCt method as a comparative quantitative method.

【Table 1】

Strains used and culture conditions

Species name Storage institution number, etc. Culture medium temperature Aerobic/Anaerobic Streptococcus thermophilus OLS3289(NCIMB 8510 ^T ) M17 37℃ Anaerobic Streptococcus thermophilus 1131※ M17 37℃ Anaerobic Streptococcus thermophilus OLS3294(NITE BP-00077) M17 37℃ Anaerobic Streptococcus thermophilus OLS3615(NITE BP-01696) M17 37℃ Anaerobic

※It can be separated from Meiji Bulgarian Yogurt LB81 (Meiji Co., Ltd.).

The results are shown in Figure 1. Compared with the control group without live bacteria, the OLS3289 group, 1131 group, OLS3294 group, and OLS3615 group added with live bacteria all showed a significant increase in the expression of ghrelin mRNA 6 hours after addition. It should be noted that the effect of increasing the expression of ghrelin mRNA brought about by these four strains of Streptococcus thermophilus was higher than that of Lactobacillus paragasseri tested under the same conditions.

As mentioned above, all four strains belonging to thermophilic Streptococcus promote the expression of the appetite stimulating hormone gene, so it can be said that bacteria belonging to the genus Streptococcus, and further bacteria belonging to thermophilic Streptococcus, can be used to promote the secretion of appetite stimulating hormone. In addition, it is known that appetite stimulating hormone has the effects of regulating the autonomic nerves (non-patent document 1), promoting the secretion of growth hormone (patent documents 1, 2), improving anorexia (patent document 4), promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting digestive tract motility, causing hypersecretion of gastric acid, causing hypersecretion of pancreatic exocrine, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells (patent document 3). Therefore, it can be said that lactic acid bacteria belonging to the genus Streptococcus and further bacteria belonging to thermophilic Streptococcus can be used for any species selected from the group consisting of regulating the autonomic nervous system, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, hypersecretion of gastric acid, hypersecretion of pancreatic exocrine, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells.

[Gene analysis]

By analyzing the common sequence, the gene sequence common to the strains having the effect of promoting the secretion of appetite stimulating hormones is extracted.

In detail, the whole genome sequence of NCIMB 8510 ^T was obtained from the database of NCBI, and the whole genome sequences of other strains were obtained by genome draft analysis. The whole genome sequence of each strain was obtained in the form of a FASTA file (a file recording the whole genome sequence). For the FASTA file recording the whole genome sequence of each strain, DFAST (https://dfast.ddbj.nig.ac.jp/) was used for annotation, and a file recording the base sequence and amino acid sequence was prepared. Orthologs were extracted from the amino acid sequence information of strains having the effect of promoting the secretion of appetite stimulants, and the amino acid sequence possessed by at least one strain of the lactic acid strain that did not have the effect of promoting the secretion of appetite stimulants was removed, and the sequence obtained was determined as a "common gene possessed only by active strains". Regarding the common gene, the base sequence obtained by OLS3294 is shown as sequence numbers 5, 7 and 9 in the sequence table, and the corresponding amino acid sequence is shown as sequence numbers 6, 8, and 10 in the sequence table. In addition, the base sequence obtained from 1131 corresponding to sequence number 7 is shown in the sequence list as sequence number 11, the base sequence obtained from OLS3615 is shown in the sequence list as sequence number 13, and the corresponding amino acid sequences are shown in the sequence list as sequence numbers 12 and 14. It should be noted that the orthologs were grouped into the same ortholog group using Orthofinder (http://www.stevekellylab.com/software/orthofinder).

In addition, the nucleotide sequences obtained from strains other than the OLS3294 strain and the % homology of the corresponding amino acid sequences to the sequence of the OLS3294 strain are shown in the following table.

【Table 2】

[Production Example 1: Fermented milk]

Milk, dairy products (milk source) and water are mixed in such a manner that the non-fat milk solids of the final product are 9.5% and the milk fat component is 3.0%, thereby preparing a yogurt base mixture. The prepared yogurt base mixture is homogenized, heat sterilized at 95°C for 5 minutes, and then cooled to about 40°C. A mixed starter of bacteria of the genus Lactobacillus and thermophilic Streptococcus 1131 (Thermophilic Streptococcus OLS3615) is inoculated into the sterilized yogurt base mixture, and fermented until each thermophilic Streptococcus reaches 5×10 ⁹ or more per 100g, thereby manufacturing a yogurt for promoting the secretion of appetite stimulating hormone.

[Production Example 2: Supplement]

In skim milk powder medium, thermophilic streptococcus OLS3615 was cultured to the late stage of logarithmic growth phase, the culture solution was concentrated 25 times, and then heated at 60°C for 10 minutes. The concentrated heated bacterial solution was spray-dried to make it powder, and then mixed with dextrin at an addition rate of 3.9%, and mixed until completely uniform. The mixture was filled into a stick-shaped container in a manner of 1g/bag, thereby manufacturing a health food for promoting the secretion of appetite stimulating hormone. The number of thermophilic streptococcus OLS3615 in 1g was 2×10 ¹⁰ or more.

Industrial Applicability

The present invention can prevent or improve the poor physical condition caused by the loss of balance of the autonomic nerves by providing materials with high safety as a source of food ingredients or materials with confirmed safety to promote the secretion of appetite stimulants, etc., and can help people who are not sick to maintain/improve their health and make their work more efficient. According to the present invention, a food composition and a method for manufacturing food that help people maintain/improve their health and make their work more efficient can be provided. In addition, the present invention can achieve nutritional improvement for various groups of people, ensure healthy living, and promote well-being.

[Sequences listed in the sequence list]

Sequence number 1 Streptococcus thermophilus 1131, 16S rRNA gene

Sequence number 2 Streptococcus thermophilus NCIMB8510 ^T , 16S rRNA gene

Sequence number 3 Streptococcus thermophilus OLS3294, 16S rRNA gene

Sequence number 4 Streptococcus thermophilus OLS3615, 16S rRNA gene

SEQ ID NO: 5 DNA encoding acyl-CoA thioesterase (12170, OLS3294)

SEQ ID NO: 6 Acyl-CoA thioesterase (12170, OLS3294)

SEQ ID NO: 7 DNA encoding site-specific DNA methyltransferase (15340, OLS3294)

SEQ ID NO: 8 Site-specific DNA methyltransferase (15340, OLS3294)

Sequence number 9 DNA encoding histidine phosphatase family protein (8200, OLS3294)

SEQ ID NO: 10 Histidine phosphatase family protein (8200, OLS3294)

SEQ ID NO: 11 DNA encoding site-specific DNA methyltransferase (15340, 1131)

SEQ ID NO: 12 Site-specific DNA methyltransferase (15340, 1131)

SEQ ID NO: 13 DNA encoding site-specific DNA methyltransferase (15340, OLS3615)

Sequence number 14 site-specific DNA methyltransferase (15340, OLS3615).

Claims (9)

1. Use of a bacterium belonging to the genus Streptococcus (Streptococcus) for the manufacture of a composition for stimulating appetite stimulating hormone secretion.

2. The use according to claim 1, wherein the bacteria belonging to the genus streptococcus have any one selected from the group consisting of: a polynucleotide comprising the sequence of SEQ ID NO. 5 or an ortholog thereof (ortholog), a polynucleotide comprising the sequence of SEQ ID NO. 7 or an ortholog thereof, and a polynucleotide comprising the sequence of SEQ ID NO. 9 or an ortholog thereof.

3. Use of a bacterium belonging to the genus streptococcus for the manufacture of a composition for any one selected from the group consisting of regulating autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation or inhibiting weight loss, inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, enhancing gastric acid secretion, enhancing pancreatic exocrine secretion, inhibiting the sympathetic nervous system, dilating blood vessels, increasing cardiac contractility and protecting myocardial cells.

4. The use according to claim 3, wherein any one selected from the group consisting of regulating autonomic nerves, promoting growth hormone secretion, improving loss of appetite, promoting fat accumulation (inhibiting weight loss), inhibiting insulin secretion, inhibiting the function of brown adipose tissue, promoting gastrointestinal motility, hyperacidity, sympathetic nervous system inhibition, vasodilation, increasing cardiac contractility, and protecting myocardial cells is based on stimulating appetite secretion.

5. The use according to any one of claims 1 to 4, wherein the bacteria belonging to the genus streptococcus are bacteria belonging to the genus streptococcus (s.thermophilus).

6. The use according to any one of claims 1 to 4, wherein the bacteria belonging to the genus streptococcus are any one selected from the group consisting of streptococcus thermophilus NCIMB 8510 ^T, streptococcus thermophilus 1131, streptococcus thermophilus NITE BP-00077 and streptococcus thermophilus NITE BP-01696.

7. The use according to claim 1, wherein the composition is for administration of bacteria belonging to the genus streptococcus at 1.3 x 10 ¹⁰ or more per day.

8. Use according to claim 1, wherein the composition is in the form of a fermented milk free of bacteria belonging to the genus lactobacillus paracasei.

9. Use of bacteria belonging to the genus streptococcus for the purpose of non-disease treatment for promoting the secretion of appetite stimulating hormones.