JP7054111B2 - Lactic acid bacteria, hypoglycemic agents derived from the lactic acid bacteria, diabetes therapeutic agents, and foods and drinks - Google Patents

Description

NPMD NITE BP-02309

The present invention comprises a novel lactic acid bacterium, an α-glycosidase inhibitor and a hypoglycemic agent containing the novel lactic acid bacterium as an active ingredient, a live bacterium, a dead bacterium, or a treated product, and a diabetes preventive therapeutic agent and a food or drink containing the hypoglycemic agent. Regarding.

Currently, the onset of obesity due to excessive calorie intake and lifestyle-related diseases such as diabetes that develops thereafter has become a problem. It is said that it is very important to be careful not to raise the blood sugar level on a daily basis in order to suppress the onset of lifestyle-related diseases. Diet and exercise therapy are said to be effective in preventing lifestyle-related diseases. In the diet, the calorie intake is limited so that the postprandial blood glucose level does not rise.
However, it is often difficult for patients to continue such a diet, so the development of foods containing substances that suppress the rise in blood glucose levels is effective in implementing a continuous diet. It is believed that there is.

Sucrose is one of the major sweeteners added in various foods. Sucrose is broken down into glucose and fructose by α-glycosidase in the intestinal tract, and they are absorbed from the intestinal tract, leading to an increase in blood glucose level. Acarbose and voglibose, which are inhibitors of α-glycosidase, have an effect of inhibiting an increase in postprandial blood glucose level, and are used as therapeutic agents for diabetes. Therefore, foods containing substances having an inhibitory effect on α-glycosidase are expected to suppress an increase in postprandial blood glucose due to excessive feeding of sucrose.

Lactic acid bacteria are gram-positive bacteria that grow on MRS agar and produce lactic acid. Certain lactic acid bacteria grow in milk and are used in the production of yogurt. Acarbose, an α-glycosidase inhibitor, is produced by a gram-positive bacterium called Actinoplanes sp. SE 50/110 (Non-Patent Document 1). In addition, it has been reported that when Lactobacillus rhamnosus, which is a kind of lactic acid bacterium, is administered to mice fed with sucrose, the increase in blood glucose concentration after 1 hour is suppressed (Non-Patent Document 2). Furthermore, it has been reported that the heat-treated bacterial cell fraction of a certain lactic acid bacterium has an α-glycosidase inhibitory activity (Non-Patent Document 3).

In addition, conventionally, mammals such as mice and rats have been used for evaluation of lactic acid bacteria that suppress postprandial hyperglycemia. However, it has been pointed out that there are problems not only in terms of cost but also in terms of animal welfare when a large number of mammals are used for experiments.

Schwientek P et al., BMC Genomics, 2012 Honda K et al., J. Clin. Biochem. Nutr., 2012 Panwar H et al., Eur J Nutr, 2014

An object of the present invention is to provide a novel lactic acid bacterium having a high hypoglycemic effect, and further, a hypoglycemic agent containing the lactic acid bacterium or a killed bacterium or a treated product of the lactic acid bacterium as an active ingredient, the lactic acid bacterium or the lactic acid bacterium. It is an object of the present invention to provide a food or drink containing a hypoglycemic agent derived from.
Another object of the present invention is to provide a diabetes preventive and therapeutic drug containing a substance derived from a novel lactic acid bacterium as an active ingredient.

The present inventors have found a method for searching for a substance having α-glycosidase inhibitory activity using silk moth. Since it is possible to breed a large number of individuals in a narrow space compared to mammals such as mice and there are few ethical problems, by using an evaluation system using silk moth, hyperglycemia due to sucrose feeding It was found that a functional lactic acid bacterium having an effect of suppressing sucrose can be searched for.

In addition, the present inventors identified a lactic acid bacterium that suppresses hyperglycemia due to sucrose feeding by using an in vivo evaluation method using silk moth. Yogurt produced using the lactic acid bacterium strain showed an effect of suppressing an increase in blood glucose level in a human sucrose load test. In addition, as a result of evaluation using the above-mentioned evaluation method for silk moth, it was found that the lactic acid bacterium has an effect of suppressing postprandial hyperglycemia and reducing the onset of diabetes.

Furthermore, the above-mentioned lactic acid bacterium having a hypoglycemic effect is a novel lactic acid bacterium strain belonging to the genus Enterococcus (hereinafter, "lactic acid bacterium 0831-07" or "lactic acid bacterium #" as a result of analysis of its properties and analysis of the base sequence of 16S rDNA. It may be abbreviated as "Ef-1"), and the present invention has been completed.

That is, the present invention is a lactic acid bacterium belonging to the genus Enterococcus whose accession number is NITE BP-02309 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), or its natural or natural. It is an artificially mutated lactic acid bacterium and provides a lactic acid bacterium having a hypoglycemic effect.
Further, the present invention provides a lactic acid bacterium isolated or purified from the above lactic acid bacterium.

The present invention also provides the lactic acid bacterium, which has an α-glycosidase inhibitory activity and is used for suppressing an increase in blood glucose due to sucrose ingestion.

Further, the present invention is an α-glycosidase inhibitor containing the lactic acid bacterium, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the lactic acid bacterium is selected from the group consisting of a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and an extract from the culture of the lactic acid bacterium. It provides an α-glycosidase inhibitor characterized by being at least one treated product.

Further, the present invention is a hypoglycemic agent containing the above-mentioned lactic acid bacterium, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the lactic acid bacterium is a culture of lactic acid bacterium; a concentrate; a paste; a spray-dried product, a freeze-dried product, a vacuum-dried product, a dried product such as a drum-dried product; a liquid product; a diluted product; a crushed product; a sterilized processed product. ; And; To provide a hypoglycemic agent characterized by being at least one treated product selected from the group consisting of extracts from the culture.

The present invention also provides a diabetes preventive and therapeutic agent, which comprises the hypoglycemic agent as an active ingredient.

The present invention also provides a food or drink containing the above-mentioned lactic acid bacterium or the above-mentioned blood glucose lowering agent.

According to the present invention, it is possible to provide a novel lactic acid bacterium having an extremely high blood glucose lowering effect. In particular, it is possible to provide a novel lactic acid bacterium having an effect of suppressing an increase in blood glucose due to ingestion of sucrose.
Further, a novel hypoglycemic agent having an extremely high hypoglycemic effect containing the novel lactic acid bacterium, the dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient is provided, and the lactic acid bacterium or the hypoglycemic agent is contained. It is possible to provide foods and drinks having an excellent blood glucose lowering effect (derived from).

Further, according to the present invention, it is possible to provide a diabetes preventive and therapeutic agent containing the hypoglycemic agent as an active ingredient, and the diabetes preventive and therapeutic agent can prevent or treat diabetes by oral administration.

The lactic acid bacterium of the present invention can be used not only for the production of general foods and drinks, health foods, drugs, fermented foods and drinks, probiotics, etc., but also for the prevention and treatment of diseases by suppressing the rise in blood glucose. .. As fermented foods and drinks, it is particularly suitable for use as fermented milk, lactic acid bacteria beverages, yogurt, pickles, lactic acid bacteria starters for pickle production, and the like. In addition, because it is resistant to acid, it does not decompose in the stomach and reaches the small intestine.

It is a graph which shows the inhibitory effect of the increase of glucose concentration in the body fluid of silk moth by the feeding of the sucrose-containing feed by the addition of lactic acid bacteria. It is a graph which shows the inhibitory effect of the addition of the lactic acid bacterium # Ef-1 strain on the increase of glucose concentration in the body fluid of silk moth by feeding a sucrose-containing feed. It is a graph which shows the inhibitory effect of the addition of the lactic acid bacterium # Ef-1 strain on the increase of the glucose concentration in the body fluid of silk moth by feeding sucrose or glucose-containing feed. It is a graph which shows the inhibitory effect of the increase of the glucose concentration in the body fluid of the silk moth by the feeding of the sucrose-containing feed by the addition of the heat-treated cells of the lactic acid bacterium # Ef-1 strain. It is a graph which shows the inhibitory effect of the increase in the glucose concentration in humans in the sucrose load test by feeding yogurt prepared with the lactic acid bacterium # Ef-1 strain. It is a graph which shows the inhibitory effect of the increase of the glucose concentration in the body fluid of the silk moth by the feeding of the sucrose-containing feed (a) or the glucose-containing feed (b) by the addition of the killed lactic acid bacterium (# Ef-1 strain). (A) It is a figure which shows the experimental scheme of the sugar transfer evaluation system in the silk moth intestinal tract in vitro. (B) It is a graph which shows the inhibitory effect of acarbose on the sucrose transport from the excised intestinal tract to the outside of the intestinal tract. It is a graph which shows the inhibitory effect of sucrose transport from the excised intestinal tract to the outside of the intestinal tract by the addition of lactic acid bacteria (# Ef-1 strain). (A) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition of lactic acid bacteria (# Ef-1 strain). (B) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition amount of lactic acid bacteria (# Ef-1 strain). (C) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition of the heat-treated bacterium of lactic acid bacterium (# Ef-1 strain). (D) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition amount of the heat-treated cells of lactic acid bacteria (# Ef-1 strain). It is a graph which shows the inhibitory effect of glucose transport by the addition of a viable lactic acid bacterium (# Ef-1 strain). (A) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition of viable lactic acid bacterium (# Ef-1 strain). (B) It is a graph which shows the inhibitory effect of the increase in glucose concentration in the extraintestinal fluid of silk moth by the addition of the heat-treated bacterium of lactic acid bacterium (# Ef-1 strain). (A) It is a graph which shows the α-glycosidase activity inhibitory effect of the silk moth intestinal tract by the addition of lactic acid bacteria (# Ef-1 strain). (B) It is a graph which shows the α-glycosidase activity inhibitory effect of a rat intestinal tract by addition of a lactic acid bacterium (# Ef-1 strain). (A) It is a graph which shows the result of having measured the uptake of fluorescence in the Caco-2 cell which added the viable lactic acid bacterium (# Ef-1 strain). (B) It is a graph which shows the result of having measured the uptake of fluorescence in Caco-2 cells by adding the viable lactic acid bacterium (# Ef-1 strain) and the heat-treated bacterial cell fraction. (C) It is a schematic diagram which shows the postprandial hyperglycemia suppressing effect by a lactic acid bacterium (# Ef-1 strain). It is a schematic diagram which shows the schedule of the sucrose tolerance test in human by feeding of lactic acid bacterium (# Ef-1 strain). It is a graph which showed the blood glucose level in a viable cell suspension ingestion group, a heat-treated cell suspension ingestion group, and a non-ingestion group for each time after sucrose loading. It is a graph which shows the result of the sucrose addition test in a viable cell suspension ingestion group, a heat-treated cell suspension ingestion group, and a non-ingestion group.

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of the technical idea.

<Lactic acid bacteria>
The present invention is a lactic acid bacterium belonging to the genus Enterococcus whose accession number is NITE BP-02309 at the Patented Microbial Deposit Center (NPMD) of the National Institute of Technology and Evaluation (NITE), or a natural or artificial lactic acid bacterium thereof. It is a lactic acid bacterium mutated in the above and has a hypoglycemic effect.

Hereinafter, a novel lactic acid bacterium strain Enterococcus faecalis 0831-07 (hereinafter, may be abbreviated as “lactic acid bacterium 0831-07”) belonging to the genus Enterococcus will be described in detail.
The lactic acid bacterium 0831-07 of the present invention was isolated for the first time using centipede (Chilopod) as an isolation source.

Gram stain result: Positive Bacterial shape: Spherical Aerobic / anaerobic: Anaerobic Lactic acid-producing ability: Yes

Physiological properties: The physiological and chemical taxonomic properties of the lactic acid bacterium 0831-07 of the present invention are as follows.
(1) Catalase:-
(2) Acid phosphatase: +
(3) Alkaline phosphatase: +
(4) Naphthol-AS-BI-phosphohydrolase: +
(5) Esterase (C4): +
(6) α-galactosidase:-
(7) Esterase lipase (C8): +
(8) β-galactosidase:-
(9) Lipase (C14):-
(10) β-glucuronidase:-
(11) Leucine allyl amidase: +
(12) α-Glucosidase: +
(13) Valine allyl amidase:-
(14) β-Glucosidase:-
(15) Cystine allyl amidase:-
(16) N-Acetyl-β-Glucosaminidase:-
(17) Trypsin:-
(18) α-Mannosidase:-
(19) α-chymotrypsin: +
(20) α-fucosidase:-

(21) Ability to generate acid and gas from the following sugars, etc. Glycerol: +
Erythritol:-
D-Arabinose:-
L-Arabinose:-
D-Ribose: +
D-Xylose:-
L-Xylose:-
D-Adonitol:-
β-Methyl-D-xylopyranoside:-
D-Galactose: +
D-Glucose: +
D-Fructose: +
D-Mannose: +
L-Sorbose:-
L-Rhamnose:-
Dulcitol:-
Inositol: +
D-Mannitol: +
D-Sorbitol: +
α-Methyl-D-mannopyranoside:-
α-Methyl-D-glucopyranoside:-
N-Acetyl glucosamine: +
Amygdalin: +
Arbutin: +
Esculin ferric citrate: +
Salicin: +
D-Cellobiose: +
D-Maltose: +
D-Lactose:-
D-Melibiose: +
D-Sucrose: +
D-Trehalose: +
Insulin:-
D-Melezitose: +
D-Raffinose:-
Starch:-
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Turanose: +
D-Lyxose:-
D-Tagatose: +
D-Fucose:-
L-Fucose:-
D-Arabitol:-
L-Arabitol:-
Gluconate: +
2-Keto-gluconate:-
5-Keto-gluconate:-

Molecular Biological Analysis Results: The analysis results of lactic acid bacterium 0831-07 regarding 16SrDNA used as an index of molecular biological phylogenetic classification are as shown in the attached sequence sheet.
That is, as a result of amplifying the base sequence of the 16SrDNA region by PCR from the genomic DNA of lactic acid bacterium 0831-07 and analyzing it with a sequencer, a base sequence corresponding to almost the entire length of 16SrDNA was found.
When this nucleotide sequence was subjected to a homology search by BLAST analysis of NCBI, the nucleotide sequence of the 16SrDNA region of lactic acid bacterium 0831-07 was homologous to the nucleotide sequence of Enterococcus faecalis V583 strain (registration number: NR_074637.1) of the genus Enterococcus. Lactic acid bacterium 0831-07 belongs to Enterococcus faecalis because it showed 99% sex.
However, the lactic acid bacterium 0831-07 of the present invention is a lactic acid bacterium strain different from the above-mentioned strain because it does not completely match even when only the 16SrDNA regions are compared.

The physiological and chemical taxonomic properties of the above-mentioned lactic acid bacterium 0831-07 are compared with the classification by Bergey's Manual of Systematic Bacteriology (vol.3 1989) and the contents of other literatures. Further, as a result of determination in consideration of the result of the above 16S rDNA analysis, the "lactic acid bacterium 0831-07" of the present invention is a novel microorganism belonging to the genus Enterococcus.
In addition, there is no microorganism having a base sequence of 16SrDNA region that matches the base sequence of 16SrDNA region of lactic acid bacterium 0831-07, and it shows a higher hypoglycemic effect than known strains belonging to the genus Enterococcus. As a result of the above examination, it was determined that the lactic acid bacterium 0831-07 was a novel isolated microbial strain.

Lactobacillus 0831-07 is a patented microorganism deposit of the Natural Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture. It is a microorganism that has been deposited domestically at the Center (NPMD) and has been deposited under the deposit number: NITE P-02309 (deposit date: July 26, 2016).
Lactobacillus 0831-07 subsequently submitted the original deposit application to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 2-5-8 122 Kazusakamatari, Kisarazu City, Chiba Prefecture. Then, an application for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty was submitted (transfer date (international deposit date): May 16, 2017), and survival was proved. As a result of receiving the transfer application to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02309" has been received.

As a general property of a bacterium, its properties as a strain are liable to change, so that lactic acid bacterium 0831-07 may not stay within the range of the physiological properties shown above. It goes without saying that such "mutations" include both natural and artificial mutations.

The method for culturing lactic acid bacteria 0831-07 will be described below. The method for culturing lactic acid bacteria 0831-07 may be carried out according to a general culture method for microorganisms of the genus Enterococcus.
Culturing is preferably carried out under anaerobic conditions. Examples of carbon sources in the medium include D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amigdalin, arbutin, esculin, salicin, and D-cellobiose. , D-maltose, schucrose, D-trehalose, gentiobiose, sugar syrup, water candy, oils and fats and other organic carbon compounds are used, and the nitrogen source is meat extract, casein, peptone, yeast extract, dried yeast, germ, soybean. Organic / inorganic nitrogen compounds such as powder, urea, amino acids, and ammonium salts can be used.
As the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt are appropriately added as needed. Further, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil and the like in terms of increasing the production amount of the target product.
Further, a defoaming agent such as silicone oil or a surfactant may be added. As the prepared medium, for example, MRS medium, GAM medium and the like are preferably used.

As described above, the culture conditions may be the same as the general culture conditions performed for the microorganisms of the genus Enterococcus. If it is a liquid culture method, static culture is desirable. If the scale is small, a static culture method using a glass bottle with a lid may be used.
The culture temperature is preferably maintained between 25 ° C. and 43 ° C., and more preferably 30 ° C. to 42 ° C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, the culture temperature, etc., but in the case of lactic acid bacteria 0831-07, a sufficient amount of the target substance is secured in preferably 12 to 72 hours, more preferably 24 to 48 hours. can do.
It is also preferable to pick up the colonies obtained by culturing and perform single colonization on the medium again.

The novel lactic acid bacterium 0831-07 of the present invention is preferably a lactic acid bacterium used for suppressing an increase in blood glucose due to ingestion of sucrose by having an α-glycosidase inhibitory activity.

<Α-Glucosidase inhibitor>
The novel lactic acid bacterium 0831-07 of the present invention has an α-glucosidase inhibitory activity as the lactic acid bacterium itself and as a naturally or artificially mutated lactic acid bacterium of the lactic acid bacterium.

"Lactic acid bacterium 0831-07 or a naturally or artificially mutated lactic acid bacterium", "killed lactic acid bacterium", and "treated product of the lactic acid bacterium" all have α-glucosidase inhibitory activity.
Here, the "processed product of lactic acid bacteria" comprises a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and an extract from the culture. At least one processed product selected from the group may be mentioned. Here, examples of the "dried product" include a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and the like.
Another aspect of the present invention is "an α having the above-mentioned lactic acid bacterium of the present invention (lactic acid bacterium 0831-07 or a naturally or artificially mutated lactic acid bacterium), a killed lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient. -A glucosidase inhibitor, the treated product of the lactic acid bacterium is an extraction of the lactic acid bacterium from a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and a culture. It is an α-glucosidase inhibitor characterized by being at least one treated product selected from the group consisting of products.

The α-glucosidase inhibitor of the present invention can contain the above-mentioned lactic acid bacterium of the present invention, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium in various states. For example, a state containing a suspension, a lactic acid bacterium cell, a culture supernatant, a medium component, and the like can be mentioned.
As the lactic acid bacteria, live cells, wet bacteria, dried bacteria and the like can be appropriately used. Further, it may be a killed bacterium that has been sterilized, that is, has undergone heat sterilization treatment, radiation sterilization treatment, crushing treatment, or the like.

The content of lactic acid bacteria, killed bacteria of the lactic acid bacteria, and treated products of the lactic acid bacteria, which are the active ingredients in the α-glucosidase inhibitor of the present invention, with respect to the entire α-glucosidase inhibitor is not particularly limited and may be used according to the purpose. It can be appropriately selected, but when the total amount of the hypoglycemic agent is 100 parts by mass, it is contained in 0.001 to 100 parts by mass as "the total amount of lactic acid bacteria, killed bacteria of the lactic acid bacteria, and processed product of the lactic acid bacteria". It is preferably contained in an amount of 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and further preferably 1 to 90 parts by mass.

In addition, any one of the active ingredients may be used alone, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each active ingredient in the α-glucosidase inhibitor is not particularly limited and can be appropriately selected depending on the intended purpose.

The α-glucosidase inhibitor of the present invention contains the lactic acid bacterium, the killed lactic acid bacterium, or the treated product of the lactic acid bacterium as an active ingredient, and may contain "other ingredients" in addition to the active ingredient. ..
The "other components" of the α-glucosidase inhibitor are not particularly limited and can be appropriately selected depending on the intended purpose within the range not impairing the effect of the present invention, and are pharmaceutically acceptable, for example. Examples thereof include carriers that can be used.
The carrier is not particularly limited and may be appropriately selected depending on, for example, a dosage form described later. Further, the content of "other components" in the α-glucosidase inhibitor is not particularly limited and can be appropriately selected depending on the intended purpose.

<Blood sugar lowering agent>
The novel lactic acid bacterium 0831-07 of the present invention has a blood glucose lowering effect as the lactic acid bacterium itself and as a naturally or artificially mutated lactic acid bacterium of the lactic acid bacterium.

"Lactic acid bacterium 0831-07 or a naturally or artificially mutated lactic acid bacterium", "killed lactic acid bacterium", and "treated product of the lactic acid bacterium" all have a blood glucose lowering effect.
Here, the "processed product of lactic acid bacteria" comprises a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and an extract from the culture. At least one processed product selected from the group may be mentioned. Here, examples of the "dried product" include a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and the like.
Another aspect of the present invention is "a blood glucose containing the above-mentioned lactic acid bacterium of the present invention (lactic acid bacterium 0831-07 or a naturally or artificially mutated lactic acid bacterium), a killed lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient. A lowering agent, the treated product of the lactic acid bacterium is obtained from a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, a sterilized processed product, and an extract from the culture of the lactic acid bacterium. It is a hypoglycemic agent characterized by being at least one treated product selected from the group.

The hypoglycemic agent of the present invention can contain the lactic acid bacterium of the present invention, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium in various states. For example, a state containing a suspension, a lactic acid bacterium cell, a culture supernatant, a medium component, and the like can be mentioned.
As the lactic acid bacteria, live cells, wet bacteria, dried bacteria and the like can be appropriately used. Further, it may be a killed bacterium that has been sterilized, that is, has undergone heat sterilization treatment, radiation sterilization treatment, crushing treatment, or the like.

The content of lactic acid bacteria, dead bacteria of the lactic acid bacteria, and the treated product of the lactic acid bacteria, which are the active ingredients in the hypoglycemic agent of the present invention, with respect to the entire hypoglycemic agent is not particularly limited and may be appropriately selected according to the purpose. However, when the total amount of the hypoglycemic agent is 100 parts by mass, it may be contained in 0.001 to 100 parts by mass as "the total amount of lactic acid bacteria, dead bacteria of the lactic acid bacteria, and processed product of the lactic acid bacteria". It is preferably contained in an amount of 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and even more preferably 1 to 90 parts by mass.

In addition, any one of the active ingredients may be used alone, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each active ingredient in the hypoglycemic agent is not particularly limited and can be appropriately selected according to the purpose.

The hypoglycemic agent of the present invention contains the lactic acid bacterium, the killed lactic acid bacterium, or the treated product of the lactic acid bacterium as an active ingredient, and may contain "other ingredients" in addition to the active ingredient.
The "other components" in the hypoglycemic agent are not particularly limited and can be appropriately selected depending on the intended purpose within a range that does not impair the effects of the present invention, and are pharmaceutically acceptable, for example. Examples include carriers.
The carrier is not particularly limited and may be appropriately selected depending on, for example, a dosage form described later. Further, the content of "other components" in the blood glucose lowering agent is not particularly limited and can be appropriately selected depending on the purpose.

<Diabetes preventive and therapeutic drug>
The lactic acid bacterium of the present invention, the killed lactic acid bacterium, and the treated product of the lactic acid bacterium are useful as a preventive and therapeutic agent for diabetes because they have or have a hypoglycemic effect, and are particularly useful as a preventive and therapeutic agent for diabetes for oral administration. It is useful.
Therefore, another aspect of the present invention is a diabetes preventive and therapeutic agent, which comprises the above-mentioned hypoglycemic agent as an active ingredient.

<Pharmaceuticals; Food and drink; Health foods, etc.>
The lactic acid bacterium of the present invention and the hypoglycemic agent of the present invention derived from the lactic acid bacterium can be blended in foods and drinks having specifications such as pharmaceuticals, quasi-drugs, general foods and drinks, health foods, and powdered milk. It can be applied to various medicines, foods and drinks regardless of their forms.
Above all, foods and drinks produced by using the above-mentioned step of fermenting with the lactic acid bacterium of the present invention, and among them, fermented milk is easy to exert the usual effect of the lactic acid bacterium and the above-mentioned effect peculiar to the present invention. preferable.

The dosage form of the α-glucosidase inhibitor, hypoglycemic agent, or diabetes preventive and therapeutic agent of the present invention is not particularly limited, and can be appropriately selected, for example, according to a desired administration method as described later.
Specifically, for example, oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), injections (solvents, suspensions, etc.) Agents, etc.), ointments, patches, gels, creams, external powders, sprays, inhalation sprays, etc.

As the oral solid preparation, for example, an excipient and, if necessary, an additive such as a binder, a disintegrant, a lubricant, a colorant, a flavoring / flavoring agent, etc. are added to the active ingredient, and a conventional method is used. Can be manufactured by

Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinylpyrrolidone and the like. Be done.
Examples of the disintegrant include dried starch, sodium alginate, canten powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearate monoglyceride, lactose and the like.
Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like.
Examples of the colorant include titanium oxide, iron oxide and the like.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

As the oral solution, for example, it can be produced by a conventional method by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to the active ingredient.

Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include tragant, gum arabic, gelatin and the like.

As the injection, for example, a pH regulator, a buffer, a stabilizer, an tonicity agent, a local anesthetic, etc. are added to the active ingredient, and the injection is subcutaneously, intramuscularly, or intravenously used by a conventional method. Etc. can be produced.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium metabisulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.

As the ointment, for example, a known base, stabilizer, wetting agent, preservative and the like can be blended with the active ingredient and mixed by a conventional method to produce the ointment.
Examples of the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.

As the patch, for example, a cream, a gel, a paste, or the like as the ointment can be applied to a known support by a conventional method to produce the patch. Examples of the support include cotton, rayon, woven fabric made of chemical fibers, non-woven fabric, soft vinyl chloride, polyethylene, polypropylene, polyurethane and other films, foam sheets and the like.

The α-glucosidase inhibitor, hypoglycemic agent, and diabetes preventive and therapeutic agent of the present invention can be suitably used, for example, for individuals who require hypoglycemic activity, individuals who seek acquired immunity against bacteria, and the like.
Specifically, for example, by administering to individuals who need to maintain their health or recover from fatigue; individuals who need prevention or treatment of cancer or lifestyle-related diseases; individuals infected with bacteria, fungi, viruses, etc. Can be used.

The target animal to which the α-glucosidase inhibitor, hypoglycemic agent, or diabetes preventive treatment agent of the present invention is administered is not particularly limited, but for example, humans; experimental animals such as mice and rats; monkeys; horses; cows, pigs, and the like. Livestock such as goats and chickens; pets such as cats and dogs; and the like.

The method for administering the α-glucosidase inhibitor, the hypoglycemic agent or the diabetes preventive and therapeutic agent is not particularly limited, and can be appropriately selected depending on, for example, the above-mentioned dosage form, oral administration, and peritoneal administration. Examples include oral administration, injection into blood, and injection into the intestine. Of these, oral administration is preferable because it is simple and exerts the above-mentioned effects.

The dose of the α-glucosidase inhibitor, the hypoglycemic agent or the diabetes preventive and therapeutic agent is not particularly limited, and may be appropriately selected according to the age, body weight, degree of desired effect, etc. of the individual to be administered. However, for example, the daily dose to an adult is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g as the amount of the active ingredient.
Further, the administration time is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may be administered prophylactically or therapeutically.

Lactic acid bacteria, hypoglycemic agents, or foods and drinks containing the lactic acid bacteria of the present invention, killed or treated products of the lactic acid bacteria, or foods and drinks containing a hypoglycemic agent (hereinafter, may be abbreviated as "food and drink of the present invention"). The content of the diabetes preventive and therapeutic agent is not particularly limited and can be appropriately selected depending on the purpose and the mode (type) of the food or drink. However, when the total amount of the food or drink is 100 parts by mass, the above total The content is preferably 0.001 to 100 parts by mass, more preferably 0.01 to 100 parts by mass, and particularly preferably 0.1 to 100 parts by mass.

Further, any one of the above may be used alone, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each substance in the food or drink is not particularly limited and can be appropriately selected according to the purpose.

The food and drink of the present invention has a blood glucose lowering effect.
The food or drink of the present invention may further contain "other components" in addition to the above-mentioned lactic acid bacteria and blood glucose lowering agent of the present invention.

The above-mentioned "other ingredients" are not particularly limited and may be appropriately selected depending on the intended purpose within the range not impairing the effect of the present invention, and examples thereof include various food raw materials. The content of the "other components" is not particularly limited and may be appropriately selected depending on the intended purpose.

The food and drink of the present invention includes drinks and foods ingested by humans. The food and drink of the present invention also includes beverages for ingestion by livestock, poultry, pets and the like.

The lactic acid bacterium of the present invention can be used for the production of general foods and drinks, health foods, drugs, fermented foods and drinks, probiotics and the like. As fermented foods and drinks, it is particularly suitable for use as fermented milk, lactic acid bacteria beverages, yogurt, pickles, lactic acid bacteria starters for pickle production, and the like.

The type of the food or drink is not particularly limited and may be appropriately selected depending on the purpose. For example, confectioneries such as jelly, candy, chocolate, biscuits, and gummy; green tea, black tea, coffee, soft drinks, etc. Favorite beverages; dairy products such as fermented milk, yogurt, ice cream, lacto ice; vegetable beverages, fruit beverages, processed vegetables and fruits such as jams; liquid foods such as soups; processed grain products such as breads and noodles; Seasoning; etc. Among them, dairy products such as yogurt and fermented milk are preferable.
The method for producing these foods and drinks is not particularly limited, and for example, they can be appropriately produced according to the usual methods for producing various foods and drinks.

Further, the food or drink may be manufactured as an oral solid preparation such as tablets, granules or capsules, or an oral liquid preparation such as an internal liquid preparation or a syrup preparation. The method for producing the oral solid preparation and the oral liquid preparation is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the oral solid preparation and the oral liquid preparation can be manufactured according to the above-mentioned method for producing the oral solid preparation and the oral liquid preparation. can.

The food and drink of the present invention is particularly useful as a functional food, a health food, etc. for the purpose of activating the blood glucose lowering mechanism and preventing diabetes.
When the lactic acid bacterium of the present invention, the dead bacterium, the treated product, or the like is used for producing a food or drink, the production method can be carried out by a method well known to those skilled in the art. If you are a person skilled in the art, a step of mixing the (dead) cell or processed product of the lactic acid bacterium of the present invention with other components, a molding step, a sterilization step, a fermentation step, a baking step, a drying step, a cooling step, a granulation step, It is possible to make the desired food and drink by appropriately combining the packaging processes and the like.

Further, when the lactic acid bacterium of the present invention is used for producing various fermented milks, it can be produced by a method well known to those skilled in the art. For example, foods and drinks produced by using the step of adding a required amount of the lactic acid bacterium of the present invention to fermented milk as a dead bacterium, and food and drink manufactured by using the step of fermenting the lactic acid bacterium of the present invention as a lactic acid bacterium starter. Can be mentioned.
When fermentation is carried out using the lactic acid bacterium of the present invention as the lactic acid bacterium starter, it can be carried out under the same conditions as the culture conditions of the lactic acid bacterium of the present invention.

Hereinafter, the present invention will be described in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the following Examples and the like. “%” Indicates “mass%” unless otherwise specified.

<Materials and methods>
<< Deposit >>
As described above, the "lactic acid bacterium 0831-07" used in the examples is isolated from the centipede (Chilopod).
Lactic acid bacterium 0831-07 belonging to Enterococcus faecalis is a patented microorganism deposit center (NPMD) of the National Institute of Technology and Evaluation (NITE) (Room 2-5-8 122, Kazusakamatari, Kisarazu City, Chiba Prefecture). (Deposit number: NITE P-02309, deposit date July 26, 2016).
Lactobacillus 0831-07 subsequently submitted the original deposit application to the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 2-5-8 122 Kazusakamatari, Kisarazu City, Chiba Prefecture. Then, an application for transfer from domestic deposit (original deposit date: July 26, 2016) to deposit based on the Butapest Treaty was submitted (transfer date (international deposit date): May 16, 2017), and survival was proved. As a result of receiving the transfer application to the deposit (international deposit) based on the Butapest Treaty, the deposit number "NITE BP-02309" has been received.

<< Types of silk moths, breeding conditions and injection conditions >>
Fertilized eggs of silkworm (hybrid fu, yo x Tsukuba, ne) were purchased from Ehime Yoseki Co., Ltd. The hatched larvae were fed with artificial feed Silkmate 2S (Nosan Corporation) at room temperature and raised to the 5th instar larvae. For the breeding container, a square type No. 2 petri dish (Eiken equipment) was used for the eggs to the second instar larvae, and a disposable plastic food pack (Food Pack FD Oofuka, Chuo Kagaku Co., Ltd.) was used for the rest. The breeding temperature was 27 ° C. Unless otherwise stated, larvae of the 5th instar and 1st day fasted after the 4th instar sleep were used in the experiment.

<< Culture of lactic acid bacteria >>
Lactic acid bacteria were cultured under anaerobic conditions on MRS agar medium containing 0.5% calcium carbonate. A zona pellucida was formed around the colonies on the MRS agar culture medium, and gram-positive bacteria were determined to be lactic acid bacteria. Species were identified for the isolated lactic acid bacteria by a sequence of genes encoding rRNA. The growth of lactic acid bacteria in a liquid medium was carried out at 30 ° C. for 1 to 3 days under static conditions.

<< Lactic acid bacteria glucose metabolism test >>
The glucose metabolism ability of the lactic acid bacterium 0831-07 strain was measured using an API 50 CH kit (Sysmex Corporation). Colonies of the lactic acid bacterium 0831-07 strain were suspended in Suspension Medium (Sysmex Corporation) and prepared to have McFarland turbidity 2. 150 μL of the prepared bacterial solution sample was added to an Api plate (Sysmex Corporation), and the cells were cultured at 30 ° C. for 48 hours. After culturing, the metabolic capacity for various sugars was determined by observing the reacted colors.

<< Blood glucose quantification method >>
The body fluid of the silk moth was collected by cutting the first abdominal limb with scissors. Glucose concentration in silk moth blood was quantified by a glucometer (Accu-Chek, Roche).

<< Manufacturing of yogurt >>
0.1 g of cells of the lactic acid bacterium 0831-07 strain grown on MRS agar medium was scraped off with Aze and suspended in 5 mL of 0.9% NaCl. 5 mL of the bacterial suspension was added to 1 L of milk (Meiji delicious milk, carton box) and stirred. The cells were cultured at 43 ° C. for 20 hours. After confirming gelation, it was stored at 4 ° C. The storage period was 3 weeks or less.

<< Clinical trials using humans >>
A glucose tolerance test was performed on the subjects using a 50% (w / v) sucrose aqueous solution before and after ingestion of yogurt, and transition data of each blood glucose level was collected. Ten subjects were divided into two groups and randomly performed in an open-label, two-group, two-stage crossover method. The criteria for selecting the subjects are as follows.

<<< Selection Criteria for Healthy Persons in Human Clinical Trials >>>
Those who meet all of the following criteria were targeted.
(1) Persons aged between 20 and 60 years old at the time of consent acquisition (2) Healthy adult men and women (3) Prior to participating in the test, receive sufficient explanation about the test product and test, and the subject's free will Person who obtained document consent

<<< Exclusion Criteria >>
Those who violate the following will not be included in the test.
(1) Those who have been diagnosed with diabetes by a doctor and are currently being treated (2) Those who are allergic to dairy products (3) Those who are taking medicines or health foods that may affect blood glucose (4) Those who have chronic diseases and regularly use medicines (5) Those who have had their stomach or intestines removed (6) Others who are responsible for the study (sharing) have judged that they are not suitable as subjects for the study. person

<<< Restrictions >>>
(1) Supper on the day before the inspection will be finished from 19:00 to 23:00, and after that, fast until the end of the inspection. Drinking water (water only) is allowed.
(2) Excessive alcohol intake on the day before the test is prohibited.
(3) Drinking water is prohibited from 1 hour before the sugar load to the end of the test.
(4) On the day of the inspection, smoking is prohibited from waking up to the end of the inspection.
(5) Exercise is prohibited during the inspection. Stay calm in the day room or on the bed.

A sucrose tolerance test with or without yogurt was carried out at intervals of 2 days or more. The time of sucrose loading in each subject was set to be the same. It was collected from the fingertips of the subject's hand using a piercing device. The blood glucose level of the subject was measured 15 minutes before the sucrose load. The yogurt was ingested 10 minutes before the sucrose load. 200 mL was ingested within 2 minutes. Then, 150 mL of a 50% (w / v) sucrose aqueous solution was drunk (the whole amount was ingested within 1 minute). Blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 minutes after sucrose loading. The blood glucose level was measured using a simple blood glucose meter (Accucheck Aviva (Roche)). The clinical trial was approved by the Institutional Review Board of Osaki Hospital Tokyo Heart Center.

<< Statistical analysis >>
Numerical data are presented as mean ± standard error of the mean (SEM). Significant differences were assessed using Student's t-test.

Example 1
<Identification of lactic acid bacteria that suppress the increase in glucose concentration in the body fluid of silk moth due to sucrose feeding>
Previous studies have shown that lactic acid bacteria with α-glycosidase inhibitory activity suppress the increase in glucose concentration in body fluids due to sucrose ingestion in silk moth (International Application No. PCT / JP2016 / 079218, Matsumoto Y et al). ., Sci Rep., 2016).
Therefore, from the lactic acid bacteria library (Table 1) possessed by the inventors, we searched for lactic acid bacteria that significantly inhibit the increase in glucose concentration in the body fluid of silk moth due to feeding of sucrose.

The silk moths on the 1st day of the 5th instar were fed with a 10% sucrose diet containing various lactic acid bacteria (25% of the total diet) in Table 1 for 1 hour. After that, the body fluid of the silk moth was collected, the glucose concentration in the body fluid was measured, and a significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 1, the vertical axis shows the glucose concentration (mg / dL). “Control” on the horizontal axis shows the results when the silk moth was fed a 10% sucrose diet containing no lactic acid bacteria. * Is P <0.05, and error bars indicate standard error (SEM). It was performed at n = 3 per group.

As a result of FIG. 1, the Enterococcus faecalis 0831-07 (Enterococcus faecalis # Ef-1) strain markedly suppressed the increase in blood glucose level of silk moth due to feeding with a 10% sucrose diet.
In the present specification and drawings, Enterococcus faecalis 0831-07 may be abbreviated as "lactic acid bacterium 0831-07" or "lactic acid bacterium # Ef-1".

Next, the silk moths on the first day of the 5th instar were fed a diet containing a 10 mass% sucrose diet supplemented with a lactic acid bacterium 0831-07 strain (0 to 50 mass% with respect to the total diet) for 1 hour. After that, the body fluid of the silk moth was collected, and the glucose concentration in the body fluid was measured. The results are shown in FIG.
In FIG. 2, the vertical axis shows the glucose concentration (mg / dL), and the horizontal axis shows the content (mass%) of the lactic acid bacterium 0831-07 strain. ** is P <0.01 and error bars indicate standard error (SEM). It was performed at n = 5 per group.

As a result of FIG. 2, the effect of suppressing the increase in blood glucose by the lactic acid bacterium 0831-07 strain was dependent on the amount of cells added to the diet.

Next, on the first day of the 5th instar, a 10 mass% sucrose diet or "a diet obtained by adding a lactic acid bacterium 0831-07 strain (25 mass% to the total diet) to a 10 mass% glucose diet" for 1 hour. Gave. The body fluid of the silk moth was collected and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 3, the vertical axis shows the glucose concentration (mg / dL). “+ Sucrose” on the horizontal axis indicates a 10% by mass sucrose diet, and “+ Glucose” indicates a 10 mass% glucose diet. *** is P <0.001, and error bars indicate standard error (SEM). It was performed at n = 7 per group.

As a result of FIG. 3, the lactic acid bacterium 0831-07 (lactic acid bacterium # Ef-1) strain remarkably suppressed "the increase in blood glucose level of silk moth due to feeding of 10% by mass sucrose diet".
In addition, the inhibitory effect of the lactic acid bacterium 0831-07 strain was also observed on the increase in glucose concentration in the body fluid when glucose was fed.
In addition, the lactic acid bacterium 0831-07 strain was able to grow in a medium containing glucose or fructose.

Next, "lactic acid bacteria 0831-07 strain (25% by mass with respect to the whole feed)" or "lactic acid bacteria 0831-07 strain heat-treated by autoclave treatment" (Lactic acid bacteria 0831-07 strain) on the 5th instar 1st day silkworm, 10% by mass sucrose diet ( A diet containing 25% by mass) ”was given for 1 hour. Then, the body fluid of the silk moth was collected, and the glucose concentration in the body fluid was measured. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 4, the vertical axis shows the glucose concentration (mg / dL). “Control” on the horizontal axis shows the results when the silk moth was fed a 10% by mass sucrose diet containing no lactic acid bacteria. "-" Indicates a lactic acid bacterium 0831-07 (# Ef-1) strain that has not been heat-treated, and "Heat-killed" indicates a lactic acid bacterium 0831-07 strain that has been heat-treated by an autoclave treatment.

As a result of FIG. 4, the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain suppressed the increase in glucose concentration in the body fluid of the silk moth due to feeding of the sucrose feed, as in the case of the untreated live bacterium (FIG. 4). ).
Therefore, it was suggested that the inhibitory effect of the lactic acid bacterium 0831-07 strain on the increase in blood glucose by feeding sucrose was due to the inhibition of α-glycosidase in the silk moth intestinal tract by the heat treatment resistant component of the lactic acid bacterium 0831-07 strain.

Example 2
<Inhibition effect of lactic acid bacterium 0831-07 strain on increase in blood glucose level due to human sucrose feeding>
Next, in order to verify whether the lactic acid bacterium 0831-07 suppresses postprandial hyperglycemia in humans, yogurt was produced from milk using the lactic acid bacterium 0831-07 strain.
A sucrose (sucrose) loading test was performed on the subjects by a crossover method between when the yogurt was not ingested and when the yogurt was ingested.
Subjects in the yogurt intake group ingested 200 mL of yogurt 10 minutes before the sucrose load. Then, both the subjects in the yogurt non-ingestion group and the yogurt ingestion group drank 150 mL of a 50% (w / v) sucrose aqueous solution. Blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 minutes after sucrose loading. The blood glucose level was measured by collecting a small amount of blood from the fingertip and using a simple blood glucose meter. A significant difference test was performed using Student's t-test. The results are shown in FIG.
In FIG. 5, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the elapsed time (minutes) after ingestion (loading) of sucrose. * Is P <0.05, and error bars indicate standard error (SEM). It was performed at n = 10 per group.

As a result of FIG. 5, the subjects who ate the yogurt produced using the lactic acid bacterium strain 0831-07 had lower blood glucose levels 45 minutes after sucrose loading as compared with the non-feeding group (FIG. 5).
Therefore, it was suggested that the bacterial cell component of the lactic acid bacterium 0831-07 strain has an effect of suppressing an increase in blood glucose level due to ingestion of sucrose (sucrose) in humans.

Example 3
<Manufacturing of hypoglycemic agents and diabetes preventive and therapeutic agents>
<< Tablets >>
The cultured lactic acid bacterium 0831-07 was sterilized at 121 ° C. for 20 minutes and then concentrated. After uniformly mixing 20.0 mg of the concentrated culture solution of lactic acid bacterium 0831-07, 40 mg of lactose, 20 mg of starch, and 5 mg of low-substitution hydroxypropyl cellulose, wet preparation using an 8% by mass aqueous solution of hydroxypropylmethyl cellulose as a binder. Granules for tableting were produced by the granulation method. To this, 0.5 mg to 1 mg of magnesium stearate necessary for imparting smoothness was added, and then the tablets were tableted using a tableting machine.

<< Liquid >>
10.0 mg of the concentrated culture solution of lactic acid bacterium 0831-07 was dissolved in 10 mL of a 2% by mass 2-hydroxypropyl-β-cyclodextrin aqueous solution to prepare an injection solution.

Example 4
<Inhibition effect of increasing glucose concentration in silk moth body fluid by feeding sucrose-containing food by adding lactic acid bacteria killed bacteria>
Heat-killed cell fraction of lactic acid bacterium 0831-07 (# Ef-1) strain or autoclaved lactic acid bacterium 0831-07 (# Ef-1) strain on a diet containing 10% by mass sucrose (Heat-killed # Ef-1) Was fed to silk moths on the 1st day of the 5th instar for 1 hour. The body fluid of the silk moth was collected and the glucose concentration in the body fluid was measured. The results are shown in FIG. 6a.

In FIG. 6a, the vertical axis shows the glucose concentration (mg / dL). “No bacteria” on the horizontal axis shows the results when the silk moth was fed a 10% by mass sucrose-containing diet (Sucrose diet) containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable # Ef-1 content" indicates the content of the unheat-treated lactic acid bacterium 0831-07 strain, and "Heat-killed # Ef-1 content" indicates the content of the heat-treated lactic acid bacterium 0831-07 strain.
*** is P <0.001, ** is p <0.01, and error bars indicate standard error (SEM). The results were performed at n = 11 to 14 per group.

As a result of FIG. 6a, it was found that the effect of suppressing the increase in blood glucose by the lactic acid bacterium 0831-07 strain was enhanced depending on the amount of cells added to the diet. In addition, the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain also suppressed the increase in glucose concentration in the silk moth body fluid due to the feeding of the sucrose-containing bait in a dose-dependent manner.

Next, a heat-killed cell fraction (Heat-killed # Ef-1) of a lactic acid bacterium 0831-07 (# Ef-1) strain or an autoclaved lactic acid bacterium 0831-07 (# Ef-1) strain on a 10 mass% glucose diet. ) Was added so as to be 25% by mass based on the total diet, and the lactic acid bacteria on the first day of the 5th instar were fed for 1 hour. The body fluid of the silk moth was collected, the glucose concentration in the body fluid was measured, and a significant difference test was performed using Student's t-test. The results are shown in FIG. 6b.

In FIG. 6b, the vertical axis shows the glucose concentration (mg / dL). “No bacteria” on the horizontal axis shows the results when the silk moth was fed a 10% by mass glucose diet containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable" was given a 10% by mass glucose diet containing an unheat-treated lactic acid bacterium 0831-07 strain, and "Heat-killed" was given a 10% by mass glucose feed containing a heat-treated lactic acid bacterium 0831-07 strain. The result of the time is shown.
* Is P <0.05, ** is P <0.01, and error bars indicate standard error (SEM). It was performed at n = 6 to 7 per group.

As a result of FIG. 6b, the lactic acid bacterium strain 0831-07, which had not been heat-treated, suppressed the increase in glucose concentration in the silk moth body fluid due to the feeding of the glucose-containing food. On the other hand, no activity was found in the heat-treated cells of the lactic acid bacterium 0831-07 strain to suppress the increase in glucose concentration in the silk moth body fluid after glucose feeding.

The above results show that the viable lactic acid bacterium 0831-07 (# Ef-1) suppresses the increase in glucose concentration in the silk moth body fluid after ingestion of sucrose and glucose, and that the lactic acid bacterium 0831-07 (# Ef-1) It was suggested that the heat-treated cells of the strain remained active in suppressing the increase in glucose concentration in the body fluid of silk moth after ingestion of sucrose.

Example 5
<Construction of experimental scheme for sugar transfer evaluation system in silk moth in vitro>

Next, we attempted to elucidate the mechanism by which the lactic acid bacterium strain 0831-07 suppresses the increase in blood glucose of silk moths after feeding sucrose. In mammals, sucrose is known to be decomposed into glucose and fructose by α-glycosidase in the intestinal tract and absorbed from the intestinal tract. First, an experimental system was constructed to analyze the process by which sucrose in the intestinal tract of silk moth is decomposed by α-glycosidase and glucose is transferred to the outside of the intestinal tract.
The intestinal tract of the silk moth on the first day of the 5th instar was removed and tied with a thread so that the solution could be put into it. A sucrose solution was added into the intestinal tract of the silk moth and tied with a thread so that the solution did not leak. Then, it was incubated in PBS to quantify glucose transported out of the intestinal tract.
"Experimental scheme of sugar transfer evaluation system in silk moth in vitro" is shown in FIG. 7a.

Using the above experimental scheme, a sucrose solution in the intestinal tract of the silk moth or a sample of acarbose (40 mg / mL) added to the sucrose solution is placed in the intestinal tract of the silk moth, incubated at 27 ° C., and glucose in the extraintestinal fluid over time. The concentration was measured. The results are shown in FIG. 7b.
In FIG. 7b, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

As a result of FIG. 7b, it was found that the glucose concentration in the extraintestinal fluid increased in a time-dependent manner (black circles in FIG. 7b). In addition, the increase in glucose concentration in the extraintestinal fluid was suppressed by adding acarbose, which is an α-glycosidase inhibitor (white circle in FIG. 7b). This result suggests that in the intestinal tract of silkworm, sucrose is decomposed into glucose and fructose by α-glycosidase and further transported out of the intestinal tract.

Example 6
<Mechanical analysis of postprandial hyperglycemia suppression after eating sucrose by lactic acid bacterium 0831-07 strain>
Using the experimental scheme constructed in Example 5, a sucrose solution or a sample obtained by adding a sucrose solution to a sucrose solution with a lactic acid bacterium 0831-07 strain (250 mg (wet weight) / mL) was placed in the sucrose intestine, and 27 Incubation was carried out at ° C, and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 8a.
In FIG. 8a, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

In addition, a sucrose solution in the intestinal tract of the silk moth, or a sample obtained by adding a lactic acid bacterium 0831-07 strain (31 mg, 63 mg, 125 mg, 250 mg (wet weight) / mL) to the sucrose solution is placed in the intestinal tract of the silk moth and incubated at 27 ° C. , The glucose concentration of the extraintestinal fluid after 60 minutes was measured. The results are shown in FIG. 8b.
In FIG. 8b, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the amount (mg / mL) of the lactic acid bacterium 0831-07 (# Ef-1) strain. * Is P <0.05, ** is p <0.01, and error bars indicate standard error (SEM). It was performed at n = 3 to 5 per group.

As a result of FIGS. 8a and 8b, when the viable lactic acid bacterium 0831-07 strain was added to the sucrose solution in the intestinal tract of silk moth, the increase in glucose concentration in the extraintestinal fluid was suppressed. It was also found that the inhibitory effect was enhanced depending on the amount of cells (FIG. 8b).

Next, a sucrose solution or a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (250 mg (wet weight) / mL) of an autoclaved lactic acid bacterium 0831-07 strain was added to the sucrose solution in the intestinal tract of silk moth. The sample was placed in the intestinal tract of the silk moth, incubated at 27 ° C., and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 8c.
In FIG. 8c, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

In addition, a sucrose solution in the intestinal tract of silk moth, or a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (31 mg, 63 mg, 125 mg, 250 mg (wet weight)) of a lactic acid bacterium 0831-07 strain autoclaved into a sucrose solution / The sample to which mL) was added was placed in the intestinal tract of silk moth, incubated at 27 ° C., and the glucose concentration of the extraintestinal solution after 60 minutes was measured. The results are shown in FIG. 8d.
In FIG. 8d, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the autoclaved lactic acid bacterium 0831-07 strain. ** is p <0.01 and error bars indicate standard error (SEM). It was performed at n = 3 to 5 per group.

As a result of FIGS. 8c and 8d, the increase in glucose concentration in the extraintestinal fluid was suppressed even when the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain was added. It was also found that the inhibitory effect was enhanced depending on the amount of cells (FIG. 8d).
From these results, it was suggested that the heat resistance factor of the lactic acid bacterium 0831-07 strain inhibits the process of transfer of the molecule (glucose) obtained by decomposing sucrose in the intestinal tract of silk moth to the outside of the intestinal tract.

Next, a glucose solution in the intestinal tract of the silk moth, or a sample obtained by adding a lactic acid bacterium 0831-07 strain (250 mg (wet weight) / mL) to the glucose solution, is placed in the intestinal tract of the silk moth, incubated at 27 ° C., and the intestinal tract over time. The glucose concentration of the external solution was measured. The results are shown in FIG. 9a.
In FIG. 9a, the vertical axis shows the glucose concentration (mg / dL). The horizontal axis shows the incubation time (minutes).

As a result of FIG. 9a, even when the glucose solution was encapsulated in the intestinal tract of the silk moth, permeation of glucose out of the intestinal tract was observed with the passage of time.

In addition, a sample in which a glucose solution or a heat-treated bacterial cell fraction (Heat-killed # Ef-1) (250 mg (wet weight) / mL) of an autoclaved lactic acid bacterium strain 0831-07 was added to the glucose solution in the intestinal tract of silk moth. Was placed in the intestinal tract of silk moth and incubated at 27 ° C., and the glucose concentration of the extraintestinal fluid was measured over time. The results are shown in FIG. 9b.
In FIG. 9b, the vertical axis shows the glucose concentration (mg / dL). "No bacteria" on the horizontal axis shows the result of a glucose solution containing no lactic acid bacterium 0831-07 (# Ef-1) strain. "Viable" shows the result of the glucose solution to which the heat-treated lactic acid bacterium 0831-07 strain was added, and "Heat-killed" shows the result of the glucose solution to which the heat-treated lactic acid bacterium 0831-07 strain was added. * Is p <0.05, and error bars indicate standard error (SEM). It was performed at n = 3 to 4 per group.

It was found that the addition of live lactic acid bacteria 0831-07 strain into the intestinal tract suppressed the increase in glucose concentration in the extraintestinal fluid (FIGS. 9a and 9b). On the other hand, when the heat-treated bacterial cell fraction of the lactic acid bacterium 0831-07 strain was added, the increase in glucose concentration in the extraintestinal fluid was not suppressed (FIG. 9b).
These results suggest that the heat-sensitive factor of the lactic acid bacterium strain 0831-07 inhibits glucose transport from the intestinal tract to the outside of the intestinal tract of the silk moth.

Example 7
<Inhibition of α-glycosidase activity in the intestinal tract of silk moth or rat by lactic acid bacterium 0831-07 strain>
Next, it was examined whether the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibits the α-glycosidase activity of the silk moth intestinal tract. The measurement of α-glycosidase was carried out in the same manner as in Example 2.
The silk moths on the first day of the fifth instar were fed with normal food for one day. The α-glycosidase activity was measured by adding a cell disruption fraction obtained by crushing the intestinal tract of the silk moth by ultrasonic treatment and a heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain. The results are shown in FIG. 10a.
In FIG. 10a, the vertical axis shows the concentration (nmol) of “Produced pNP”. The higher the concentration (nmol) of "Produced pNP", the higher the α-glycosidase activity. The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain.

As a result of FIG. 10a, α-glycosidase activity was observed in the cell disruption fraction of the silk moth intestinal tract, and it was found that the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibited this activity in a dose-dependent manner.

Next, the rat intestinal acetone extract fraction and the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain were added to measure the α-glycosidase activity, and a significant difference was obtained using Student's t-test. The test was performed. The results are shown in FIG. 10b.
In FIG. 10b, the vertical axis shows the concentration (nmol) of "Produced pNP". The higher the concentration (nmol) of "Produced pNP", the higher the α-glycosidase activity. The horizontal axis shows the amount (mg / mL) of the heat-treated bacterial cell fraction (Heat-killed # Ef-1) of the lactic acid bacterium 0831-07 strain. *** is p <0.001, and error bars indicate standard error (SEM). It was performed at n = 3 per group.

As a result of FIG. 10b, the heat-treated fraction of the lactic acid bacterium 0831-07 strain also inhibited the α-glycosidase activity in the rat intestinal lysate in a dose-dependent manner. Therefore, the heat-treated fraction of the lactic acid bacterium 0831-07 strain inhibits the α-glycosidase activity in the intestinal tract of silk moths and mammals, and the sucrose in the intestinal tract is decomposed into glucose and fructose, and they are transferred to the outside of the intestinal tract. It was suggested that it would be inhibited.

Example 8
<Inhibitory effect on glucose uptake by lactic acid bacterium 0831-07 strain>
Caco-2 cells are cultured cells derived from the human intestinal tract, and methods for quantifying glucose uptake of these cells have been established (Yamabe N., et al., Am J Physiol. Endocrinol. Metab., 2015). .. Using this method, the inhibitory effect of the lactic acid bacterium strain 0831-07 on glucose uptake of Caco-2 cells was verified.
First, in the uptake system of 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] -D-glucose) in Caco-2 cells, lactic acid bacteria 0831- Live bacteria of 07 strain were added, the uptake of the tendency in Caco-2 cells was measured, and a significant difference test was performed using Student's t-test. The results are shown in FIG. 11A.
In FIG. 11A, the vertical axis shows the amount of glucose uptake, and the ratio when the control (the amount of the lactic acid bacterium 0831-07 strain is 0%) is 100% is shown. The horizontal axis shows the amount (mg / ml) of the lactic acid bacterium 0831-07 strain. *** is p <0.001, and error bars indicate standard error (SEM). It was performed at n = 3 per group.

As a result of FIG. 11A, it was found that the lactic acid bacterium strain 0831-07 inhibits glucose uptake by Caco-2 cells.

Next, in the uptake system of 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino] -D-glucose) in Caco-2 cells, lactic acid bacteria 0831 A viable cell fraction of -07 strain and a heat-treated cell fraction were added to measure the tendency uptake in Caco-2 cells, and a significant difference test was performed using Student's t-test. The results are shown in FIG. 11B.
In FIG. 11B, "No bacteria" on the horizontal axis shows the results when the lactic acid bacterium 0831-07 (# Ef-1) strain is not contained. "Viable" shows the result of the lactic acid bacterium 0831-07 strain which has not been heat-treated, and "Heat-treated" shows the result of the lactic acid bacterium 0831-07 strain which has been heat-treated. * Is P <0.05, and error bars indicate standard error (SEM). It was performed with n = 3 per group.

As a result of FIG. 11B, it was found that the glucose uptake inhibitory activity of Caco-2 cells in the lactic acid bacterium 0831-07 strain was reduced by the heat treatment of the cells.

From the above results, it was suggested that the heat-sensitive component derived from the lactic acid bacterium 0831-07 strain inhibits glucose uptake by Caco-2 cells. FIG. 11C shows a schematic diagram showing the postprandial hyperglycemia suppressing effect of the lactic acid bacterium strain 0831-07.
In FIG. 11C, "Lumen" is "lumen", "Degradation" is "degradation", "Transport" is "transport", "Outside of intestine" is "outside the intestinal tract", "Sucrose" is "sucrose", and "Glucose". "Is glucose", "Fructose" is "fructose", and "E. faecalis (# Ef-1)" is "lactic acid bacterium 0831-07".

Example 9
<Suppressive effect of lactic acid bacterium 0831-07 strain on increase in blood glucose level due to human sucrose intake>
It was investigated whether the lactic acid bacterium strain 0831-07 has an effect of suppressing an increase in blood glucose level after ingestion of sucrose in humans. A sucrose loading test was carried out on 14 healthy subjects in the live bacterial suspension ingestion group, the heat-treated bacterial cell suspension ingestion group, and the non-intake group. Subjects ingested 50 mL of sample suspended in saline 15 minutes prior to sucrose loading. Then, the subject drank 150 mL of a 50% (w / v) sucrose aqueous solution.
The blood glucose levels of the subjects at 0, 15, 30, 45, 60, 90, and 120 minutes after sucrose loading were compared between the groups. The blood glucose level was measured by collecting a small amount of blood from the fingertip and using a simple blood glucose meter.
FIG. 12A shows the experimental schedule of this example. First, a sucrose tolerance test was carried out in the heat-treated bacterial cell suspension ingestion group. Seven days later, a sucrose tolerance test was performed in the non-ingestion group. Further, 7 days later, a sucrose tolerance test was carried out in the viable cell suspension ingestion group.

FIG. 12B shows blood glucose in the live bacterial suspension ingestion group (Viable # Ef-1), the heat-treated bacterial cell suspension ingestion group (Heat-treated # Ef-1), and the non-intake group (Control) for each subject. It is a graph which showed the value for each time after sucrose loading. The vertical axis shows the blood glucose level (mg / dL). * Is p <0.05, and n = 14 per group.
FIG. 12C shows the results of the sucrose loading test in the live bacterial suspension ingestion group (Viable # Ef-1), the heat-treated bacterial cell suspension ingestion group (Heat-treated # Ef-1), and the non-intake group (Control). Is shown. The vertical axis shows the blood glucose level, and the horizontal axis shows the time (minutes) after ingesting sucrose. * Is p <0.05, and error bars indicate standard error (SEM). It was performed at n = 14 per group.

As a result of FIGS. 12B and 12C, it was obtained that the blood glucose level of the live bacterium suspension ingested group of the lactic acid bacterium 0831-07 strain was lower than that of the non-feeding group 45 and 60 minutes after the sucrose loading. On the other hand, there was no difference in the blood glucose level after sucrose loading between the heat-treated bacterial cell suspension ingested group and the non-fed group.
From the above results, it was shown that the lactic acid bacterium strain 0831-07 has an inhibitory effect on the increase in blood glucose level after ingestion of sucrose in humans, and that the blood glucose increase inhibitor is heat-sensitive.

<Summary of Examples>
From the above examples, it was shown that yogurt prepared from the lactic acid bacterium 0831-07 (lactic acid bacterium # Ef-1) strain using the silk moth evaluation system suppresses postprandial hyperglycemia due to feeding of human sucrose. Therefore, the lactic acid bacterium 0831-07 strain obtained in this example is a functional lactic acid bacterium that suppresses an increase in blood glucose level due to ingestion of sucrose (sucrose). Yogurt made with the lactic acid bacteria is expected to be more effective in the diet of obese and diabetic patients and their reserve humans.

In order to evaluate the effectiveness of food, it is necessary to evaluate using individual animals. Mammals such as mice and rats, which have been conventionally used as experimental animals, are expensive to carry out screening using a large number of individuals. On the other hand, silk moths do not require a large breeding space and can breed a large number of individuals at low cost. Furthermore, from the viewpoint of animal welfare, experiments using mammals are conducted in accordance with the international principles of the 3Rs, namely Replacement (development of alternative methods), Reduction (reduction of animal numbers), and Refinement (reduction of animal pain). Must be (Russell et al., 1959).
The use of silk moth as a substitute animal is consistent with the idea of Relative Replacement in the 3Rs. The use of silk moth in the search for functional foods could reduce the number of mammals sacrificed and solve problems in terms of cost and animal welfare.

In addition, the lactic acid bacterium strain 0831-07 was found to have an activity of inhibiting α-glycosidase in the silk moth intestinal tract and an activity of inhibiting the transport of glucose from the intestinal tract to the outside of the intestinal tract. It was suggested that these activities possessed by the bacterium suppress the increase in blood glucose level of silk moths fed with sucrose.

In addition, the heat-treated cells of the lactic acid bacterium 0831-07 strain retained the activity of suppressing postprandial hyperglycemia caused by feeding sucrose of silk moth. This fraction inhibited the activity of α-glycosidase, an enzyme that degrades sucrose in the silk moth intestine, but not glucose transport in the intestinal tract. Therefore, it was suggested that the effect of the lactic acid bacterium 0831-07 strain on suppressing the increase in blood glucose of silk moths ingested sucrose was mainly due to the inhibition of α-glycosidase activity.

The novel lactic acid bacterium of the present invention and the treated product have an effect of suppressing postprandial hyperglycemia due to feeding of sucrose, and further have an effect of preventing and treating diabetes. Therefore, it is possible to provide a drug or food or drink containing a blood glucose lowering agent using the lactic acid bacterium of the present invention, and it can be widely used in the pharmaceutical industry, the food industry and the like.

This application is based on Japanese Patent Application No. 2016-159555, which is a Japanese patent application filed on August 16, 2016, and all the contents of those applications are cited herein as disclosure of the specification of the present invention. It is something that is taken in.

NITE BP-02309

SEQ ID NO: 1 is a base sequence corresponding to almost the entire length of 16S rDNA of an unknown strain belonging to the genus Enterococcus.

Claims (9)

A lactic acid bacterium belonging to Enterococcus faecalis whose accession number is NITE BP-02309 at the Patented Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE) and has a hypoglycemic effect. The lactic acid bacterium according to claim 1, which has an α-glycosidase inhibitory activity and is used for suppressing an increase in blood glucose due to sucrose ingestion. The lactic acid bacterium according to claim 1 or 2, which has the base sequence of the 16SrDNA region shown by SEQ ID NO: 1 in the sequence listing. An α-glycosidase inhibitor containing the lactic acid bacterium according to any one of claims 1 to 3, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the lactic acid bacterium is at least one treated product selected from the group consisting of a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, and a sterilized processed product of the lactic acid bacterium. An α-glycosidase inhibitor characterized by being present. A hypoglycemic agent containing the lactic acid bacterium according to any one of claims 1 to 3, a dead lactic acid bacterium, or a treated product of the lactic acid bacterium as an active ingredient.
The treated product of the lactic acid bacterium is at least one treated product selected from the group consisting of a culture, a concentrate, a paste, a dried product, a liquefied product, a diluted product, a crushed product, and a sterilized processed product of the lactic acid bacterium. A hypoglycemic agent characterized by being present. A diabetes preventive and therapeutic agent comprising the hypoglycemic agent according to claim 5 as an active ingredient. A food or drink containing the lactic acid bacterium according to any one of claims 1 to 3. A food or drink containing the hypoglycemic agent according to claim 5. A food or drink produced by the step of fermenting with the lactic acid bacterium according to any one of claims 1 to 3.

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