WO2012014971A1 - Lactic bacterium having an effect of ameliorating metabolic syndrome - Google Patents

Abstract

Provided is a novel lactic bacterium capable of ameliorating metabolic syndrome by means of a novel screening method. Specifically disclosed are: a Lactobacillus which increases the production of adiponectin in 3T3-L1 cells and increases the production of an anti-inflammatory cytokine in bone-marrow-derived dendritic cells and/or macrophage; a culture of Lactobacillus; and a processed product of said culture. Also disclosed are a composition for foods and a pharmaceutical composition which contain the aforementioned Lactobacillus, culture, or processed product.

Description

Lactic acid bacteria with metabolic syndrome improvement effect

The present invention relates to a lactic acid bacterium having an improvement effect on metabolic syndrome, and a pharmaceutical composition and a food composition containing the lactic acid bacterium.

In recent years, various approaches have been sought for improving metabolic syndrome including obesity and diabetes, and adiponectin is attracting attention as one of them. Adiponectin is expressed specifically in adipocytes, and secretion of adiponectin from adipocytes is significantly suppressed in obese and diabetics (Non-Patent Documents 1 to 3). Adiponectin secreted from adipocytes acts on muscle cells to enhance sugar transport through activation of IRS-1 and PI3-kinase, and expression of fatty acid transport protein type 1 (FATP-1) and AMP-kinase It is known that insulin sensitivity is increased by increasing fatty acid oxidation and excretion (Non-patent Document 4). Adiponectin is also known to have an anti-inflammatory effect on the chronic inflammatory state of adipose tissue, and is known to improve insulin resistance by suppressing the production of TNFα (Non-patent Document 5). Therefore, increasing the secretion of adiponectin from adipocytes is thought to be important for improving metabolic syndrome including obesity and diabetes. In recent years, research targeting the functional control of adipocytes has attracted attention. It has become a target for the function of food factors (Non-Patent Document 6).

On the other hand, the relationship between cytokines and metabolic syndrome has also been studied. For example, in an animal model of metabolic syndrome, an increase in IFNγ in adipose tissue worsens metabolic syndrome, but an increase in IL-10 improves metabolic syndrome. (Non-Patent Documents 7 to 11). Further, regarding the relationship between adiponectin and IL-10, it has been shown that when adiponectin is added to human primary cultured macrophage cells, the amount of IFNγ secretion decreases and the amount of IL-10 secretion increases (non-patent document). 12).

Food-derived ingredients and food materials that affect the production of adiponectin have also been studied. For example, naringenin chalcone derived from tomato extract (Patent Document 1), proanthocyanidins derived from grape extract (Patent Document 2), apple extraction It has been suggested that product-derived polyphenols (Patent Document 3) and the like affect the tendency to increase adiponectin production.

In addition, the effects of lactic acid bacteria on adiponectin production have been studied, for example, Lactobacillus
It has been reported that some strains of gasseri and Lactobacillus helveticus promote or decrease the blood adiponectin concentration (Patent Document 4 and Patent Document 5). However, these reports only indicate that a strain affected adiponectin production in vivo, and only a one-sided evaluation of the effect on metabolic syndrome. .

JP 2008-115163 A JP 2006-182706 A JP 2006-193502 A JP 2008-63289 A JP 2009-107956 A

When searching for lactic acid bacteria that improve metabolic syndrome, the present inventors have a stronger improvement effect by adding not only adiponectin as an index but also an index indicating suppression of inflammation induced by metabolic syndrome. I thought it could be expected. Then, it discovered that it was necessary to establish the new test system for searching the target lactic acid bacteria. The present inventors then evaluated the ability of adipocytes to produce anti-inflammatory cytokines such as IL-10 secreted from immune system cells in addition to the ability to produce adiponectin in an in vitro test system. As a result of finding that probiotics capable of improving metabolic syndrome including insulin resistance can be searched by controlling the function of, and further earnestly researching it, the present invention has been completed.

That is, the present invention relates to a genus Lactobacillus that increases adiponectin production for adipocytes and increases production of inflammatory immunity-related cytokines for bone marrow-derived dendritic cells and / or macrophages.
The present invention also relates to the aforementioned Lactobacillus genus, wherein the inflammatory immunity-related cytokine is IL-10.
Furthermore, this invention relates to the said Lactobacillus genus microbe which is Lactobacillus plantarum.
The present invention also relates to a Lactobacillus plantarum OLL2712 strain (Accession Number: FERM BP-11262).
Furthermore, this invention relates to the culture of the said microbe, or its processed material.
The present invention also relates to a pharmaceutical composition comprising one or more selected from the group consisting of the above-mentioned fungus, the above-mentioned culture and its processed product.
Furthermore, this invention relates to the said pharmaceutical composition for suppressing accumulation | storage of a visceral fat.
Moreover, this invention relates to the foodstuff composition containing the 1 type (s) or 2 or more types selected from the group which consists of said microbe, said culture, and its processed material.
Furthermore, this invention relates to the said food composition for suppressing accumulation | storage of a visceral fat.

The Lactobacillus genus of the present invention can increase adiponectin production with respect to adipocytes, and can increase anti-inflammatory cytokine production with respect to bone marrow-derived dendritic cells and / or macrophages. In other words, in addition to increasing the production of adiponectin, it is also possible to increase the production of anti-inflammatory cytokines independent of adiponectin, and to improve the metabolic syndrome with a highly reliable complex approach Play.

FIG. 1a is a graph showing the results of the expression level of adiponectin mRNA when pioglitazone (Pio) and / or TNFα (TNF) is added to 3T3-L1 cells. FIG. 1b is a graph showing the results of mRNA expression level of Cu, Zn-SOD when pioglitazone (Pio) and / or TNFα (TNF) is added to 3T3-L1 cells. FIG. 2a is a graph showing the result of the expression level of adiponectin mRNA in 3T3-L1 cells when insulin resistance was induced by addition of TNFα. FIG. 2b is a graph showing the results of mRNA expression levels of Cu, Zn-SOD in 3T3-L1 cells when insulin resistance was induced by addition of TNFα. FIG. 2c is a graph showing the results of the expression level of IL-6 mRNA in 3T3-L1 cells when insulin resistance was induced by addition of TNFα. FIG. 2d is a graph showing the results of mRNA expression level of MCP-1 in 3T3-L1 cells when insulin resistance was induced by addition of TNFα. FIG. 3a is a graph showing the results of adiponectin mRNA expression level in 3T3-L1 cells when insulin resistance was not induced by addition of TNFα. FIG. 3b is a graph showing the results of mRNA expression levels of Cu, Zn-SOD in 3T3-L1 cells when insulin resistance was not induced by addition of TNFα. FIG. 3c is a graph showing the results of the expression level of IL-6 mRNA in 3T3-L1 cells when insulin resistance was not induced by addition of TNFα. FIG. 4a is a graph showing the results of the effect of dead lactic acid bacteria on IL-10 production of mouse bone marrow-derived dendritic cells (BMDC). FIG. 4b is a graph showing the results of the effect of dead lactic acid bacteria on IL-12 (p70) production of mouse bone marrow-derived dendritic cells (BMDC). FIG. 5a is a graph showing the results of the effect of dead lactic acid bacteria on IL-6 production of mouse macrophages J774.1. FIG. 5b is a graph showing the results of the effect of dead lactic acid bacteria on IL-10 production of mouse macrophages J774.1. FIG. 5c is a graph showing the results of the effect of dead lactic acid bacteria on IL-12 (p40) production of mouse macrophages J774.1. FIG. 6 is a graph showing the effect of lactic acid bacteria skim milk culture on blood adiponectin concentration in diabetic / obese model mice (KKAy mice). Mean values ± SE are shown. FIG. 7 is a graph showing the effect of lactic acid bacteria skim milk culture on the amount of adiponectin mRNA in visceral adipose tissue-derived adipocytes of diabetic / obese model mice (KKAy mice). Mean values ± SE are shown. FIG. 8 is a graph showing the results of the effect of lactic acid bacteria skim milk culture on visceral fat weight of diabetic / obese model mice (KKAy mice). Mean values ± SE are shown. FIG. 9 is a graph showing the results of the effects of lactic acid bacteria skim milk culture on blood triglyceride levels in diabetic / obese model mice (KKAy mice). Mean values ± SE are shown. FIG. 10 is a graph showing the results of the influence of a lactic acid bacteria skim milk culture on blood HbA1c values in diabetic / obese model mice (KKAy mice). Mean values ± SE are shown.

The Lactobacillus genus of the present invention increases adiponectin production relative to adipocytes. Here, the adipocytes are not particularly limited as long as they are established in vitro (in vitro). For example, mouse fibroblast-derived 3T3-L1 cells known as adipocyte models are known. Can be mentioned.

The Lactobacillus genus of the present invention usually increases the production of inflammatory immunity-related cytokines to cells that do not produce adiponectin, such as bone marrow-derived dendritic cells and macrophages. Preferably, the production of inflammatory immune related cytokines is increased against bone marrow derived dendritic cells and / or macrophages.
It is confirmed in vitro that the Lactobacillus genus of the present invention increases adiponectin production to adipocytes and / or increases production of inflammatory immune-related cytokines to cells that do not produce adiponectin, respectively. be able to.

Here, the inflammatory immunity related cytokine includes all cytokines related to inflammatory reaction. Inflammatory immunity-related cytokines can be broadly classified into cytokines that have the property of promoting inflammatory responses and cytokines that have the property of suppressing inflammatory responses. Cytokines having the property of promoting an inflammatory response include, but are not limited to, for example, IL-1α, IL-1β, IL-6, IL-7, IL-8, IL-12, IL-13 Inflammatory cytokines such as IL-17, IL-18, IFNγ, MCP-1, TNFα, and LIF. Cytokines having the property of suppressing inflammatory reactions include, but are not limited to, for example, anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β, and antiviral cytokines such as IFNα and IFNβ Etc.

Preferred inflammatory immunity-related cytokines are those that are associated with metabolic syndrome, such as IL-10 and IL-6, and particularly preferably IL-10.
In the present invention, “production increased” means adiponectin (protein, mRNA) expressed by a target cell upon stimulation of a test cell (live or dead) or a sample such as a culture or culture supernatant thereof. This means that the value serving as an index such as the expression level of inflammatory immune-related cytokines increases beyond the range of statistical errors. For the statistical analysis, any statistical analysis method known to those skilled in the art may be used, and it is not limited thereto, and examples thereof include Student's t test and Mann-Whitney U test.

Examples of the genus Lactobacillus included in the present invention include, but are not limited to, for example, Lactobacillus delbrueckii subsp. Burgaricus, Lactobacillus
delbrueckii subsp. lactis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus
acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus
gallinarum, Lactobacillus gasseri, Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus
fermentum, Lactobacillus brevis, Lactobacillus
plantarum and the like, preferably Lactobacillus amylovorus, Lactobacillus plantarum and the like, particularly preferably Lactobacillus
plantarum.

The Lactobacillus genus of the present invention is preferably resistant to gastric acid or bile acids. Due to such resistance, for example, as a food composition containing lactic acid bacteria, it can reach the intestine with live bacteria without using an enteric coating material, etc., improving the intestinal environment and continuously improving metabolic syndrome And / or a preventive effect.

Lactobacillus plantarum OLL2712 (also referred to as strain 23 in the present specification) of the present invention is deposited at the National Institute of Advanced Industrial Science and Technology under the accession number: FERM BP-11262, and has the following characteristics:
It is a plantarum fungus.
(A) Morphological properties Neisseria gonorrhoeae (b) Culture properties Medium name: Lactobacilli MRS Broth (Difco, Ref. No. 288130)
pH: Unadjusted culture temperature: 37 ° C
Culture time: 18 hours (1) Shape: Circular (2) Diameter: 1-2mm
(3) Color tone: White (4) Raised state: Hemispherical (5) Periphery: Full edge (6) Surface shape: Smooth (7) Transparency: Opaque (8) Viscosity: Butter-like (c) Physiological properties ( 1) Gram staining: Positive (2) Lactic acid fermentation format: Homolactic fermentation (3) Oxygen requirement: facultative anaerobic (4) Growth temperature: 15 ° C +, 45 ° C-

In the present invention, the subject for improving and / or preventing metabolic syndrome is an animal suffering from or possibly having a disease associated with metabolic syndrome including obesity and diabetes (for example, human, domestic animal) Species, wild animals, pets, etc.).
Further, the metabolic syndrome to be improved and / or prevented in the present invention includes, for example, metabolic syndrome such as diabetes, obesity (particularly visceral fat obesity), high blood pressure, high blood sugar, dyslipidemia, arteriosclerosis and the like. It may include diseases that are implicated.

When the Lactobacillus genus of the present invention is used for pharmaceuticals or foods for the improvement and / or prevention of metabolic syndrome, live bacteria, killed bacteria, cultures and processed products thereof can be used. The culture is a culture supernatant or a medium component after completion of cultivation of the Lactobacillus genus of the present invention, and the processed product is not particularly limited as long as it is derived from the culture. And the like obtained by processing such as concentration, pasting, spray drying, freeze drying, vacuum drying, drum drying, liquefaction, dilution and crushing. For these processes, known methods can be used as appropriate. In the culture or the processed product, the genus Lactobacillus of the present invention may be live or dead.
The Lactobacillus genus of the present invention, its culture, or its processed product, for example, a medium component, an additive suitable for oral tube feeding, and a solvent such as water, carbohydrates, proteins, lipids, vitamins Biopharmaceutical trace metals, fragrances, pharmaceutically acceptable carriers, food additives, and other optional components can be added to form pharmaceutical compositions and food compositions.

The pharmaceutical composition of the present invention typically increases the production of adiponectin on adipocytes and increases the production of inflammatory immune-related cytokines on bone marrow-derived dendritic cells and / or macrophages 1 type or 2 types or more selected from the culture and the processed product. Such a pharmaceutical composition improves the intestinal environment of an ingested individual and has an improving effect on metabolic syndrome. Furthermore, the pharmaceutical composition of the present invention can be used for effectively suppressing the accumulation of visceral fat.
In addition, the route of administration of the pharmaceutical composition is not particularly limited, and includes oral or parenteral administration, and examples thereof include oral administration, tube administration, and enteral administration. Oral administration is preferred from the viewpoint of convenience and safety. The dosage form is not particularly limited, and can be appropriately selected depending on the administration route. For example, aerosol, liquid, extract, elixir, capsule, granule, pill, eye ointment, transdermal Absorption-type preparations, suspensions, emulsions, suppositories, powders, spirits, tablets, syrups, soaking agents, injections, patches, tinctures, eye drops, troches, ointments, poultices, aromatic water Agents, liniments, limonade agents, fluid extracts, and lotions.

Oral preparations can be made into various known dosage forms, such as granules, powders, tablets, pills, capsules, solutions, syrups, emulsions, suspensions, lozenges, and the like. can do. In addition, by making an enteric preparation, it can be more efficiently transported to the intestine without receiving the effect of gastric acid.
Examples of parenteral administration include administration in the form of injections. Moreover, the Lactobacillus genus bacteria of this invention, its culture, or its processed material can also be locally administered to the area | region which wants to treat. For example, it can be administered by local injection during surgery or by use of a catheter.

Carriers that can be used in the pharmaceutical composition of the present invention include surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, and disintegrants. , Lubricants, fluidity promoters, flavoring agents and the like are listed as pharmaceutically acceptable carriers, and other commonly used carriers can be used as appropriate. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, and inorganic salts.

When the Lactobacillus genus of the present invention is used in a pharmaceutical composition, the cell concentration is not particularly limited, but when used as a concentrate, 2 × 10 ¹⁰ cells / g or more, when used as a dry product, 3 × 10 ^It is preferable to set it to ¹¹ pieces / g or more.
In the pharmaceutical composition of the present invention, the amount of Lactobacillus spp., Its culture or processed product thereof is not particularly limited, but can be appropriately adjusted according to the dosage form, symptoms, body weight, use and the like.
The daily intake of the pharmaceutical composition of the present invention is not particularly limited, but can be appropriately adjusted according to age, symptoms, body weight, use and the like. Typically, 0.1 to 10000 mg / kg body weight can be taken, preferably 0.1 to 1000 mg / kg body weight, more preferably 0.1 to 300 mg / kg body weight.

The food composition of the present invention typically increases the production of adiponectin on adipocytes and increases the production of inflammatory immune related cytokines on bone marrow-derived dendritic cells and / or macrophages 1 type or 2 types or more selected from the culture and the processed product. Such a food composition improves the intestinal environment of the ingested individual and has an improvement effect on the metabolic syndrome. Furthermore, the food composition of the present invention can be used to effectively suppress visceral fat accumulation.
The food composition of the present invention further includes carbohydrates, proteins, lipids, vitamins, biologically essential trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.), fragrances, and other ingredients as long as they do not interfere with the growth of lactic acid bacteria. Things can be included.

Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like.
Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, egg protein, meat protein and other animal and vegetable proteins, hydrolysates thereof; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components.

Examples of lipids include animal oils such as lard and fish oil, fractionated oils thereof, hydrogenated oils and transesterified oils; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils.
Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium.

There are no restrictions on the category or type of the food composition of the present invention, and it may be a functional food, a food for specified health use, a food for specific use, a functional nutrition food, a health food, a food for nursing care, and a confectionery, lactic acid bacteria beverage, cheese, It may be a dairy product such as yogurt or a seasoning. There is no limitation on the shape of the food and drink, and it can take any form of food or drink that can be distributed normally, such as solid, liquid, liquid food, jelly, tablet, granule, capsule, and various foods (milk, Soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, formula milk, liquid foods, food for the sick, nutritional foods, frozen foods, processed foods and other commercial foods) . Manufacture of the said food / beverage products can be performed by those skilled in the art.

As described above, the Lactobacillus genus of the present invention, its culture, or its processed product can be processed into general foods and drinks including dairy products and fermented milk, as well as starters for producing dairy products and fermented milk such as yogurt and cheese. It is also possible to use as. In the case of a starter, other microorganisms may be mixed as long as there is no hindrance to the survival and growth of the Lactobacillus genus of the present invention and as long as there is no hindrance to dairy production. For example, Lactobacillus delbrueckii subsp. Bulgaricus, Streptococcus, which are the main bacterial species as lactic acid bacteria for yogurt
It may be mixed with thermophilus, Lactobacillus acidophilus, etc. In addition, it can be mixed with bacterial species generally used for yogurt or cheese to form a starter. Production of dairy products and fermented milk using the starter can be performed according to a conventional method. For example, plain yogurt can be produced by mixing the above starter with milk or a dairy product cooled after heating, mixing, homogenizing, and sterilizing, and then fermenting and cooling.

The starter is appropriately selected according to the dairy product and fermented milk to be produced. Specifically, for example, a lactic acid bacterium or bifidobacteria belonging to the genus Streptococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus is used. Can be used.
More specifically, Streptococcus lactis, Streptococcus
cremoris, Streptococcus diacetylactis, Streptococcus thermophilus, Enterococcus
faecium, Enterococcus faecalis, Lactobacillus
acidophilus, Lactobacillus brevis, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus
delbrueckii subsp. lactis, Lactobacillus gasseri, Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus plantarum, Lactobacillus oris, Lactobacillus reuteri and Lactobacillus
Lactic acid bacteria such as rhamnosus, Bifidobacterium longum, Bifidobacterium bifidum and Bifidobacterium
A microorganism such as Bifidobacterium such as breve can be used as a starter. More preferably, Lactobacillus
delbrueckii subsp.bulgaricus, Streptococcus
thermophilus and Lactobacillus delbrueckii subsp. lactis can be used as starters. When these microorganisms are used as starters, they can be used alone or in combination of two or more as required.

A method for isolating these microorganisms from nature and fermented milk is known. Alternatively, already isolated microorganisms can be obtained by distribution from a cell bank or the like. Furthermore, lactic acid bacteria starters are commercially available. Several products are sold depending on the pH and physical properties of the produced fermented milk. The physical properties of fermented milk refer to hardness, smoothness, and the like.

In the present invention, the microorganism added to the raw milk as a mixed starter can be selected from microorganisms deposited in the cell bank. Examples of desirable strains that can be utilized in mixed starters include Lactobacillus bulgaricus JCM 1002T and Streptococcus
Streptococcus, a lactic acid bacteria starter consisting of a mixed culture of thermophilus ATCC 19258
thermophilus OLS 3059 (FERM BP-10740), Streptococcus thermophilus OLS3294 (NITE P-77), Streptococcus thermophilus OLS3059 (FERMP-15487), Lactobacillus delbrueckii subspecies bulgaricus OLL 1073R-1 (FERM BP-10741), Lactobacillus delbrueckii
and a lactic acid bacteria starter comprising a mixed culture of subspecies bulgaricus OLL 1255 (NITE BP-76) and Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 (FERM P-17227).

In general production of fermented milk, raw milk (yogurt base mix) may be added with one or more selected from lactic acid bacteria and yeasts other than these lactic acid bacteria. In the present invention, Lactobacillus bulgaricus (L. bulgaricus) and Streptococcus thermophilus (S.
thermophilus) is preferably used. Furthermore, when adding additional microorganisms, additional microorganisms can also be mixed in this mixing starter in consideration of the fermentation temperature and fermentation conditions of the target fermented milk. Other microorganisms such as Lactobacillus gasseri (L. gasseri), Lactobacillus plantarum, and Bifidobacterium can be shown as microorganisms to be additionally mixed in the mixed starter.

When the Lactobacillus genus of the present invention is used in a food composition, the cell concentration is not particularly limited, but when used as a concentrate, 2 × 10 ¹⁰ cells / g or more, when used as a dry product, 3 × 10 ^It is preferable to set it to ¹¹ pieces / g or more. In addition, the content of the Lactobacillus genus of the present invention in the food composition (as the amount of dry substance) is 0.01 to 100 w / w%, more preferably 1 to 10% in the solid content of the food composition. 80 w / w%, more preferably 10 to 40 w / w%.
In the food composition of the present invention, the amount of Lactobacillus sp., Its culture or processed product thereof is not particularly limited, but can be appropriately adjusted according to the dosage form, symptoms, body weight, use and the like.
The daily intake of the food composition of the present invention is not particularly limited, but can be appropriately adjusted according to age, symptoms, body weight, use and the like. Typically, 0.1 to 10000 mg / kg body weight can be taken, preferably 0.1 to 1000 mg / kg body weight, more preferably 0.1 to 300 mg / kg body weight. Further, the dry substance amount of the Lactobacillus genus of the present invention is 0.1 to 100 mg / kg body weight and 0.5 to 10 mg / kg body weight, and the number of the Lactobacillus genus of the present invention is 10 ⁶ to 10 ^12. Examples are 10 ⁷ to 10 ¹¹ , and 10 ⁸ to 10 ¹⁰ .

Hereinafter, the present invention will be further described based on examples, but these examples are exemplifications of the present invention and do not limit the present invention.

Example 1: Construction of screening system using mouse fibroblast-derived 3T3-L1 cells Screening system for lactic acid bacteria having anti-obesity / anti-diabetic activity using mouse fibroblast-derived 3T3-L1 cell line which is a model of adipocytes Was established.
(1) Test strains From about 270 strains isolated from healthy human feces, 20 strains (strain numbers 1 to 19, 23) having excellent resistance to gastric acid or bile acids were selected and used. In addition to these, a total of 23 strains shown in Table 1 below were used, in which 3 strains (strain numbers 20 to 22) having high immunomodulating activity were added from many strains isolated from various fermented milks.

(2) Culture of lactic acid bacteria and preparation of culture supernatant Lactic acid bacteria are prepared from skim milk powder medium (Table 2) and MRS medium (Lactobacilli MRS Broth (Difco,
No. 288130)) and cultured at 37 ° C.
The culture supernatant was prepared by first cultivating lactic acid bacteria in skim milk powder medium and MRS medium at 37 ° C. for 18 hours, collecting the supernatant, and then passing the collected supernatant through a 0.22 μm filter. It was. The culture supernatant was stored at −20 ° C.

(3) Assay using 3T3-L1 cells The 3T3-L1 cell line (purchased from DS Pharma Biomedical) was seeded in a 24-well plate at 2 × 10 ⁵ cells / dish and DMEM containing 10% CS (calf serum). Dulbecco's Modified Eagle Medium (Dulbecco's
modified Eagle medium)) for 2 days, replaced with 10% FCS (fetal calf serum) -containing DMEM, and further 2 days later, differentiation medium (10% FCS-containing DMEM, 10 μg / ml insulin, 2.5 μM dexamethasone, 3 -Isobutyl-1-methylxanthine (0.5 mM). After culturing in the differentiation medium for 48 hours, the cells were further cultured in the maintenance medium (10 μg / ml insulin in 10% FCS-containing DMEM) for 48 hours, and thereafter cultured in DMEM containing 10% FCS. Cells 7 days after replacement with maintenance medium were used for the assay. In addition, the adipocyte differentiation / maintenance reagent (AdipoInducer Reagent (for animal cell)) was purchased from Takara Bio Inc. The composition of the added reagent for differentiation medium was an insulin solution (10 μg / ml), a dexamethasone solution (2.5 μM), and a 3-isobutyl-1-methylxanthine solution (0.5 mM). The composition of the addition reagent for the maintenance medium was an insulin solution (10 μg / ml).
The medium was replaced with a medium supplemented with 1% of lactic acid bacteria culture supernatant 22 hours before collecting the cells, and 2 hours later, 10 ng / ml of TNFα (purchased from Sigma-Aldrich) was added. Also, instead of the culture supernatant of lactic acid bacteria, pioglitazone (pioglitazone, sold by Wako Pure Chemical Industries, Ltd., manufacturer: Alexis) used as a therapeutic agent for diabetes was added to a concentration of 10 μM to serve as a positive control. Thereafter, the cell lysate was collected with TRIzol reagent (Invitrogen, purchased from Life Technologies) and real-time
PCR system (Applied Biosystems, Life
The gene expression level was measured by Technologies).

(4) Results In 3T3-L1 cells, adiponectin and Cu, Zn-SOD (Cu, Zn-Superoxide
The amount of each mRNA of Dismutase) was significantly increased by pioglitazone. In addition, the amount of Cu, Zn-SOD mRNA was shown to decrease in the metabolic syndrome animals, and it was shown to correlate significantly with the increased expression of adiponectin.
Moreover, it was significantly suppressed by the addition of 10 ng / ml of TNFα, and was significantly recovered by co-addition of 10 μM pioglitazone and 10 ng / ml of TNFα (FIGS. 1a and b). In FIG. 1a-b, “control” is 3T3-L1 cells to which nothing is added, “TNF10 ng / ml” is “Pio 10 μM for 3T3-L1 cells to which TNFα is added at 10 ng / ml. “Is for 3T3-L1 cells supplemented with 10 μM pioglitazone, and“ Pio + TNF ”is for 3T3-L1 cells co-added with 10 μM pioglitazone and 10 ng / ml TNFα.
Similarly, significant effects were confirmed for catalase, which is another representative antioxidant factor (data not shown).

Example 2: Anti-obesity and anti-diabetic activity of various lactic acid bacteria skim milk culture supernatants Adiponectin in which the skim milk milk culture supernatants and MRS culture supernatants of 22 strains of lactic acid bacteria of Nos. 1-21 and 23 in Table 1 are 3T3-L1 cells The effect on the expression level was evaluated.
As a result of the primary screening, a skim milk powder culture supernatant of Lactobacillus amylovorus MEP222812 (hereinafter referred to as strain 12), Lactobacillus plantarum MEP222817 (hereinafter referred to as strain 17), and Lactobacillus plantarum OLL2712 (hereinafter referred to as strain 23) of number 23 was obtained. It showed the effect of significantly increasing the expression level of adiponectin in 3T3-L1 cells. Furthermore, as a result of confirming reproducibility for these three strains, it was found that two strains, strain 17 and strain 23, showed stable effects.

In addition, Lactobacillus delbrueckii subsp. Bulgaricus MEP222822 (hereinafter referred to as strain 22), which has been confirmed to have anti-inflammatory effects such as markedly inducing IL-10 production in mouse bone marrow-derived BMDC cells, is cultured on skim milk powder. Qing was also evaluated at the same time.
The results of evaluation in a system in which insulin resistance was induced by TNFα (10 ng / ml) are shown in FIGS. In FIGS. 2a to d, “control” is 3T3-L1 cells to which nothing has been added, and “skimmed milk powder + TNF” is a skim milk powder medium and 3T3-L1 cells to which TNFα is added at 10 ng / ml. In this case, “strain 22 + TNF” is a non-fat dry milk culture supernatant obtained by culturing strain 22 and 3T3-L1 cells supplemented with 10 ng / ml TNFα, and “strain 17 + TNF” is a non-fat dry milk culture supernatant obtained by culturing strain 17 In the case of 3T3-L1 cells to which 10 ng / ml of TNFα is added, “strain 23 + TNF” is “pio + TNF” in the case of the skim milk culture supernatant obtained by culturing strain 23 and 3T3-L1 cells to which 10 ng / ml of TNFα is added. "Is the case of 3T3-L1 cells co-added with 10 μM pioglitazone and 10 ng / ml TNFα.

Both the skim milk culture supernatants of strains 17 and 23 significantly increased the expression level of adiponectin in 3T3-L1 cells. Furthermore, strain 23 significantly suppressed IL-6 expression and significantly increased Cu, Zn-SOD expression. On the other hand, strain 22 significantly increased the expression level of IL-6, but did not affect the expression levels of other genes.
Pioglitazone did not suppress IL-6 but significantly suppressed MCP-1. On the other hand, the skim milk culture supernatant of strain 23 suppressed IL-6 and did not affect MCP-1. The culture supernatant of strain 22 significantly increased IL-6.

From the above results, it was suggested that the skim milk culture supernatant of strain 23 increases the adiponectin expression level by a mechanism different from that of pioglitazone. The mechanism of action of pioglitazone is a ligand for the nuclear receptor type transcription factor PPARγ, and changes the expression of genes that are transcriptionally regulated by PPARγ such as adiponectin. Since PPARγ is highly expressed especially in adipocytes, it is considered that adipocytes are the main target cells of pioglitazone. As a result of pioglitazone acting on fat cells, it suppresses the production of TNFα and free fatty acid (free fatty acid: FFA), while increasing the secretion of adiponectin, resulting in improved insulin resistance of fat cells and skeletal muscles. To do.

The results of a similar study in a system that does not induce insulin resistance by the addition of TNFα are shown in FIGS. 3a to 3c. In FIGS. 3a to 3c, “control” is 3T3-L1 cells to which nothing is added, “skim milk powder” is 3T3-L1 cells to which skim milk medium is added, and “strain 17” is In the case of 3T3-L1 cells added with the skim milk powder culture supernatant obtained by culturing the strain 17, “strain 23” is the case of 3T3-L1 cells added with the skim milk powder culture supernatant obtained by culturing the strain 23.
The culture supernatant of strain 23 significantly increased the expression levels of adiponectin and Cu, Zn-SOD in 3T3-L1 cells even in the system without addition of TNFα. Further, although the expression level of IL-6 was not significant, the average value was suppressed by about 20%. On the other hand, strain 17 had no significant effect on any gene expression level.

As a result, the non-fat dry milk culture supernatant of strain 23 increased the adiponectin expression level and suppressed the IL-6 expression level, suggesting the possibility of having anti-obesity / anti-diabetic effects. The non-fat dry milk culture supernatant of strain 22 has no such activity, and it was shown that strain 23 has excellent characteristics in terms of utilization of non-fat dry milk culture as an anti-obesity / anti-diabetic material.

Example 3 Assay Using Mouse Bone Marrow-Derived Dendritic Cells BMDC BMDC was extracted from femurs of 7-week-old male ICR. Bone marrow fluid was passed through a 70 μm cell strainer and then hemolyzed, and rabbit IgG was added to prevent nonspecific adsorption. Furthermore, biotin-labeled anti-CD4 antibody, anti-CD8 antibody, and IA ^d (MHCII marker) antibody were added, and the mixture was allowed to stand on ice for 30 minutes. Furthermore, streptoavidin magnetic beads and anti-B220 antibody magnetic beads were added. After passing through a 40 μm cell strainer again, the negative fraction was collected by auto MACS DEPLETE. By this operation, T cells, B cells and antigen-presenting cells were removed from cells in the bone marrow fluid, and only immature dendritic cells were separated.

10% GM-CSF (Granulocyte Macrophage
RPMI (Roswell Park Memorial) with colony-stimulating factor
Institute) -medium 1640 was cultured in 10 ml, and after 3 days, 5 ml of RPMI-medium 1640 containing GM-CSF was added. Five days later, floating cells considered to be BMDC were collected, seeded in a 96-well plate at 1 × 10 ⁵ cells / well, and lactic acid bacteria killed at each concentration were added simultaneously. After 24 hours, the culture supernatant was collected, and the concentrations of IL-10 and IL-12 (p70) were quantified using a mouse ELISA kit. Antibodies and ELISA kits were purchased from Becton, Dickinson and Company.

We investigated the effect of lactic acid bacteria on the cytokine production ability of dendritic cells and macrophages.
In addition to strain 17 and strain 23, the skim milk culture supernatant showed stable and significant activity against 3T3-L1 cells. In addition, strain 12 showed significant activity several times in 3T3-L1 cells. The effect of the three freeze-dried dead cells on the cytokine production ability of BMDC cells was examined. As comparative objects, a strain 22 having known activity and a dead cell of Lactobacillus gasseri MEP222804 (hereinafter referred to as strain 4) were used. Fig. 4 shows the results of measuring the production of IL-10 and IL-12 (p70) after 48 hours by ELISA using lactic acid bacteria dead cells (0.5, 1, 5, 10 µg / ml) added to BMDC. Shown in 4a-b. 4A and 4B, “strain 12” is a BMDC cell to which a dead cell of strain 12 is added, and “strain 17” is a BMDC cell to which a dead cell of strain 17 is added. 23 ”is a BMDC cell to which a dead cell of strain 23 is added,“ Strain 22 ”is a BMDC cell to which a dead cell of strain 22 is added,“ Strain 4 ”is a dead cell of strain 4 For BMDC cells supplemented with

The dead cells of strain 23 showed IL-10 and IL-12 production-inducing activity comparable to that of strain 22. The dead cells of strain 17 had very low IL-10 and IL-12 (p70) production-inducing activity compared to these, and the dead cells of strain 12 showed little activity.

Example 4: Assay using mouse macrophage J774.1 cell line Mouse macrophage J774.1 cells (purchased from Riken Cell Bank (RCB)) were cultured in RPMI medium containing 10% FCS and passaged every 3 days. Cell suspension prepared to 1 × 10 ⁶ cells / ml was seeded on a 48-well plate at 250 μl / well, and freeze-dried cells of various lactic acid bacteria and LPS (Wako) were added at the same time. Qing was recovered. Lyophilized dead cells were adjusted to 10 mg / ml with PBS, diluted with RPMI medium to each concentration, and added to cells at 125 μl / well. LPS was adjusted to 1 mg / ml with distilled water, adjusted to 4 μg / ml with RPMI medium, and added to cells at 125 μl / well to a final concentration of 1 μg / ml.

The IL-6, IL-10, and IL-12 (p40) concentrations in the collected supernatant were quantified using a mouse ELISA kit (Becton, Dickinson, and Company). Since the active form of IL-12 (p70) is not expressed in J774.1 cells, IL-12 (p40) was measured instead.

In addition to strain 17 and strain 23, the effect of three freeze-dried cells of strain 12 on the cytokine production ability of J774.1 cells was examined. In addition, dead cells of strain 4 significantly enhance the ability of J774.1 cells to produce IL-12p40. Strain 22 was predicted to have anti-inflammatory effects such as enhanced IL-10 production from the results of BMDC. Therefore, dead cells (1, 10 μg / ml) of the above 5 strains were added to J774.1 cells, and the production of IL-6, IL-10, IL-12 (p40) after 48 hours was measured by ELISA. The results are shown in FIGS. In FIGS. 5a to 5c, “control” is J774.1 cells to which nothing is added, “strain 12” is J774.1 cells to which dead cells of strain 12 are added, “strains” “17” is a J774.1 cell to which a dead cell of strain 17 is added, “strain 23” is a J774.1 cell to which a dead cell of strain 23 is added, “strain 22” is strain 22 In the case of J774.1 cells to which the dead cells were added, “strain 4” is the case of J774.1 cells to which the dead cells of strain 4 were added.

Strain 4 had the highest production induction ability for any cytokine. Strain 23 was found to have the same ability to induce IL-10 and IL-12 (p40) production as strain 22.

It was revealed that the dead cells of strain 23 exhibited a similar IL-10 production-inducing activity to BMDC as strain 22 which is an anti-inflammatory lactic acid bacterium with high immune activity. In addition, it was revealed that dead cells of strain 23 showed IL-10 production-inducing activity comparable to that of strain 22 against J774.1 cells. That is, the strain 23 is considered to be an anti-inflammatory lactic acid bacterium having a high immune activity that is comparable to the strain 22.

From the above results, strain 23 significantly increased adiponectin production relative to 3T3-L1 cells compared to other strains, and both in mouse bone marrow-derived dendritic cells and mouse macrophage J774.1 cells. It was revealed that the production of anti-inflammatory cytokines such as IL-10 was significantly increased compared to other strains. From this result, strain 23 not only improves the metabolic syndrome through enhancing the ability of adipocytes to produce adiponectin, but also enhances the ability to produce anti-inflammatory cytokines such as IL-10 secreted from immune system cells. This suggests an improvement effect on metabolic syndrome. This suggests that the fermentation product using strain 23 has an anti-metabolic syndrome effect.

Example 5. Efficacy Test Using Diabetes / Obesity Model Mice As the diabetes / obesity model mice, KKAy mice in which the obesity-onset gene AY was introduced into KK mice were used. KKAy mice are known to develop obesity, insulin resistance, and hyperlipidemia from a young age.
(Subject)
-Lactic acid bacteria culture: Strain 23 (Lactobacillus plantarum OLL2712, (Accession number: FERM BP-11262)) cultured in the skim milk medium described in Table 2 was used for administration as it was. The number of strains 23 contained in the lactic acid bacteria culture was 2 × 10 ⁸ cfu / ml. The dosage of strain 23 culture in this study is 1 × 10 ⁸ cfu / body / day (3.7 × 10 ⁹ cfu / kg / day).
-Positive control drug: Pioglitazone hydrochloride (Pioglitazone Hydrochloride, Funakoshi Co., Ltd.) was dissolved in carboxymethylcellulose sodium (Wako) solution, diluted with distilled water to 1 mg / ml, and used for administration. Pioglitazone hydrochloride is a therapeutic agent for type 2 diabetes, which has been confirmed to suppress TNFα expression in adipose tissue, improve insulin resistance, and promote sugar uptake and utilization. The CasNo. Of pioglitazone hydrochloride is 112529-15-4 and the chemical name is (5RS) -5- {4- [2- (5-Ethylpyridin-2-yl) ethoxy] benzyl} thiazolidine-2,
4-dione monohydrochloride. The dose of pioglitazone hydrochloride in this study is 0.5 mg / body / day. This dose was set with reference to the article by Mohapatra et al. (Mohapatra J, et
al., Pharmacology. 84-4: 203, 2009).

(Animal test)
Five-week-old male KKAy mice (Claire Japan) were individually housed in a microvent breeding device (Allentown) and acclimated for 1 week. After acclimatization, the mice were divided into 3 groups (control group, lactic acid bacteria administration group, positive control group, each n = 4) based on blood glucose level, HbA1c level, and body weight (day 0). At this time, the average value of the weight of each group was about 27 g. Furthermore, for 3 weeks from day 1 to day 21, the skim milk powder medium shown in Table 2 is given to the control group, the lactic acid bacteria culture is given to the lactic acid bacteria administration group, and the positive control drug is given to the positive control group once a day at 0.5 ml / body. did. During the administration period of the test substance, water was ad libitum and food was ad libitum CRF-1.
During the test administration period, blood was collected from the tail vein once a week (day 0, day 7, day 14 and day 21) and fasted for 3.5 hours, and blood glucose level and hemoglobin A1c level were measured. After blood collection, CRF-1 was ingested freely. In addition, body weight and food intake were measured twice a week (day 0, day 4, day 7, day 11, day 14, day 18, day 21) during the administration period of 3 weeks.
After the administration, the patient was dissected after euthanasia by cervical dislocation, and the perirenal adipose tissue and epididymal adipose tissue were collected, each wet weight (g) was measured, and the total was taken as the visceral fat weight (g). Furthermore, the adipose tissue was separated into a mature adipocyte fraction (MAF fraction) and a stromal blood vessel fraction (SVF fraction) by centrifugation, and the MAF fraction was analyzed for the expression level of adiponectin mRNA.

(Measuring method)
-Blood glucose level: Measured with a blood glucose measurement device (Breeze 2, Bayer Yakuhin).
Blood hemoglobin A1c (HbA1c): The concentration of hemoglobin A1c in blood was measured using a Hemoglobin A1c Testing Analyzer (DCA2000 system, Bayer Medical).
Blood adiponectin: The concentration of adiponectin in blood was measured using a mouse adiponectin ELISA kit (Otsuka Pharmaceutical).
Blood triglyceride: The blood triglyceride concentration was measured using Fuji Dry Chem System and Fuji Dry Chem Slide TG-PIII (Fuji Film).
-Adiponectin mRNA expression of MAF fraction: Total RNA was extracted from MAF fraction using TRizol reagent (invitrogen), cDNA was synthesized using PrimeScript RT reagent kit (Takara), and ABI 7300 real time PCR system (ABI) Was used to measure the expression level of adiponectin mRNA in the MAF fraction.

(result)
• There was no difference between groups in food intake and body weight changes during the study period.
In the lactic acid bacteria administration group (strain 23) and the positive control group (pio), the blood adiponectin concentration increased during the test period in all individuals, but half of the control group (skim milk powder) decreased (FIG. 6, day 21) The value obtained by subtracting the blood adiponectin concentration on day 0 from the blood adiponectin concentration was calculated as the amount of change in blood adiponectin concentration.)
-The adiponectin gene expression level of visceral adipose tissue-derived adipocytes tended to increase in the lactic acid bacteria administration group (Fig. 7). (P = 0.08 vs. control group, Mann-Whitney U test). When the amount of adiponectin mRNA increases in adipocytes in adipose tissue, it is considered that adiponectin production increases and sugar uptake in whole body tissues is promoted. Adiponectin is also considered to suppress fat accumulation because it promotes fat burning.
-Visceral fat weight was significantly low in the lactic acid bacteria administration group (FIG. 8). (P = 0.02 vs. control group, Mann-Whitney U test)
-Blood triglyceride levels tended to be suppressed in the lactic acid bacteria administration group (Fig. 9). (P = 0.08 (day 21) vs. control group, Mann-Whitney U test, Student's t-test)
・ In many cases, the increase in blood HbA1c level was suppressed in the lactic acid bacteria-administered group and the positive control group (Figure 10, change in blood HbA1c by subtracting day 0 blood HbA1c value from day H2 blood HbA1c value) Calculated as a quantity).

Example 6: Production of yogurt A yogurt base mix was prepared according to a conventional method and mixed starter (Lactobacillus
bulgaricus and Streptococcus thermophilus) and the mixed starter strain 23 (Lactobacillus plantarum OLL2712, (Accession number: FERM
BP-11262)) was added and the inoculated one was fermented to produce yogurt.
As a result, it was shown that the yogurt obtained by adding and inoculating the strain 23 had equivalent flavors and physical properties comparable to or higher than the yogurt obtained without addition.

According to the genus Lactobacillus of the present invention, in addition to increasing the production of adiponectin, it is also possible to increase the production of inflammatory immunity-related cytokines independent of adiponectin, a highly reliable complex Since the metabolic syndrome can be improved by a simple approach, it can be ingested easily and effectively by containing it in a pharmaceutical composition or food composition, and the metabolic syndrome can be improved.

[Supplement under Rule 26 16.08.2011]

Claims (9)

1. Lactobacillus spp. That increase adiponectin production for adipocytes and increase production of inflammatory immunity related cytokines for bone marrow-derived dendritic cells and / or macrophages.
2. The Lactobacillus spp. According to claim 1, wherein the inflammatory immunity related cytokine is IL-10.
3. The Lactobacillus genus according to claim 1 or 2, which is Lactobacillus plantarum.
4. Lactobacillus plantarum OLL2712 strain (Accession number: FERM BP-11262).
5. The culture or processed product of the fungus according to any one of claims 1 to 4.
6. A pharmaceutical composition comprising one or more selected from the fungus according to any one of claims 1 to 4, the culture according to claim 5, and a processed product thereof.
7. 7. The pharmaceutical composition according to claim 6, for suppressing the accumulation of visceral fat.
8. A food composition comprising one or more selected from the fungus according to any one of claims 1 to 4, the culture according to claim 5, and a processed product thereof.
9. The food composition according to claim 8 for suppressing visceral fat accumulation.

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