KR20180060928A - Composition for preventing and treating degenerative brain disease using novel lactic acid bacteria - Google Patents

Abstract

The present invention relates to a composition containing Lactobacillus reuteri ELF, which is novel lactic acid bacteria, and 5-aminolevulinic acid, thereby having excellent on the prevention and treatment of degenerative brain diseases and anti-diabetes and anti-obesity. Thus the composition of the present invention can be used to provide various pharmaceutical compositions and health foods using thereof.

Description

[0001] The present invention relates to a composition for preventing and treating degenerative brain diseases using novel lactic acid bacteria,

The present invention relates to a composition for preventing and treating degenerative brain diseases using novel lactic acid bacteria.

Lactic acid bacteria produce various kinds of metabolites by metabolism. Among these metabolites, it is known that they inhibit the growth of harmful bacteria in the intestines, inhibit the generation of harmful substances, improve the inflammatory diseases, improve the cardiovascular diseases, lower the cholesterol Lactic acid bacteria have been used for the production of health foods and medical preparations.

Lactic acid bacteria may degrade certain harmful substances in the body by metabolism depending on the kind, may produce specific substances depending on the substances used for metabolism, or may increase the absorption rate of specific substances in the body. For example, Korean Patent Publication No. 2004-0037011 discloses a food composition containing Lactobacillus lutea for prevention of obesity or diabetes, and Korean Patent No. 1426275 discloses an anti-obesity composition containing a mixture of several lactic acid bacteria .

5-aminolevulinic acid is an intermediate in the process of biosynthesis of porphyrin, and porphyrin is a metal ion complex immobilized in a tetrapyrrole ring and is used to perform important functions in vivo . For example, chlorophyll, hemoglobin or myoglobin correspond to complexes of different metal ions with porphyrin as the nucleus. 5-aminolevulinic acid is dehydrated by the dehydrating enzyme 5-aminolevulinic acid dehydratase to form porphobilinogen as a dimer and the porphobilinogen is converted to a porphyrin (protoporphyrin) via uroporphyrin.

The protoporphyrin produced from 5-aminolevulinic acid can be used as an agent for the treatment of acne or in the photodynamic therapy of skin cancer, and 5-aminolevulinic acid is used for the production of protoporphyrin. However, since the intermediate product, porfofibilogen, is an expensive compound due to unstable properties, difficulty in synthesis and purification, and since it is expected that the conversion of the phosphopyranogen also proceeds to protoporphyrin, there is little research on it to be.

We have isolated and identified new lactic acid bacteria to study new uses of lactic acid bacteria and new uses of pyrrole compounds, including pooforbinogen. In addition, it has been found that a novel lactic acid bacterium and its metabolites are remarkably effective for treating degenerative brain diseases, diabetes and obesity while 5-aminolevulinic acid and new lactic acid bacteria are treated together.

Korean Patent Publication No. 2004-0037011 Korea Patent No. 1426275

The present invention provides a composition for health promotion using a novel strain, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP).

The present invention provides a composition for treating diseases using a novel strain, Lactobacillus luteri ELF.

The present invention provides a composition for improving health comprising a novel strain, Lactobacillus luteri ELF and 5-aminolevulinic acid.

The present invention provides a composition for treating diseases comprising a novel strain, Lactobacillus luteri ELF and 5-aminolevulinic acid.

And a novel strain, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP). The present invention also provides a health functional food composition for preventing or treating degenerative brain diseases.

And a novel strain, Lactobacillus reuteri ELF (accession number: KCTC 13154BP). The present invention also provides a pharmaceutical composition for preventing or treating degenerative brain diseases.

And a novel strain, Lactobacillus reuteri ELF (accession number: KCTC 13154BP), for preventing or treating obesity.

And a novel strain, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP), for the prevention or treatment of obesity.

And a novel strain, Lactobacillus reuteri ELF (accession number: KCTC 13154BP). The present invention also provides a health functional food composition for prevention or treatment of diabetes.

And a novel strain, Lactobacillus reuteri ELF (accession number: KCTC 13154BP). The present invention also provides a pharmaceutical composition for preventing or treating diabetes.

A health functional food composition for the prevention or treatment of degenerative brain diseases comprising a novel strain Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid is provided.

There is provided a pharmaceutical composition for the prevention or treatment of degenerative brain diseases comprising a novel strain Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid.

A health functional food composition for the prevention or treatment of obesity comprising a novel strain Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid is provided.

There is provided a pharmaceutical composition for the prevention or treatment of obesity comprising a novel strain, Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) and 5-aminolevulinic acid.

The present invention provides a health functional food composition for prevention or treatment of diabetes comprising a novel strain Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid.

There is provided a pharmaceutical composition for the prevention or treatment of diabetes comprising a novel strain, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid.

The composition comprising the novel strain of the present invention, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP), is easy to ingest and has no side effects.

The composition comprising the novel strain Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) of the present invention is highly effective in the prevention and treatment of degenerative brain diseases. In particular, it can be very effective in the prevention and treatment of Alzheimer's disease among degenerative brain diseases.

The composition comprising the novel strain Lactobacillus luteri ELF of the present invention is very effective for prevention and treatment of diabetes.

The composition comprising the novel strain of the present invention, Lactobacillus lutei elf, is highly effective in the prevention and treatment of obesity.

The novel strain of the present invention, Lactobacillus luteri ELP, can produce pyrrole compounds at a significantly higher efficiency than the known Lactobacillus luteri.

A novel strain of the present invention, Lactobacillus lutei elf, is produced from 5-aminolevulinic acid by the metabolism of 5-aminolevulinic acid, porphobilinogen, hydroxymethylbilane, porphyrinogen, Hepta-carboxylate porphyrinogen, hexa-carboxylate porphyrinogen, pentaerythritol, pentaerythritol, pentaerythritol, pentaerythritol, pentaerythritol, A variety of other compounds such as penta-carboxylate porphyrinogen, coproporphyrinogen, protoporphyrin, protoporphyrinogen, heme, chlorophyll and derivatives thereof. The roll-type compound can be produced with high efficiency. Particularly, 5-aminolevulinic acid can be produced with high efficiency and can be effectively used for prevention and treatment of degenerative brain diseases, diabetes and obesity.

Figure 1 shows the weight change by Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP).
Figure 2 shows the freezing time change by Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) in a contextual fear conditioning experiment (WT-Cont; normal fed diet Mouse, WT-Elf; normal mouse fed Lactobacillus luteal elf; TG-Cont; Alzheimer-induced mouse fed normal diet; TG-Elf; Alzheimer-induced mouse fed Lactobacillus luteal elf).
Figure 3 is a graph showing the effect of phosphorylated tau protein (p-Tau), tau protein, beta-amyloid (A [beta]), postsynaptic density protein-95 in normal mouse (WT) and Alzheimer- -95, PSD-95), synaptophysin, and a housekeeping gene, GAPDH.
Fig. 4 shows the expression of beta-amyloid (A [beta]) in Alzheimer-induced mice by Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP).
Figure 5 shows the expression of postsynaptic density protein-95 (PSD-95) in normal mice and Alzheimer-induced mice by Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) .
FIG. 6 shows the expression of synaptophysin in Lactobacillus reuteri ELF (accession number: KCTC 13154BP) in normal mice and Alzheimer-induced mice.
Figure 7 shows the expression of phosphorylated tau protein (p-Tau) in Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) in normal mice and Alzheimer-induced mice.
Figure 8 shows the expression of tau protein (Tau) in Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) in normal mice and Alzheimer-induced mice.
FIG. 9 shows the expression ratio of phosphorylated tau protein (p-Tau) and tau protein (Tau) in normal mouse and Alzheimer-induced mouse by Lactobacillus reuteri ELF (Accession No. KCTC 13154BP).
FIG. 10 shows the expression of glial fibrillary acidic protein (GFAP) expressed in Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) in normal mice and Alzheimer-induced mice.
11 is a graph showing the effect of Glial fibrillary acidic protein (GFAP) on DG (Dentate gyrus) of a mouse and Alzheimer-induced mouse hippocampus fed with Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) ) (Scale bar = 100 탆).
Figure 12 shows the change of beta-amyloid (A [beta]) plaques by Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) in Alzheimer-induced mice.
Figure 13 shows beta-amyloid (A [beta]) staining in DG (Dentate gyrus) of Alzheimer-induced mouse hippocampus receiving Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) 100 탆).
14 shows the frequency change of CD3 T cells according to Example 10. Fig.
Fig. 15 shows frequency changes of Th lymphocytes according to Example 10. Fig.
16 shows frequency changes of CD4 ⁺ CD25 ⁺ cells according to Example 10. FIG.
FIG. 17 shows frequency changes of Gr-1 / CD11b cells according to Example 10. FIG.
FIG. 18 shows frequency changes of NKG2D, an indicator of NK cell activity according to Example 10. FIG.
Fig. 19 shows changes in IL-2 production in splenocytes according to Example 10. Fig.
20 shows the change in IL-4 production in the splenocytes according to Example 10. Fig.
21 shows the change in IFN-y production in the splenocytes according to Example 10. Fig.
22 shows a change in the production amount of nitric oxide in macrophages according to Example 11. Fig.
23 shows changes in total lymphocyte cells according to Example 11. Fig.
24 shows the activity change of cytochrome c oxidase (COX) in the liver according to Example 12. Fig.
25 shows the activity of cytochrome c oxidase (COX) in the brain according to Example 13. Fig.
26 shows a change in body weight of the mouse according to Example 14. Fig.
Fig. 27 shows the results of the glucose oral load test according to Example 15. Fig.
28 shows the expression pattern of the AMPK-alpha 1 gene according to Example 16. Fig.
29 shows the expression pattern of the UCP-2 gene according to Example 17. Fig.
30 shows the expression pattern of the adiponectin gene according to Example 18. Fig.
31 is a 16S rRNA gene sequence sequence of Lactobacillus reuteri ELF (accession number: KCTC 13154BP).
32 shows the phylogenetic tree of Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP).

Hereinafter, the present invention will be described in detail. In the description and drawings of the present invention, it is to be understood that the present invention may be practiced otherwise without departing from the spirit and scope of the present invention. For the purpose of facilitating understanding of the present invention, some of the drawings may be exaggerated or omitted, It is to be understood that the terminology used herein is to be interpreted in a general sense to a person having ordinary skill in the art to which the present invention belongs.

The present invention relates to a novel strain, Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) and compositions comprising same.

The present invention relates to a composition comprising a new strain, Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP) and 5-aminolevulinic acid.

The inventors of the present invention separated strains of lactic acid bacteria from a woman's breast milk using a lactic acid screening medium, and then selected strains having excellent acid resistance, biliary properties and antibiotic resistance, and then sorted the selected strains into 16S rRNA sequence- Analysis was performed to isolate and identify Lactobacillus luterium strains. Lactobacillus reuteri ELF ( Lactobacillus reuteri ELF) was isolated and identified and deposited on Nov. 22, 2016 with KCTC13154BP at the Microbiological Resource Center of Korea Research Institute of Bioscience and Biotechnology.

The present invention provides a health functional food composition for prevention or treatment of diseases comprising Lactobacillus reuteri ELF (Accession No. KCTC 13154BP).

The present invention provides a pharmaceutical composition for preventing or treating diseases comprising Lactobacillus reuteri ELF (Accession No. KCTC 13154BP).

The present invention relates to a pharmaceutical composition for treating a disease comprising Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) and 5-aminolevulinic acid (hereinafter, 5-ALA can be used interchangeably) Or a pharmaceutically acceptable salt thereof.

The present invention relates to a pharmaceutical composition for treating a disease comprising Lactobacillus reuteri ELF (Accession No. KCTC 13154BP) and 5-aminolevulinic acid (hereinafter, 5-ALA can be used interchangeably) Or a pharmaceutically acceptable salt thereof.

In the present invention, " treatment " means all actions that advantageously modify a subject having a disease, such as improving or alleviating symptoms of a disease using the composition of the present invention.

&Quot; Prevention " in the present invention means any action that inhibits or delays the onset of a disease using the composition of the present invention.

The disease of the present invention may be one or more selected from degenerative brain diseases, obesity and diabetes.

In the present invention, the degenerative brain diseases are selected from the group consisting of Alzheimer's disease, deemia, Parkinson's disease, Huntington's chorea, Creutzf- eldtjakob disease, Type 3-diabete (Alzheimer's disease) and Pick's disease (Alzheimer's disease).

Lactobacillus reuteri ELF (Accession No. KCTC13154BP) is a strain isolated and identified in breast milk of women and is a harmless strain to the living body. It has good biocompatibility, is not cytotoxic, and there is no risk of side effects due to strain ingestion.

The composition of the present invention preferably includes, but is not limited to, a Lactobacillus luteriol strain. For example, the microorganism of Lactobacillus lutei elf, the dried bacterium of Lactobacillus luteri ELP, the broth of Lactobacillus luteri ELP, the lysate of Lactobacillus luteri ELP, the extract of Lactobacillus luteri ELP, the enrichment of Lactobacillus luteri ELP Water, a culture broth of Lactobacillus luteal elef, a culture suspension containing Lactobacillus luteal elef, a filtrate of a Lactobacillus luteal elef culture, and a filtrate obtained by removing the strain after centrifugation of the Lactobacillus luteal elef culture. Composition can be provided.

The composition of the present invention is particularly effective for the treatment and prevention of degenerative brain diseases including Alzheimer's.

According to one embodiment of the present invention, a composition comprising Lactobacillus luteri ELF can reduce beta amyloid (A [beta]) and postsynaptic density protein (PSD), which may be effective in Alzheimer's treatment and prevention have. Also, a composition containing Lactobacillus luteal elef can significantly inhibit plaque formation of beta amylod. In addition, the composition containing Lactobacillus luteal elef does not directly affect the phosphorylation of tau protein or the decrease of phosphorylated tau protein, but it can reduce tau protein and thus may be effective for the treatment and prevention of Alzheimer's disease. That is, the composition of the present invention can reduce both beta amyloid and tau protein, which are major causes of Alzheimer's disease, and can be very effective for the treatment and prevention of Alzheimer's disease.

According to another embodiment of the present invention, a composition containing Lactobacillus lutealys can inhibit an increase in the expression of glial fibrillary acidic protein (GFAP), which is increased in expression in Alzheimer's patients. These effects also show that the composition containing the Lactobacillus lutei elf of the present invention is effective for the treatment and prevention of Alzheimer's.

Lactobacillus reuteri ELF (Accession No. KCTC13154BP), which is a novel strain of the present invention, has a remarkably high productivity of pyrrole compounds by itself. In addition, a specific pyrrole-based compound can be produced with high efficiency by metabolizing a precursor compound capable of producing a pyrrole-based compound such as 5-aminolevulinic acid. Particularly, the content of phosphbilinogen (hereinafter, 'PBG' can be used in the same meaning) in the pyrrole-based compound can be remarkably improved. Phosphobilinogen can increase the functional aspect of lactic acid bacteria so that a composition containing Lactobacillus lutei elf can treat and prevent disease more effectively than a composition containing other lactic acid bacteria. According to one embodiment of the present invention, the Lactobacillus luteri ELP can produce at least six times more pyrrole compound than the known Lactobacillus luteri.

In the present invention, 5-aminolevulinic acid is known to be biosynthesized from glycine and succinyl-CoA by the 5-aminolevulinic acid-producing enzyme and can be represented by the formula C ₅ H ₉ NO ₃ and 5 - aminolevulinic acid derivatives may also be used. 5-Aminolevulinic acid can be converted to porphobilinogen by a phospholipid biline synthase and converted to various substances therefrom. For example, it can be converted from protoporphyrinogen to protoporphyrin IX via the uroporphyrinogen intermediate and further converted to heme or chlorophyll.

The present invention can obtain 5-aminolevulinic acid and various biosynthetic compounds which are converted therefrom with high efficiency through metabolism of lactobacillus luteri ELF, which is a novel lactic acid bacterium. The Lactobacillus luteal elef and its metabolites do not have toxicity or side effects, and it is not necessary to separate Lactobacillus luteal elef or a specific metabolite separately. That is, a composition containing Lactobacillus lutei elf and a metabolite thereof as it is can be ingested to obtain a preventive and therapeutic effect of degenerative brain diseases without any other side effect. In addition, a composition containing Lactobacillus luteolymph and a metabolite thereof as it is can be taken to obtain an anti-obesity and anti-diabetic effect without any other side effects.

In the present invention, the novel lactic acid bacterium, Lactobacillus luteri ELF, can metabolize 5-aminolevulinic acid to produce various compounds which can be converted from 5-aminolevulinic acid as a metabolite. A composition comprising a metabolite resulting from Lactobacillus lutein elef and 5-aminolevulinic acid has been found to be particularly effective in the treatment of degenerative brain diseases, anti-obesity, antidiabetic and immune enhancement.

Since the composition of the present invention increases COX (cytochrome c oxidase) activity of brain mitochondria, it can be very effective in the treatment of degenerative brain diseases including Alzheimer's.

Generally, the blood brain barrier (BBB) is present in the brain, so that the pharmaceutical composition can not reach the brain, so that most of the brain can not act. However, according to one embodiment of the present invention, when the composition of the present invention is administered, the activity of COX in the mitochondria of brain cells may be abruptly increased, so that the composition of the present invention can act directly on the brain. The degree of COX activity is directly related to the degenerative brain disease, especially Alzheimer's. In Alzheimer's patients, the activity of COX is lower than that of the general population. In particular, the degree of COX activity in the hippocampus is reduced by 30% or more, resulting in an increase in free radicals resulting in a direct abnormality in the brain, which can lead to Alzheimer's disease. In addition, platelets in patients with Alzheimer's disease cause COX reduction and increased reactive oxygen species. And it is already well known that it suppresses COX that occurs during inflammation and thus helps to improve cognitive function. In addition, COX inhibitors have been shown to be effective in psychosis.

That is, the composition of the present invention can directly increase the COX activity of brain mitochondria, and thus can be very effective for the treatment and prevention of degenerative brain diseases including Alzheimer's.

According to one embodiment of the present invention, a composition containing Lactobacillus lutealys can increase long-term memory and may be effective in the prevention and treatment of degenerative brain diseases including Alzheimer's. It may also be effective in the treatment of congnitive impairment such as memory deficits, aphasia, real verification, and memory impairment. Cognitive disorders may include cognitive deficits due to degenerative brain diseases such as Alzheimer's, Alzheimer's Dementia, senile dementia and Parkinson's disease, cognitive deficits due to aging, cognitive deficits due to trauma or psychological shock, autism or attention deficit, It is not.

And, overweight or obesity may be an important cause of Alzheimer's disease because it raises the level of nerve fiber thickener or forms a plaque-forming protein, so a composition having an anti-obesity effect may be more effective for Alzheimer's treatment or prevention .

In addition, obesity and diabetes are likely to be accompanied together. Type 3 diabetes caused by insulin resistance of the brain in diabetes is highly related to the onset of Alzheimer's disease. In addition to mitochondrial dysfunction, oxidative stress, DNA damage, and beta amyloid deposition, brain insulin deficiency and insulin resistance can also cause degenerative brain diseases such as Alzheimer's. Thus, treatment of type 3 diabetes by insulin resistance in the brain can treat and prevent degenerative brain diseases including Alzheimer's.

According to one embodiment of the present invention, the composition of the present invention can significantly reduce the body weight and glucose concentration of blood, and significantly increase AMPK-alpha 1, UCP-2 and adiponectin gene expression, which are highly associated with obesity and diabetes . This effect means that the composition of the present invention is effective not only for anti-obesity and anti-diabetic but also for the treatment and prevention of degenerative brain diseases including Alzheimer's. According to another embodiment of the present invention, the composition of the present invention can remarkably increase the COX-activating enzyme of the brain mitochondria, and is highly effective in the prevention and treatment of degenerative brain diseases including Alzheimer's caused by type 3 diabetes Lt; / RTI >

In addition, the continuous formation and deposition of beta amyloid aggregates, a major cause of Alzheimer's disease, impairs the chronic activation of the immune system and the ability to remove anti-inflammatory agents. In addition, immune enhancement is directly related to the treatment or prevention of Alzheimer's, such as the increase of beta amyloid accumulation when the three major immune cells lack T cells, B cells and NK cells.

According to one embodiment of the present invention, the composition of the present invention may increase the proliferation and activity of T lymphocytes, B lymphocytes and NK cells. In addition, various cytokine production by T lymphocytes, B lymphocytes and NK cells may be increased. In addition, the activity of macrophages can be increased. Specifically, the proliferation and activity of CD3 T cells, Th lymphocytes, CD4 ⁺ CD25 ⁺ cells, Gr-1 / CD11b cells, NK cells and macrophages can be enhanced. In addition, lymphocytes in the abdominal cavity can be increased. In addition, the production of NKG2D, IL-2, IL-4 and IFN-y by immunocompetent cells can be increased. This effect means that the composition of the present invention is effective not only for improving immunity but also for treating and preventing degenerative brain diseases including Alzheimer's. Considering the increased COX activity of the brain mitochondria, antidiabetic, anti-obesity, and immuno-enhancing effects and the relationship between Alzheimer's treatment and prevention, the composition of the present invention may be highly effective in the treatment of Alzheimer's disease.

According to the present invention, the new Lactobacillus strain Lactobacillus lutei elf has a higher content of pyrrole compounds than other Lactobacillus lutei species. Also, the ability to produce a pyrrole compound including phosphobilinogen from 5-aminolevulinic acid is significantly superior to other Lactobacillus luterian species.

In the present invention, metabolites produced from 5-aminolevulinic acid by lactic acid metabolism include 5-aminolevulinic acid, porphobilinogen, hydroxymethylbilane, porphyrinogen derivatives And porphyrin derivatives. For example, the metabolites may include 5-aminolevulinic acid, porphobilinogen, hydroxymethylbilane, porphyrinogen, preuroporphyrinogen, But are not limited to, for example, hydroxyapatite, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, polyimide, And may include at least one selected from coproporphyrinogen, protoporphyrin, protoporphyrinogen, heme and chlorophyll. And includes I to IV depending on the morphology of the right porphyrinogen and co-porphorphyrinogen, and the protoporphyrin includes Protoporphyrin IX which is predominant in nature. It may also contain derivatives of each of the metabolites or salts thereof.

According to one embodiment of the present invention, when all of the metabolites of 5-aminolevulinic acid produced by Lactobacillus lutealis and Lactobacillus luteolig metabolism are contained, When the 5-aminolevulinic acid alone is metabolized under the same conditions as Elfman, the effects associated with degenerative brain disease treatment, immune enhancement, anti-obesity and anti-diabetic effects may be better than in the case of 5-aminolevulinic acid alone.

In a composition comprising a metabolite produced from 5-aminolevulinic acid by the Lactobacillus luteri ELP and Lactobacillus luteolig metabolism of the present invention, each of the components contained in the metabolite is used for the treatment of degenerative brain diseases, And the mechanism of action specific to the antidiabetic effect is unclear. However, it appears that some or all of the metabolites produced from 5-aminolevulinic acid by the Lactobacillus luteal elef may work synergistically with the Lactobacillus lutei elf. For example, posophobilinogen, which is known to be unstable as one of the metabolites, can be produced stably and with high efficiency by Lactobacillus luteri ELF, and this is one of the important effects on the effect of the composition of the present invention see.

The health functional food composition of the present invention can freely form solid / liquid forms. The form thereof is preferably used in the form of beverage, pill, tablet, powder, encapsulation, and the like. And, it can be manufactured by incorporating various types of food additives into health functional foods. Specific examples of the functional food include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen and other noodles, gums and ice cream, various soups, drinks, tea, drinks, And the like.

The pharmaceutical composition of the present invention can be prepared by self-culture of lactic acid bacteria or by adding 5-aminolevulinic acid to lactic acid bacteria and culturing according to a conventional culture method of lactic acid bacteria. Depending on the type of lactic acid bacteria used, the culture conditions can be adjusted for the preferred culture. For example, distilled water in which dissolved oxygen has been removed by bubbling with an inert gas is prepared in accordance with an embodiment of the present invention. After 5-aminolevulinic acid is added to distilled water, pH is adjusted to 8.5 to 9.5 And then the mixture is stirred at 25 to 40 for 10 to 24 hours. The mixture is stirred and centrifuged to obtain a metabolic material produced by lactic acid bacteria and lactic acid bacteria metabolism. In addition, it may further comprise a freeze-drying step after obtaining the metabolites produced by lactic acid bacteria and lactic acid bacteria metabolism.

In the present invention, the lactic acid bacteria and the metabolites produced by the lactic acid bacteria metabolism are obtained by stirring and then centrifuging to obtain the precipitated lactic acid bacteria and their metabolites. Alternatively, the lactic acid bacteria can be removed or the metabolites alone can be used without isolation or purification .

The composition may be prepared by removing a certain amount of water by obtaining lactic acid bacteria and metabolites thereof. However, the composition may be freeze-dried to produce a composition which is more effective and easy to process, and may be used in various forms.

The composition of the present invention may contain a pharmaceutically acceptable carrier as an active ingredient. Carriers which may be included in the present invention include, but are not limited to, starch, acacia rubber, water, syrup, cellulose, gelatin, mineral oil, talc, In addition to the carrier, sweeteners, flavors, preservatives, wetting agents, and the like may be further included.

The pharmaceutical composition of the present invention can be formulated by a person skilled in the art as a pharmaceutically acceptable carrier or excipient or a mixture thereof. The form of the formulation is not limited and includes, for example, solutions, emulsified forms, powders, granules, tablets, capsules, and may further contain stabilizers or dispersants.

The composition of the present invention is readily available and can be manufactured as a health functional food or included in food.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to the following examples. However, it should be understood that the present invention is not limited to the following examples.

Lactobacillus Luteri Elf ( Lactobacillus reuteri ELF)

The cultured anaerobic strain isolated from healthy female breast milk was plated on MRS medium containing 1.5% agar and cultured at 37 ° C for 24 hours. The 16S rRNA gene sequence (SEQ ID NO: 1) was analyzed for identification and classification of lactic acid bacteria. The strain was identified as Lactobacillus reuteri with 99% or more rRNA homology with the Luterian standard strain. Lactobacillus luteri strains having the same 16S rRNA base sequence were not found.

The 16S rRNA sequence of the isolated Lactobacillus lutealis strain was analyzed by the National Center for Biotechnology Information (NCBI) for the homology of the genomic information database and the nucleotide sequence of the Lactobacillus reuteri standard strain Respectively. The phylogenetic tree was constructed based on the Neighbor-joining method using the Jukes-Cantor model (Fig. 32).

The isolated Lactobacillus lutealis strain was compared with Lactobacillus luteri ATCC23272 and the mycological characteristics are shown in Table 1. The Lactobacillus luteal elef was able to produce more pyrrole compounds including phosphobilinogen than the same Lactobacillus luteal elf.

Mycological characteristic Lactobacillus Luteal Elves Lactobacillus luterian ATCC23272
Pyrrole compound production ≥1.7 1.0 Production of ruterin ≤0.8 1.0 motility none none shape Bacillus Bacillus Gram stain positivity positivity Catalase production voice voice Oxydase production voice voice D-glucose metabolism possible possible D-Mannos Metabolism possible possible D-fructose metabolism possible possible Metabolism of lactose possible possible Metabolism of inositol possible possible Ambassador to Schrox possible possible * The production of phosphobilinogen and rutherin was measured after incubation of Lactobacillus luteri ELF and Lactobacillus luteri ATCC23272 in the same medium for 24 hours. The comparison of the yields was made by comparing the yield of Lactobacillus luteri ATCC23272 to 1.0.

The inventors named the strain as Lactobacillus reuteri ELF and deposited it on Nov. 22, 2016 with the Microbiological Resource Center of Korea Research Institute of Bioscience (Accession No. KCTC 13154BP).

Lactobacillus Luteri Elven  Culture and separation of pyrrole compounds

And a medium prepared as shown in Table 2 below based on MRS medium was prepared.

ingredient Content (g) Soy Peptone PR 10 Beef extract 10 Yeast extract 5 Lactose 20 Tween 80 One Citric acid 2 Sodium acetate 5 Magnessium sulfate 0.1 Manganese sulfate food additive 0.05 Dipotassium phosphate 2 Glutamic acid One 10% L-Carnithine base 5 Water 1L

After sterilization of the medium, Lactobacillus reuteri ELF (1.5 * 10 ⁸ CFU / ml) was inoculated at 1% (V / V) and cultured for 15 to 20 hours. The pH was adjusted to 6.5 and the temperature was maintained at 38 [deg.] C. When the culture was completed, 20 μl of the culture was taken out and added to 2 ml of Ehrlich reagent to confirm a strong red absorption at 550 nm wavelength (Absorbance λ 550 = 0.22). The bacteria were separated by centrifugation, and the filtrate was purified according to the following method to obtain a pyrrole compound. 200 ml of anion exchange resin (Trilite, SAR20MB Samyang Corp.) was prepared in a column, and an aqueous solution of 25% sodium acetate was sufficiently flowed to saturate the anion with acetic acid. At this time, when the silver nitrate aqueous solution was dropped on the eluate, the procedure was continued until no white precipitate appeared. The column was washed with distilled water and the pH of the eluate was adjusted to 7.5 or less. The prepared filtrate was then added to the column. Thereafter, it was washed with a sufficient amount of distilled water and then eluted again with 1N acetic acid. The eluted 1N acetic acid solution was lyophilized to obtain 370 mg of a purified pyrrole compound.

Lactobacillus Luteri Elf  Well-known Lactobacillus Luterian  Culture and Pyrrole Separation of the compounds

The pyrrole compound was isolated from a known Lactobacillus luteri according to the same procedure as in Example 2. The Lactobacillus luterium strain was used as a known strain, ATCC23272.

When Ellrich's reagent test was conducted as in Example 2, it was found that the absorbance was? 550 = 0.06, and the lactobacillus luteal elf produced about three times or more of pyrrole.

However, the final product obtained through the purification of the anion exchange resin was only 50 mg, which was less than one-seventh of the amount of the pyrrole compound obtained using the Lactobacillus luteri ELF.

Lactobacillus Luteri Elven  Check your weight loss effect

A high-fat diet (HFD) was administered to male 8-week-old C57BL / 6J mice (Korean BioLink) at constant temperature (25 ± 2 ° C), humidity (50 ± 5% (AIN-76A diet) and water were freely fed in an SPF environment adjusted for 7 weeks (07:00 ~ 19:00) for 2 weeks. After 10 weeks of age, when the body weight was 24g or more, a high fat diet (# D12492 60 kcal fat, Research Diets Inc, USA).

12 g of Lactobacillus lutei elf isolated by centrifugation after cultivation in Example 2 and a 300 mg metabolite pyrrole were mixed well in a high-fat diet (60% fat content) for 12 kg mice (60% fat content) The mice were divided into two groups, one on the high-fat diet mixed with Lactobacillus luteal elet and its metabolites, and the other on the regular high-fat diets and observed continuous weight changes. As a positive control, 245 mg / kg of Garsinia cambogia extract was used. Rats fed with Lactobacillus lutein elf-like effects showed similar effects to the positive control Garcinia cambosia extract (Fig. 1).

Background Fear Conditioning ( Contexture  fear conditioning

Sixteen-week-old Alzheimer-induced mouse 5XFAD (Tg6799: B6SJL genetic background) mice were divided into a first group and a control group. Ten normal mice were divided into a second group. Five mice of each of the first and second groups were fed with Lactobacillus lutei elf, which had been cultured and centrifuged in Example 2, with the general diets, and the remaining five mice of the first and second groups were mixed with Lactobacillus luteri ELF Mixed with uncommon feeds. All mice were allowed to feed and work for 5 to 7 days. 12 g of Lactobacillus luteal elf was uniformly mixed with 12 kg of a general feed (Doihe Bio, Seoul) and made into a pellet form and fed to a mouse. All mice received the same amount of feed.

The background fear conditioning experiment was carried out by appropriately modifying the known experimental conditions as follows. Four standard conditioning chambers were used. Each chamber consisted of a sound isolating partition and had a stainless steel grid floor connected with a solid-stat shock scrambler. Each scrambler was connected to an electronic constant-current shock source controlled via an interface connected to a Windows computer running FreezFrame software (Coulbourn Instruments, Allentown, PA). A digital camera was installed on the ceiling of each chamber and the behavior of the mouse was analyzed. Mouse training allows the mouse to be freely navigated for 3 minutes for adaptation to the chamber context and a footshock (1.0 Ma, 2 seconds) is placed in the conditioning chamber at intervals of 3 to 5 The After the last footshock was applied, the mice were placed in the chamber for 30 seconds. Hippocampus-dependent contextual fear memory formation and remote memory stabilization were achieved when mice were reinserted into the same conditioning chamber after 1 and 30 days of training, respectively, with no respiration and no exercise for 3 minutes Were assessed by freezing behavior. In the automated FreezeFrame system, the video signal was digitized at 4 Hz, the motion was compared frame by frame, the freezing amount was recorded, and the freeze time was converted into freezing time (%).

As a result, the transgenic mice in which the Alzheimer-induced mice had a very low freezing time, but the freezing response of the transgenic mice fed the feed containing the Lactobacillus luteal elef was restored to normal levels (FIG. 2).

Genetic modification  Using Alzheimer's Mouse Lactobacillus Luteri Elven  Check the effect

Sixteen-week-old Alzheimer-induced mouse 5XFAD (Tg6799: B6SJL genetic background) mice were divided into a first group and a control group. Ten normal mice were divided into a second group. In each of the first and second groups, 5 mice were mixed with the Lactobacillus lteriarf diets centrifuged after culturing in Example 2, and the remaining 5 mice of the first and second groups were fed with a mixture of Lactobacillus lutei elp not mixed I mixed the feeds. 12 g of Lactobacillus luteal elf was uniformly mixed with 12 kg of a general feed (Doihe Bio, Seoul) and made into a pellet form and fed to a mouse. All mice received the same amount of feed.

Mice of each group were fed for about one month and sacrificed and the brain was extracted to include the temporal cortex, midbrain, cerebellum, cerebellum and hippocampus to produce phosphorylated tau (p-Tau), tau, amyloid beta Expression of Aβ, postsynaptic density protein-95, PSD-95, synaptophysin and glial fibrillary acidic protein (GFAP) was measured. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as a control and its expression was confirmed.

Protein expression and gene expression were confirmed by appropriately modifying known methods.

Here is a brief description.

The extracted brain tissue was washed, homogenized with a buffer, and the protein concentration was quantitated. After separating into gel, it was transferred to a nitrocellulose membrane. After transcription, Western blotting was performed using antibodies against each protein.

The extracted brain tissue, including the hippocampus, was fixed in paraformaldehyde, frozen at low temperature and then sliced with a microtome. After that, the sections of the brain were treated with antibodies against amyloid beta and glial fibrillary acidic protein (GFAP), and immunostained by immunofluorescence microscopy. For the staining of glial fibrillary acidic protein (GFAP), mouse monoclonal anti-neurofilament 160 / 200kD antibody (Zymed Lab. USA) was used as the primary antibody and fluoresceinisothiocyanate (FITC) was used as the secondary antibody. Thioflavin-s was used for staining of amyloid beta.

RNA was extracted from the extracted brain tissue, cDNA was synthesized, and real-time PCR was performed to confirm the expression level of each protein. The amount of mRNA was quantified with the GAPDH gene as needed.

Experimental results showed that beta amyloid expression was reduced by about 20% in Alzheimer's mice fed a diet containing Lactobacillus luteal elef (FIG. 4). The expression of postsynaptic density protein-95 (PSD-95) was also reduced by about 15% in Alzheimer's mice fed a diet containing Lactobacillus luteri ELF (FIG. 5) . On the other hand, there was no significant change in the expression of synaptophysin (Fig. 6).

There was also no significant change in the expression of phosphorylated tau protein (p-Tau) (Fig. 7). However, the expression of tau protein was reduced by about 25% in Alzheimer's mice fed a diet containing Lactobacillus luteal elef (Figure 8).

Expression of glial fibrillary acidic protein (GFAP) was significantly increased in Alzheimer 's mice. However, the expression of GFAP in Alzheimer's mice ingested with feed containing Lactobacillus luteal elf was reduced by about 30% (FIGS. 10 and 11).

Amyloid plaque measurement using thioflavin S showed about 50% reduction in amyloid plaque formation in Alzheimer's mice fed a feed containing Lactobacillus luteal elef (FIG. 12, FIG. 13).

Lactobacillus Luteri Elves ( Lactobacillus reuteri ELF) and producing a composition comprising a 5-aminolevulinic acid.

3,000 ml of distilled water and 431 g of anhydrous sodium carbonate (Na ₂ CO ₃₎ are added to a 4 L incubator and stirred, and 130 g of cysteine hydrochlroride salt is added. Nitrogen was injected, and the mixture was stirred while bubbling to remove dissolved oxygen. Then, 200 g of 5-aminolevulinic acid was added thereto and dissolved. Hydrochloric acid and sodium carbonate to adjust the pH to be between 8.6 and 9.5. Lactobacillus reuteri ( Lactobacillus reuteri) isolated according to Example 2 ELF) were added and stirred to give a uniform suspension. And the mixture was stirred for 30 to 24 hours. Then 1,000 ml of 1 M calcium lactate solution was added and stirred for 1 hour and centrifuged (5,000 rpm, 10 minutes) to precipitate a composition containing Lactobacillus luteoligin and 5-aminolevulinic acid . The obtained composition contained lactobacillus luteal elef and 5-aminolevulinic acid as well as metabolites due to lactic acid bacteria metabolism. The precipitate obtained as necessary was freeze-dried to completely remove moisture and packaged and stored.

Lactobacillus Luteri Elf  And 5- Aminolevulinic acid  Analysis of metabolite concentration by metabolism of lactic acid bacteria in the contained composition

To 2 ml of Ehrlich reagent, 20 占 퐇 of the culture solution of Example 7 was added. When the metabolism of 5-aminolevulinic acid by metabolism in lactic acid bacteria is used as a metabolite of phospholipids such as pyrrole-containing phospholipids, strong absorbance (red color) appears at a wavelength of 550 nm. The concentration of 5-aminolevulinic acid metabolites was measured by absorbance values proportional to pyrrole (Absorbance λ ₅₅₀ = 2.3, 0.45%).

Lactic acid bacteria and 5- Aminolevulinic acid  Analysis of metabolites by metabolism of lactic acid bacteria in the included compositions

200 ml of anion exchange resin (Trilite, SAR20MB Samyang Corp.) was prepared in a column, and an aqueous solution of 25% sodium acetate was sufficiently flowed to saturate the anion with acetic acid. At this time, when the silver nitrate aqueous solution was dropped on the eluate, the procedure was continued until no white precipitate appeared. The column was washed with distilled water and the pH of the eluate was adjusted to 7.5 or less.

1 g of the lyophilized composition according to Example 7 was dispersed in 10 ml of 1N acetic acid. After centrifugation (5,000 rpm, 5 minutes), the supernatant is adjusted to pH 7 and passed over the column. Wash with a sufficient volume of distilled water, re-elute with 1N acetic acid and freeze-dry the 1N acetic acid solution that has passed through. The lyophilized material was then dissolved in D ₂ O as a solvent, and the metabolites were identified by ¹ H NMR. ¹ H NMR result peak is 5.8 (s, 1H), 4.3 (s, 2H), 3.6 (s, 2H), 2.7 (t, 2H), 2.5 one shown metabolites by (t, 2H) The capsule reportage Billy Noggen. Prior to the analysis with the column, ¹ H NMR results showed very complex peaks due to the inclusion of a wide variety of metabolites including the porphobilinogen.

To check the immunity enhancing effect PBMC 2, IL-4, IL-10 and IFN-γ were measured by T lymphocyte proliferation and T lymphocyte activation in peripheral blood mononuclear cells and spleen.

8 weeks old Balb / c mice were treated with 100 mg / kg of vehicle control group, 5-aminolevulinic acid (hereinafter referred to as 'ALA'), Lactobacillus reuteri ELF, or less that can be represented by 'LAB') 100 mg / kg , Lactobacillus ruteri Elf and 5-aminolevulinic acid mixed sample (hereinafter may be represented as 'ALA + LAB') 50 mg / kg, Lactobacillus ruteri Elf And 5-aminolevulinic acid (100 mg / kg) were orally administered for 14 days (2 weeks).

Example  10-1. PBMC and  Fluorescence of activated T lymphocytes in the spleen Flow cell  analysis

Two weeks after the administration, PBMCs from the mice were collected by cardiac catheterization using a 3 ml syringe treated with heparin. 10 mL of ACK solution (a solution of 8.3 g NH ₄ Cl and 1 g KHCO ₃ mixed in demineralized water containing 0.1 Mm EDTA) was prepared and treated at room temperature for 5 minutes to remove red blood cells. The spleen was removed and the splenocytes were separated by 100 mesh. The cells were centrifuged at 1700 rpm for 5 minutes with D-PBS, washed and passed through a cell strainer (FALCON) to remove undissolved tissues or impurities . Separated PBMCs and spleen cells were washed twice with FACS buffer using PBS containing 1% FBS (Fetal Bovine Serum), and then passed through a cell trainer to remove impurities other than the cells. PBMC cells were adjusted to 5 × 10 ⁵ cells in each test tube and subjected to immunofluorescence staining at 4 ° C. FITC, anti-CD69-FITC, anti-CD44-cy5.5-PE, anti-CTLA-4-FITC, anti-CD1- PE and anti-CD49b-FITC were added to the cells for 30 min on ice. The cells were washed 3 times with PBS and analyzed by flow cytometry using the Cell Quest program. CD4 ⁺ CD25 ⁺ , CD44 ⁺ CD69 ⁺ , CD49b ⁺ NK1.1 ⁺ , and CD25 ⁺ CTLA-4 ⁺ , respectively.

After 2 weeks of oral administration, the number of active Th lymphocyte cells in the PBMC was 27.9% in the negative control group (NC) and slightly increased in the Lactobacillus lutein elf - treated group compared to the negative control group. In the 5 - aminolevulinic acid - treated group, active lymphocyte counts increased more than 46.2% at 100 mg / kg compared to the negative control group. In the Lactobacillus luteal elf and 5-aminolevulinic acid-treated groups, active Th lymphocyte counts were increased to 15% and 36.9% at 50 mg / kg and 100 mg / kg, respectively (C and D in FIG. The frequency of CD3 T cells was increased from 100 mg / kg of 5-aminolevulinic acid to 50 mg / kg and 100 mg / kg of lactobacillus luteolymph and 5-aminolevulinic acid compared to the control group (Fig. 14A and B ) And CD19 B cells were not different between groups. The frequencies of CD4 ⁺ CD25 ⁺ (FIG. 16) and Gr-1 / CD11b (G and H in FIG. 17) cells were 100 mg / kg of 5-aminolevulinic acid, And 100 mg / kg of the mixed acidic solution. In addition, the frequency of NKG2D, an indicator of NK cell activity (FIG. 18), was significantly higher than that of the control group, with 100 mg / kg of 5-aminolevulinic acid and 50 mg / kg and 100 mg / kg of Lactobacillus luteolymph and 5-aminolevulinic acid In the control group. That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, metabolism by the 5-aminolevulinic acid metabolism of Lactobacillus luteri ELF with Lactobacillus luteriol and 5-aminolevulinic acid It was confirmed that the effect of enhancing the immunity was remarkably increased by the effect of mixing by

Example  10-2. Cultivated In splenocytes  Which is an active substance of active T cells IFN -γ, IL-2, IL-4, IL- 10 measurements

Two weeks after the administration, the spleen was removed from the mouse, and splenocytes were isolated with 100 mesh. Before measurement, anti-CD3 mAb (1 μg / ml) was coated on a 96-well plate, overnight at 4 ° C. in a refrigerator, and then washed twice with D-PBS. Separated spleen cells were cultured in 5% FBS-DMEM culture medium for 48 hours at 5 × 10 ⁵ cells in each well coated with anti-CD3 mAb after removing red blood cells with ACK solution, and centrifuged at 2,000 rpm for 3 minutes And 200 의 of culture supernatant was obtained. Levels of IFN-γ, IL-2, IL-4 and IL-10 in culture supernatants were measured by ELISA (enzyme-linked immuno-sorbent assay). 50 μl of culture supernatant was dispensed into each well, left at room temperature for 2 hours at 25 ° C, washed twice with washing buffer, and incubated with antibody-biotin-IL conjugated antibody for 2 hours Respectively. After washing twice with buffer solution, 100 μl of Avidin-HRP conjugated antibody (Avidin-HRP conjugated antibody) was treated, left at room temperature for 1 hour, and then washed again. 100 .mu.l of TMB (tetrameyhylbenzidine) substrate was dispensed and left for 30 minutes in a dark room, and treated with 100 .mu.l of stop solution. The absorbance was then measured at 450 nm in an ELISA reader.

As a result of measurement, there was no difference in the group treated with Lactobacillus lutein elf compared to the control group (NC), and it was more than 2-fold increased in the group administered with 100 mg / kg of 5-aminolevulinic acid, and 5-aminolevulinic acid and Lactobacillus luteri ELP Kg, and 100 mg / kg, respectively. The production of IL-2 (FIG. 19), IL-4 production (FIG. 20) and IFN-γ production (FIG. 21) were significantly higher in the Lactobacillus lteriarf- mg / kg group, and the dose of Lactobacillus luteolymph and 5-aminolevulinic acid mixed group was significantly increased by about 2.1 times as compared to the control group in the dose-dependent manner . That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, metabolism by the 5-aminolevulinic acid metabolism of Lactobacillus luteri ELF with Lactobacillus luteriol and 5-aminolevulinic acid It was confirmed that the effect of enhancing the immunity was remarkably increased by the effect of mixing by

The degree of increase of nitric oxide (NO) in macrophage was measured to confirm immunity enhancing effect.

Example  11-1. Measurement of nitric oxide (NO) production from macrophages

Balb / c mice were treated with Vehicle control group, 5-aminolevulinic acid 100 mg / kg, Lactobacillus reuteri ( Lactobacillus reuteri) ELF), 50 mg / kg of a mixed sample of Lactobacillus luteal elef and 5-aminolevulinic acid, and 100 mg / kg of a mixed sample of Lactobacillus lutealis and 5-aminolevulinic acid were orally administered for 14 days (2 weeks). Three days before the end of the administration, 1.5 ml of 10% aqueous solution of proteose peptone (Difco) was injected into the abdominal cavity of the mouse, and the abdomen was sterilized with alcohol. The skin of the abdomen was cut with scissors, the peritoneum was exposed, and 4.5 mL of cooled HSSB (Hanks' Balanced Salt Solution) was injected into the abdominal cavity with a syringe and the abdomen was massaged for 30 seconds and recovered with a syringe. The recovered cells were then cooled, washed twice with the same HBSS solution, suspended in RPMI 1640 medium cooled, and living cells were counted by trypan blue staining to be 2 × 10 ⁶ cells / ml Then, 200 [mu] l of each well was added to a 96-well culture dish. CO ₂ incubator for 1 to 2 hours to attach macrophages, followed by washing three times with 37 ° C HBSS solution to remove floating cells, and 100 μl of RPMI 1640 medium was added. After culturing in a CO ₂ incubator for 72 hours, the culture supernatant was collected and cryopreserved. 100 쨉 l of the culture supernatant was taken, and 200 쨉 l of Griese reagent I in which 0.5 g of sulfanilamide was dissolved was mixed with 2 ml of distilled water, 25 ml of strong hydrochloric acid and 25 ml of distilled water. 200 μl of Griese Reagent II in which 0.06 g of N-1-naphtylene amidehydrochloride was dissolved in 50 ml of distilled water was rapidly added, and the absorbance was measured at 540 nm after stirring.

As a result of the measurement, the amount of nitric oxide produced in the administration group of 100 mg / kg of 5-aminolevulinic acid, 50 mg / kg of lactobacillus luteolin and 5-aminolevulinic acid and 100 mg / , Respectively. In addition, a mixed sample of Lactobacillus luteal elef and 5-aminolevulinic acid showed a remarkable effect twice as much as that of 5-aminolevulinic acid alone (Fig. 22). That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, metabolism by the 5-aminolevulinic acid metabolism of Lactobacillus luteri ELF with Lactobacillus luteriol and 5-aminolevulinic acid It was confirmed that the effect of enhancing the immunity was remarkably increased by the effect of mixing by

Example  11-2. Abdominal cavity Cell  Measure the effect on proliferation

Balb / c mice were treated with Vehicle control group, 5-aminolevulinic acid 100 mg / kg, Lactobacillus reuteri ( Lactobacillus reuteri) ELF) 100 mg / kg, Lactobacillus luteal elf and 5-aminolevulinic acid mixed sample 50 mg / kg, Lactobacillus luteal elf and 5-aminolevulinic acid mixed sample 100 mg / kg for 14 days (2 weeks) . Two days before the end of the administration, 0.5 ml of a 2% starch physiological saline suspension is injected into mouse abdominal cavity and immunized. The mice were sacrificed and the abdomen was sterilized with alcohol. The skin of the abdomen was cut with scissors, the peritoneum was exposed, and 5 mL of cold HBSS was injected into the abdominal cavity with a syringe and the abdomen was massaged for 30 seconds, and about 3 mL was recovered with a syringe . The recovered cells were cooled, washed twice with the same HBSS solution, and the number of peritoneal cells present therein was directly measured under a microscope with a hemocytometer. After the measurement of the total number of peritoneal cells, the peritoneal wash was centrifuged at 4O < 0 > C for 10 minutes to obtain a cell suspension, which was then suspended in PBS. Immunofluorescence staining was performed at 4 ° C after adjusting the peritoneal cells to 5 × 10 ⁵ cells in each test tube. After incubation for 30 min on ice, the cells were washed 3 times with PBS. The cells were washed three times with anti-CD11b-FITC, anti-CD3-cy5.5-PE, anti-CD4-FITC, anti-CD8- Cell counts of CD11b ⁺ CD14 ⁺ , CD3 ⁺ CD4 ⁺ , and CD3 ⁺ CD8 ⁺ cells were analyzed by flow cytometry using the Cell Quest program, And the absolute number of cells of the peritoneal cavity was calculated.

As a result, lymphocyte cells were increased in both lactobacillus luteolin, 5-aminolevulinic acid, Lactobacillus luteal elef and 5-aminolevulinic acid mixed sample compared to the control group. However, more lymphocyte increase was observed in the Lactobacillus luteal elf and 5-aminolevulinic acid treated group, and a marked increase in lymphocyte was observed particularly in the group administered with 100 mg / kg of Lactobacillus luteolymph and 5-aminolevulinic acid (Fig. 23 ). That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, metabolism by the 5-aminolevulinic acid metabolism of Lactobacillus luteri ELF with Lactobacillus luteriol and 5-aminolevulinic acid It was confirmed that the effect of enhancing the immunity was remarkably increased by the effect of mixing by

COX-active enzyme measurement of liver mitochondria

Balb / c mice were treated with Vehicle control group, 5-aminolevulinic acid 100 mg / kg, Lactobacillus reuteri ( Lactobacillus reuteri) ELF) 100 mg / kg, and a mixed sample of Lactobacillus luteolymph and 5-aminolevulinic acid were orally administered for 14 days (2 weeks). To measure the activity of mitochondrial COX (cytochrome c oxidase) in mice after the administration, proteins were extracted and measured by COX activity assay kit (TA100, Toyo B Net) in mouse hepatocytes. As a result, the activity of COX was not different in the control group and Lactobacillus lutei elf-treated group (LAB), and ALA was increased more than 2 times, but statistical significance was not shown. In the ALA + LAB treated group (p < 0.01), the COX activity was increased 4 times or more statistically significantly (Fig. 24). That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, metabolism by the 5-aminolevulinic acid metabolism of Lactobacillus luteri ELF with Lactobacillus luteriol and 5-aminolevulinic acid It was confirmed that the effect of COX activity was remarkably increased by the effect of mixing by

Measurement of COX-activating enzyme in brain mitochondria

C57bl / 6J mice (vehicle control group) and obesity control (HFD control) were prepared as normal controls. A high-fat diet (HFD) was administered to male 8-week-old C57BL / 6J mice (Korean BioLink) at constant temperature (25 ± 2 ° C), humidity (50 ± 5% (AIN-76A diet) and water were freely fed in an SPF environment adjusted for 7 weeks (07:00 ~ 19:00) for 2 weeks. After 10 weeks of age, when the body weight was 24g or more, a high fat diet (# D12492 60 kcal fat, Research Diets Inc, USA) was used to produce a mouse obese (DIO) condition model. 100 mg / kg and 200 mg / kg of Lactobacillus luteal elf and 5-aminolevulinic acid mixed sample (ALA + LAB) were each administered together for 14 days (2 weeks) at 10 weeks of the high fat diet . After the oral administration, mouse brain was extracted to measure cytochrome oxidase activity in the mitochondria of mouse brain, and the COX activity of the brain was measured according to the manual of the COX activity assay kit (TA100, Toyo B Net). As a result, the COX activity of the obesity control group was lower than that of the normal control group, and the COX activity of the ALA + LAB group (p <0.01) was 1.5 times higher than that of the normal control group.

That is, in a rat administered with a mixed sample containing Lactobacillus luteoligin and 5-aminolevulinic acid, a mixture of 5-aminolevulinic acid and 5-aminolevulinic acid together with metabolites by 5-aminolevulinic acid metabolism by lactobacillus luteoligin It was confirmed that the COX activity effect significantly increased. It was also confirmed that the mixed samples could directly affect the COX activity of the brain mitochondria without being affected by the blood-brain barrier of the brain. These results indicate that a mixture containing Lactobacillus lutealis and 5-aminolevulinic acid is effective in the treatment of degenerative brain diseases caused by decreased COX activity of brain mitochondria.

Obesity Control Study of Weight Change in Rats

An obesity control (HFD) was prepared in the same manner as in Example 13, and 100 mg / kg, 200 mg / kg and milk thistle of a mixture of Lactobacillus lutei elf and 5-aminolevulinic acid (ALA + LAB) MTS) 100 mg / kg were administered together from 10 to 19 weeks of the high - fat diet. Body weight change was measured according to ① weight change: measurement / record at 9:00 every Wednesday, ② total body weight increase: final body wt - initial body wt, and ③ 1 day average body weight gain = total body wt gain / days. (P <0.01), ALA + LAB (100 mg / kg) (p <0.01) and ALA + LAB (200 mg / kg) (Fig. 26).

Oral load test using obesity control group

Glucose oral test is absorbed in the intestinal tract more than the processing ability in the liver and tissues when the glucose is ingested orally, and the blood sugar becomes the highest value in 30-60 minutes. Glucose is used in tissues to inhibit glucose release in the liver, and blood sugar levels are lowered by mechanisms such as insulin secretion enhancement and growth hormone secretion inhibition. As in Example 13, an obesity control group was prepared and each test sample was administered together with glucose. (Fig. 27) In the glucose tolerance test of obesity control group, MTS (100 mg / kg) (p <0.05) decreased slightly after 120 minutes after the blood glucose level was increased more than 3 times at 30 minutes after glucose administration ) And ALA + LAB (200 mg / kg) (p <0.05) significantly decreased blood glucose levels at 30 minutes, 60 minutes and 120 minutes. In the ALA + LAB (100 mg / kg) group, the blood glucose level was decreased at 30 minutes, 60 minutes and 120 minutes compared to the obesity control group.

A target protein of obesity and diabetes AMPK - alpha 1  Analysis of gene expression patterns

After measuring the body weight in accordance with Example 14, RNA was extracted from each tissue using RNAsolB (Tel-Test) solution in the liver tissues of the rats to which the normal control, the fat control and the test sample were administered, cDNA and real-time PCR analysis were performed using the step SYBR Green PCR kit (AB science). 500 μl of RNAzol B was added to the tissue, the tissue was ground with a homogenizer, 50 μl of chloroform (CHCl ₃ ) was added thereto, and the mixture was mixed again for 15 seconds. This was left on ice for 15 minutes and centrifuged at 13,000 rpm. About 200 μl of the supernatant was recovered, mixed with 200 μl of 2-propanol in the same amount, and then slowly shaken and left on ice for 15 minutes. After centrifugation at 13,000 rpm, the cells were washed with 80% EtOH and dried in a vacuum pump for 3 minutes to extract RNA. The extracted RNA was dissolved in 20 μl of diethyl pyrocarbonate (DEPC) and dehydrated at 75 ℃ for heating strand cDNA. For reverse transcription, 3 μg of prepared total RNA was reacted with DNase I (10 U / μl) 2 U / tube in a heating block at 37 ° C for 30 minutes and denatured at 75 ° C for 10 minutes. 2.5 μl of 10 mM dNTPs mix , 1 μl of RNase inhibitor (20 U / μl), 1 μl of 100 mM DTT, 4.5 μl of 5 × RT buffer (250 mM Tris-HCl, pH 8.3) as an RNA inhibitor, 1 μl of random sequence hexanucleotides 375 mM KCl, 15 mM MgCl 2) was added to the wells. Then, 1 μl of M-MLV RT (200 U / μl) was added again and DEPC-treated distilled water was added to a final volume of 20 μl. The 20 μl reaction mixture was mixed well, centrifuged at 2,000 rpm for 5 sec, and reacted for 45 min at 37 ° C in a heating block. The first strand cDNA was synthesized and allowed to stand at 95 ° C for 5 min. After inactivation, the synthesized cDNA was used for polymerase chain reaction (PCR). Real-time quantitative PCR was performed using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, USA). The primer sequence was 5'-AAGCCGACCCAATGACATCA-3 'as a forward primer and 5'-CTTCCTTCGTACACGCAAAT-3' as a reverse primer. As a result of analysis of AMPK-α1 mRNA gene expression expressed in the liver according to the administered sample in the mouse obesity model, the relative closure value of the experimental group was analyzed when the RQ value of the AMPK-α1 mRNA gene expression in the obesity control was 1. AMPK-α1 mRNA expressed in liver of obese control group showed AMPK-α1 mRNA gene expression similar to that of normal control (C57bl). There was no significant difference between the MTS group and the obese control group. However, there was a statistically significant 5 to 3-fold increase in ALA + LAB (200 mg / kg) (p <0.001) and ALA + LAB (100 mg / kg) ).

Heat generation  protein UCP Analysis of expression pattern of -2 gene

After the body weight measurement according to Example 14, the liver surrounding tissues of the rats to which the normal control, the fat control and the test sample were administered were treated as in Example 10 and PCR was performed. The probe used was 5'-TTCAAATGAGATTGTGGGAAAAT-3 '(sense), 5'-ACCGATACAGTACAGTACAGTA-3' (antisense). Analysis analyzed the relative closure values of the experimental group when the RQ value of the UCP-2 mRNA gene expression in the obese control group was 1 (FIG. 29). The expression of UCP-2 mRNA in the obese control group was significantly decreased compared to the normal control group. In the experimental group, ALA + LAB (200 mg / kg) (p <0.01) and ALA + LAB (100 mg / kg) / kg) treated group was slightly increased compared to the obese control group, but there was no statistical significance.

Adiponectin  Gene expression pattern experiment

Adiponectin, which is specifically expressed in adipocytes, increases insulin sensitivity in liver tissues and increases fatty acid oxidation to decrease body weight. After the body weight measurement according to Example 14, the liver surrounding tissues of the rats to which the normal control, the fat control, and the test sample were administered were treated as in Example 16 and PCR was performed. The probe used was 5'-TTCAAATGAGATTGTGGGAAAAT-3 '(sense), 5'-ACCGATACAGTACAGTACAGTA-3' (antisense). Analysis was performed on the relative closure values of the experimental group when the RQ value of the adiponectin mRNA gene expression in the obese control group (DIO-NC) was set to 1. Adiponectin mRNA expressed in liver of obese control group showed Adiponectin mRNA gene expression similar to that of normal control. Compared with the control group, the positive control group, MTS - treated group, was not significantly different from the obese control group. However, the ALA + LAB (200 mg / kg) (p <0.05) and ALA + LAB (100 mg / kg) showed a statistically significant increase 4 to 2 times.

<110> WEDEA <120> Composition for preventing and treating degenerative brain          disease using novel lactic acid bacteria <130> P17040980423 <150> KR 10-2016-0160188 <151> 2016-11-29 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 1427 <212> DNA <213> Lactobacillus reuteri <400> 1 agtcgtacgc actggcccaa ctgattgatg gtgcttgcac ctgattgacg atggatcacc 60 agtgagtggc ggacgggtga gtaacacgta ggtaacctgc cccggagcgg gggataacat 120 ttggaaacag atgctaatac cgcataacaa caaaagccac atggcttttg tttgaaagat 180 ggctttggct atcactctgg gatggacctg cggtgcatta gctagttggt aaggtaacgg 240 cttaccaagg cgatgatgca tagccgagtt gagagactga tcggccacaa tggaactgag 300 acacggtcca tactcctacg ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc 360 tgatggagca acaccgcgtg agtgaagaag ggtttcggct cgtaaagctc tgttgttgga 420 gaagaacgtg cgtgagagta actgttcacg cagtgacggt atccaaccag aaagtcacgg 480 ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggatttattg 540 ggcgtaaagc gagcgcaggc ggttgcttag gtctgatgtg aaagccttcg gcttaaccga 600 agaagtgcat cggaaaccgg gcgacttgag tgcagaagag gacagtggaa ctccatgtgt 660 agcggtggaa tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg 720 caactgacgc tgaggctcga aagcatgggt agcgaacagg attagatacc ctggtagtcc 780 atgccgtaaa cgatgagtgc taggtgttgg agggtttccg cccttcagtg ccggagctaa 840 cgcattaagc actccgcctg gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg 900 ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc 960 aggtcttgac atcttgcgct aaccttagag ataaggcgtt cccttcgggg acgcaatgac 1020 aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tgttactagt tgccagcatt aagttgggca ctctagtgag actgccggtg 1140 acaaaccgga ggaaggtggg gacgacgtca gatcatcatg ccccttatga cctgggctac 1200 acacgtgcta caatggacgg tacaacgagt cgcaagctcg cgagagtaag ctaatctctt 1260 aaagccgttc tcagttcgga ctgtaggctg caactcgcct acacgaagtc ggaatcgcta 1320 gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacaccatgg gagtttgtaa cgcccaaagt cggtggccta accttta 1427 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMPK-alpha1 forward primer <400> 2 aagccgaccc aatgacatca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AMPK-alpha1 reverse primer <400> 3 cttccttcgt acacgcaaat 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> UCP-2 forward primer <400> 4 ttcaaatgag attgtgggaa aat 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> UCP-2 reverse primer <400> 5 accgatacag tacagtacag ta 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin forward primer <400> 6 ttcaaatgag attgtgggaa aat 23 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin reverse primer <400> 7 accgatacag tacagtacag ta 22

Claims (5)

A novel Lactobacillus reuteri ELF (accession number: KCTC 13154BP).
A health functional food for preventing or treating Alzheimer's disease comprising a novel Lactobacillus reuteri ELF (Accession No: KCTC 13154BP).
3. The method of claim 2,
A health functional food for preventing or treating Alzheimer which further comprises 5-aminolevulinic acid.
A novel pharmaceutical composition for preventing or treating Alzheimer's disease comprising Lactobacillus reuteri ELF (Accession No .: KCTC 13154BP).
5. The method of claim 4,
A pharmaceutical composition for the prevention or treatment of Alzheimer which further comprises 5-aminolevulinic acid.

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