KR101087973B1 - Novel Lactobacillus helveticus, culture method thereof and use thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus helvetticus, its culture method and use, and more particularly to a novel Lactobacillus helvetticus, a culture thereof, which produces a product that inhibits the production of beta amyloid protein. It relates to a pharmaceutical composition and a culture method for preventing or treating dementia or cognitive dysfunction. The lactobacillus helvetticus or the culture thereof of the present invention is effective in preventing or treating dementia, enhancing cognitive function or preventing or treating cognitive dysfunction.

Lactobacillus Helveticus, Dementia, Cognitive Impairment, Beta Amyloid, Alzheimer's

Description

Novel Lactobacillus helveticus, culture method, etc.

The present invention relates to a novel Lactobacillus helvetticus, its culture method and use, and more particularly to a novel Lactobacillus helvetticus, a culture thereof, which produces a product that inhibits the production of beta amyloid protein. It relates to a pharmaceutical composition and a culture method for preventing or treating dementia or cognitive dysfunction.

According to the Korean Society of Dementia, dementia is a type of syndrome that causes people's intellectual and social activities to be lost due to a number of causal diseases, resulting in impairments in daily life. In other words, dementia is a brain disease caused by damage and loss of nerve cells, and severe disorders such as memory, concentration, speech, and cognition progress progressively for a long time and ultimately lead to death. The causative diseases include Alzheimer's disease, Vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease And Pick's disease are known.

The highest incidence of these causative diseases is Alzheimer's disease, which accounts for 50-70% of all dementia patients. Alzheimer's disease is classified into sporadic type and familial type depending on the cause. More than 80% of cases are sporadic and the cause of the disease is not known. Most cases are known to occur after age 65 (Physiol Rev 2001, 81 : 741-66). Familiality accounts for less than 20% of the total, and the major causes of 100% mutations in genes such as amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2), and the onset time is fast Occurs below. Alzheimer's disease is a pathological feature in which senile plaques and neurofibrillary tangles accumulate outside and inside neurons, respectively. The senile plaques are generally spherical, mainly found in the limbic system or neocortex, and peptides called beta amyloid (β) bind and deposit with each other to form nuclei and degenerated neurofibrillary axons around them. (axon), dendrites, activated microglia and astrocytes. Neuronal fiber concentrates are substances in neurons formed by binding to each other after abnormally large phosphorylation of a protein called tau is observed in various brain tissues of patients with Alzheimer's disease.

The number of dementia patients, including Alzheimer's disease, continues to grow as the population ages. Alzheimer's disease is known to be around 26 million people worldwide as of 2008 and is expected to grow to more than 100 million by 2050 due to an increase in elderly population (Carlsson, J Alz Dis, 2008, 15: 327-338). According to a survey by the Korea Institute for Health and Social Affairs, 350,000 people, or 8.3% of the population aged 65 or older, were dementia patients in 2005, and will continue to increase with the increase of the elderly population. Predicting Although the number of dementia patients is low in the total population, dementia is attracting attention because of the high socioeconomic burden of caring for dementia patients. One year of treatment and care for a person with severe dementia costs more than 100 million won, which means that as of 2005, if all patients were treated with dementia, the cost would be about 35 trillion won. In addition to this enormous economic burden, the social life of families with dementia suffers a lot. In general, to care for one dementia patient, two or more carers are needed, which leads to a limitation in the social life of the family, and furthermore, the loss of the economically active population, which causes a great loss in the country. For these reasons, a number of studies for the treatment of dementia have been conducted at the national level in developed countries including the United States, and research on dementia has been actively conducted in Korea.

To date, research has shown that the main cause of Alzheimer's disease is Aβ, a constituent of the senile plaque, which causes neuronal damage and causes Alzheimer's disease. Aβ is a peptide formed by degradation of β- and γ-secretase, one of the main pathogens of familial Alzheimer's disease, consisting of 40-42 amino acids (Nature, 1999, 402: 533-7). ). Based on these mechanisms, studies on the development of inhibitors of β- and γ-secretase and α-secretase activity promoters that inhibit the production of Aβ are underway. In addition, they inhibit accumulation of Aβ or promote the accumulation of accumulated Aβ. Drugs are being developed (Nat Rev Neurosci, 2004, 5: 677-85). Recently, efforts to develop Aβ as a vaccine have also been attempted by multinational pharmaceutical companies. However, the mechanism of action-based dementia treatment takes a long time to be commercialized, and those currently on the market are acetylcholine esterase inhibitors such as Tacrine, Donepezyl, and Rivastigmin, which have improved memory and short-term relief. , Reminyl (Galantamine) and NMDA receptor antagonist memantine (Ebixa). In addition, blood circulation improvers such as niceroline, elcarnitine, and ginkgo biloba extract are indirectly confirmed to be effective for vascular dementia.

As discussed above, the current dementia treatment mainly serves to alleviate the symptoms of early dementia, but the therapeutic effect is weak and new drugs based on the mechanism of action are still required for development and release, and candidate drugs have side effects and are expected to be effective as expected. It does not appear and is failing in the clinic (J Alz Dis. 2008, 15; 327-338). For these reasons, there is an increasing demand for development of alternative substances, such as drugs or health functional foods, which can suppress the pathogenesis and prevent long-term effects due to the prevention of Alzheimer's disease.

Lactobacillus has been used for a long time and it is recognized that Bulgarians who take Lactic Acid Bacteria fermented foods have a high rate of healthy longevity. In addition, Mechinnikov's research shows that harmful substances produced by enteric harmful bacteria enter the blood and pose a threat to health, and many studies have been conducted on the function of lactic acid bacteria in attempts to replace the intestinal flora with beneficial lactic acid bacteria. In addition, many beneficial functions such as anti-cancer effect, immune enhancing effect and anti-aging effect have been reported (Trends Biotechnol, 1997, 15; 270-274). Currently, lactic acid bacteria and fermented products are widely used as medicines or health foods.

In order to further expand the functions of these beneficial lactic acid bacteria and to use them for the purpose of preventing dementia, which has recently caused serious economic and social problems, studies on the protective effect of lactic acid bacteria on dementia have been conducted. Prior to conducting the study, research on literature and patents related to dementia of lactic acid bacteria could not confirm the relevant data.

The inventors of the present invention, while studying a new substance for the treatment of dementia, found a lactic acid strain containing a substance exhibiting an inhibitory effect on beta amyloid production by inhibiting the action of β-secretase, one of the causes of dementia, in the culture medium and its culture solution. The present invention was completed by confirming the effect of preventing or treating dementia or cognitive dysfunction by animal experiment.

It is therefore an object of the present invention to provide a Lactobacillus helvetticus which produces a product which inhibits the production of beta amyloid protein.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating dementia, which contains Lactobacillus helvetica or its culture as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction, which contains Lactobacillus helvetticus or a culture thereof as an active ingredient.

Another object of the present invention is to provide a food composition for improving cognitive function containing Lactobacillus helvetica or its culture as an active ingredient.

Still another object of the present invention is to inoculate Lactobacillus helvetikus cultured in a medium containing a dairy material; (b) removing the lactic acid bacteria from the culture; (c) Lactobacillus helvetticus culture having a dementia preventive or therapeutic effect comprising the step of precipitating and separating the active ingredient by adding a solvent selected from the group consisting of acetone or an alcohol having 1 to 6 carbon atoms, the culture from which the lactic acid bacteria have been removed It is to provide a method for producing water.

In order to achieve the above object, the present invention provides a Lactobacillus helveticus (Accession Number: KCTC-11332BP) to produce a product that inhibits the production of beta amyloid protein.

In order to achieve the another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating dementia, which contains Lactobacillus helvetticus or a culture thereof as an active ingredient.

In order to achieve the other object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction, which contains Lactobacillus helvetticus or a culture thereof as an active ingredient.

In order to achieve the other object of the present invention, the present invention provides a food composition for improving cognitive function containing Lactobacillus helvetticus or a culture thereof as an active ingredient.

In order to achieve another object of the present invention, the present invention comprises the steps of: (a) inoculating the culture medium containing lactobacillus helveticus containing the raw material; (b) removing the lactic acid bacteria from the culture; (c) Lactobacillus helvetticus culture having a dementia preventive or therapeutic effect comprising the step of precipitating and separating the active ingredient by adding a solvent selected from the group consisting of acetone or an alcohol having 1 to 6 carbon atoms, the culture from which the lactic acid bacteria have been removed Provided are methods for preparing water.

Hereinafter, the present invention will be described in detail.

The Lactobacillus Helveticus IDCC 3801 strain of the present invention is a novel microorganism isolated from the feces of breastfeeding infants.

The microorganism of the present invention is a strain belonging to the genus Lactobacillus in the molecular phylogenetic analysis based on the 16S rDNA sequencing strain showing the highest molecular systematic flexibility to the standard strain of Lactobacillus helveticus (Lactobacillus helveticus) Was identified as Lactobacillus helveticus IDCC 3801, and was deposited on May 8, 2008 with the Korea Microbiological Conservation Center (www.kccm.or.kr), an international microbial depositing institution for patent applications. (Accession number: KCTC-11332BP).

The inventors of the present invention have been shown to be effective in inhibiting the production of beta amyloid by inhibiting the action of β-secretase in the culture of Lactobacillus helvetticus and the culture is excellent in preventing or treating dementia. Animal experiments confirmed that. Such a dementia preventive or therapeutic effect of the Lactobacillus helvetticus culture of the present invention is an effect first known by the present invention.

The Lactobacillus helvetticus according to the present invention can be cultured in large quantities by a conventional method for culturing Lactobacillus sp. As the culture medium, a medium consisting of a carbon source, a nitrogen source, vitamins and minerals or whey medium using milk components, skim milk medium, and the like can be used. Available as the carbon source in the medium for the production of the culture of the present invention is at least one selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose and combinations thereof, Preferably it is at least one selected from the group consisting of sucrose, fructose, glucose, galactose, arabinose and lactose, more preferably glucose. The nitrogen source available in the culture medium for producing a culture of the present invention is one selected from the group consisting of yeast extract, soytone, peptone, bee extract, tryptone, cachetone and combinations thereof Or more, preferably one or more selected from the group consisting of yeast extract, peptone, tryptone and soyton, and more preferably yeast extract or soyton.

On the other hand, the present invention provides a pharmaceutical composition for preventing or treating dementia or cognitive impairment or a food composition for improving cognitive function, which contains Lactobacillus helvetticus or a culture thereof as an active ingredient.

The culture of the present invention can also be used to remove the cells with a culture medium cultured Lactobacillus helvetikus. The Lactobacillus helvetticus can be any of the same kind of lactic acid bacteria, and the range includes all subspecies or variants of the Lactobacillus helveticus species. Preferably it may be Lactobacillus helveticus IDCC 3801 of accession number KCTC-11332BP.

In order to prepare a culture solution of the present invention having an effect of inhibiting the production of beta amyloid, the lactobacillus helveticus of the present invention may be cultured with a culture medium containing dairy material. Dairy ingredients are milk components derived from mammals. The dairy ingredients are sterilized in a liquid state and can be used as they are or powdered by a method such as lyophilization. Dairy ingredients can be taken directly from animals or purchased from commercially available products. Examples of dairy ingredients include, but are not limited to, milk, goat's milk, goat's oil, camel oil or their skim milk, whole milk, and the like.

For example, a liquid medium added with glucose and milk can be used to prepare a Lactobacillus helvetticus strain culture that inhibits the production of beta amyloid protein. The culture can be carried out under conventional lactic acid bacteria culture conditions, for example, can be cultured for about 24 hours at about 37 ℃. Centrifugation or filtration may be performed to recover the cells in the culture and to separate the culture medium, and this step may be performed according to the needs of those skilled in the art. The concentrated cells can be preserved so as not to lose their activity by freezing or lyophilizing according to a conventional method. For example, it may be stored at minus 80 ℃ containing glycerol components or suspended in sterile 10% skim milk can be lyophilized. In addition, the microorganism according to the present invention may be improved or improved to have equivalent activity or better activity by conventional physicochemical mutation methods and the like known in the art.

Cultures that inhibit the production of the beta amyloid protein of the present invention can be purified and used to increase the efficacy and to be used as a pharmaceutical composition. The purification process may be performed by any method as long as the activity of the culture of the present invention is maintained. Preferably, the impurities are removed by centrifugation, filtration, and ultrafiltration, and then the fraction containing the active ingredient is precipitated by an organic solvent. Can be precipitated. The organic solvent used may be any one as long as it can precipitate the active ingredient in the culture of the present invention, preferably acetone or an alcohol having 1 to 6 carbon atoms. The amount of organic solvent used for precipitation may be any amount as long as the activity of the culture is maintained and may vary depending on the type of precipitation solvent, but preferably, if the precipitation solvent is acetone or alcohol, it is more than 1 times the volume of the culture. It can be added up to 4 times and mixed, and centrifuged to separate the active ingredient. Precipitation using an organic solvent may be performed once, or may be repeated one or more times with the same or different types of organic solvents or with the same or different volume ratio with the culture. When repeated, the object may be either a solution of a precipitate from a previous precipitation or a supernatant from a previous precipitation. The precipitated active ingredient can be used by itself or in other solvents.

On the other hand the present invention (a) inoculating the culture medium containing lactobacillus helveticus containing the raw material; (b) removing the lactic acid bacteria from the culture; (c) Lactobacillus helvetticus culture having a dementia preventive or therapeutic effect comprising the step of precipitating and separating the active ingredient by adding a solvent selected from the group consisting of acetone or an alcohol having 1 to 6 carbon atoms, the culture from which the lactic acid bacteria have been removed Provided are methods for preparing water.

In step (a), lactobacillus helveticus is inoculated in a medium containing dairy material, and the culture is fermented. The Lactobacillus helvetticus can be any of the same kind of lactic acid bacteria, and the range includes all subspecies or variants of the Lactobacillus helveticus species. Preferably it may be Lactobacillus helveticus IDCC 3801 of accession number KCTC-11332BP. Inoculation of Lactobacillus helvetticus is carried out by adding the appropriate amount of Lactobacillus helticus to the culture medium so that Lactobacillus helvetus can be fully grown under culture conditions. The inoculated amount of Lactobacillus helveticus may be added to the culture medium in a pre-cultured Lactobacillus helvetticus culture medium in an amount of about 1 to 5% (v / v). Cultivation and fermentation of Lactobacillus helveticus can be made according to the culture medium and the culture medium containing milk materials known in the art. This process can be easily adjusted and used by those skilled in the art according to the strain selected. Such various methods are disclosed in various documents (eg, James et al., Biochemical Engineering, Prentice-Hall International Editions). Suspension cultures and adherent cultures are classified into batch, fed-batch and continuous cultures according to the culture method.

 In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, in order to maintain an aerobic state, oxygen or an oxygen-containing gas (eg, air) may be injected. The temperature at the time of culture is usually 25 ℃ to 40 ℃, preferably 35 ℃ to 40 ℃, can be incubated for 7 hours to 40 hours.

In step (b), lactic acid bacteria are removed from the fermented product fermented and cultured in step (a). After the end of the culture may be subjected to centrifugation (filtration) or filtration process to separate, remove the lactic acid bacteria cells in the culture, such steps can be performed by those skilled in the art as needed. At this time, removal of the bacteria means that the lactic acid bacteria cells have been substantially removed through a method for removing conventional cells.

Step (c) may be precipitated and separated as described above.

On the other hand, the production method of the lactic acid bacteria culture of the present invention may further comprise the step of ultrafiltration (ultrafiltration) of a substance having a molecular weight of 5,000 or less for smooth separation and concentration of the active ingredient. In the production method of the present invention (a) inoculating the culture medium containing lactobacillus helvetikus in the medium containing the raw material; (b) centrifuging the culture to remove lactic acid bacteria; (c) obtaining a substance having a molecular weight of 5,000 or less in the culture in which the lactic acid bacteria were removed; And (d) adding a solvent selected from the group consisting of acetone and an alcohol having 1 to 6 carbon atoms to the obtained material to precipitate and separate the active ingredient, thereby preventing the dementia of Lactobacillus helvetticus culture. It may be a manufacturing method.

Steps (a), (b), and (d) can be performed as described above.

In the above (c) step (b) to obtain a substance with a molecular weight of 5,000 or less in the culture from the lactic acid bacteria removed. This may be preferably performed by a method of ultrafiltration using a filter that filters only a substance having a molecular weight of 5,000 or less, to obtain an ultrafiltration solution, but is not limited thereto.

In an embodiment of the present invention, nine kinds of lactic acid bacteria are isolated from feces of breastfeeding infants and precultured in MRS medium, using SG medium containing 10% skim milk powder and 0.5% glucose for 37 ° C. for 25 hours. The culture was obtained by stationary culture. The culture was centrifuged at 15,000 rpm for 10 minutes to remove the strain by the method of taking a supernatant, followed by ultrafiltration using a filter membrane that filtered first with a 0.22um syringe filter and filtered molecular weight 5,000 or less. The filtered solution was added to acetone or alcohol by 1 times the volume, and then centrifuged to obtain a 1-fold precipitate, and to the remaining supernatant, 1-fold volume of the ultrafiltrate was added to secure a 2-fold precipitate in the same manner. . A four-fold precipitate was also secured in the same way. The recovered precipitate was dissolved in 0.5% trifluoroacetic acid (TFA) and used (see Examples 1-1 and 1-2).

The recovered acetone precipitate (precipitated by adding twice the acetone compared to the ultrafiltrate) was injected with β-secretase and wild-type APP695 gene, treated with BA-3, a HEK293 cell line overexpressed, and cultured. Lactobacillus having a β-secretase activity inhibitory effect was selected by measuring the amount of APPα and APPβ in the interior (see Example 1-3). As a result, it was confirmed that the Lactobacillus Helveticus IDCC 3801 strain of the present invention has an excellent effect of inhibiting β-secretase activity, and the strain was identified as belonging to the Lactobacillus Helveticus species in May 2008. It was deposited at the Korea Microorganism Conservation Center on 8th day (Accession Number: KCTC-11332BP) (see Example 1-4).

In another embodiment of the present invention, the conditions for obtaining a culture that inhibits the β-secretase activity of the present invention were investigated. As a result, it was confirmed that the Lactobacillus helvetticus of the present invention has an effect of inhibiting β-secretase activity when cultivated in SG medium containing skim milk powder rather than culturing in MRS which is a lactic acid bacteria culture medium. In comparison, it is possible to precipitate using alcohol, which is harmless to the human body, and it was confirmed that β-secretase activity of the precipitate was best suppressed, especially when precipitated by adding alcohol twice as large as the culture (see Example 2). ).

In another embodiment of the present invention, the memory test through the Y-maze and the amount of Aβ40 in the brain tissue using an Alzheimer's disease animal model to confirm whether the culture of the present invention is effective against Alzheimer's disease, which is a dementia or a related disease, It was. As a result, it was found that the Y-maze test resulted in memory improvement of about 44% when the fermentation product of the present invention was treated, and Aβ40 was reduced by about 20%. As a result, it was found that the culture of Lactobacillus helvetticus of the present invention showed an effect of improving dementia in an animal model of Alzheimer's disease (see Example 4).

On the other hand, the present invention provides a pharmaceutical composition for the prevention or treatment of dementia containing Lactobacillus helvetica or its culture as an active ingredient. The Lactobacillus helvetticus can be any of the same kind of lactic acid bacteria that inhibit the production of beta amyloid protein disclosed in the present invention, and the range includes all subspecies or variants of the Lactobacillus helveticus species. Preferably it may be Lactobacillus helveticus IDCC 3801 of accession number KCTC-11332BP.

The dementia of the present invention refers to a disease in which cognitive impairment and personality change occur after maintaining normal intellectual level. Dementia causes the brain nerves to be destroyed by various causes, resulting in general disorders of mental function such as memory impairment, speech impairment, urinary incontinence, paranoid thinking, aphasia, and psychiatric symptoms such as depression, personality disorder, and aggression. May be accompanied. Diseases classified as dementia include, but are not limited to, Alzheimer's dementia, vascular dementia, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia, Creutzfeldt-Jakob dementia, and Pix's disease. Dementia, senile dementia and dementia due to head injury.

The composition of the present invention inhibits the activity of β-secretase, has the activity of promoting the activity of α-secretase and ultimately inhibiting the production of beta amyloid, which was first revealed by the present inventors. .

In Alzheimer's disease, Aβ and tau proteins are the main causes of senile plaques and nerve fibers, which are formed inside and outside nerve cells, respectively, causing brain tissue atrophy and memory disorders. Aβ and tau are closely related to direct neuronal cell death, with Aβ suggesting that activation of the Aβ-producing pathway in APP causes early symptoms and tau protein involvement in subsequent disease acceleration. (Small and Duff, Neuron, 2008, 5: 591-598). Therefore, in order to prevent Alzheimer's disease, improve symptoms or treat, it is considered that the blocking of Aβ production pathway is important. Aβ is cleaved by C-terminal fragment β (CTFβ) that contains APPβ and Aβ amino acid sequences by β-secretase, and then γ-secretase acts on CTFβ, resulting in ACID (APP intracellular domine). ) And A [beta] are formed, thus inhibiting the activity of [beta] -secretase is one method of preventing, ameliorating or treating Alzheimer's disease. Α-secretase also inhibits the production of Aβ by competitively breaking down APP with β-secretase, and APP, one of the products degraded by α-secretase, induces APPα production. Increasing the activity of is another way of preventing, ameliorating or treating Alzheimer's disease.

The composition of the present invention is an A [beta] production blocker, which is compatible with the above-mentioned object of developing Alzheimer's disease therapeutic agent that effectively blocks the A [beta] production pathway and promotes the production of APP [alpha], which shows a neuroprotective effect.

In one embodiment of the present invention, the composition of the present invention promotes the production of APPα, and confirmed by Western blot through cell experiments that inhibit the production of APPβ (see Example 2).

In another embodiment of the present invention it was confirmed by the beta amyloid quantitative kit through the animal experiment that the composition of the present invention reduces the beta amyloid levels of the brain in animal models of dementia (see Example 4-2). In addition, it was confirmed that the composition of the present invention improves or treats memory and cognitive decline, which is one of the main symptoms of dementia in a dementia animal model, through a Y-shaped maze test in dementia-induced rats to which the composition of the present invention is administered. (See Example 4-1).

As a result, the composition of the present invention effectively blocks the A [beta] production pathway, promotes the production of APPα that exhibits neuronal protective effects, and has been found to have an excellent effect on preventing, improving or treating dementia.

The present invention also provides a pharmaceutical composition for preventing or treating cognitive dysfunction, which contains Lactobacillus helvetticus or a culture thereof as an active ingredient.

The Lactobacillus helvetticus can be any of the same type of lactic acid bacteria that are effective in cognitive dysfunction symptoms, and the range includes all subspecies or variants of the Lactobacillus helveticus species. Preferably it may be Lactobacillus helveticus IDCC 3801 of accession number KCTC-11332BP.

Cognitive impairment is a mental or behavioral disorder caused by brain disease, brain injury, or addiction, or an mental disorder caused by endocrine disorders, metabolic or nutritional abnormalities, or drugs. It is a type of brain disease that occurs not only in the case of forgetfulness (anmesia), which is a temporary short-term memory disorder, but also in dementia, which causes general impairment of mental ability and judgment according to the severity of symptoms.

Cognitive deficits or cognitive dysfunctions include cognitive functions or cognitive domains such as work memory, attention and vigilance, language learning and memory, visual learning and memory, reasoning and problem solving such as executive functions, processing speed Or lowering of social cognition. In particular, cognitive deficits or cognitive impairments integrate attention deficit, disrupted thinking, slow thinking, difficulty understanding, poor concentration, problem solving disorders, poor memory, difficulty in expressing thoughts and / or thoughts, emotions and behaviors. Difficulty in doing, or the disappearance of irrelevant thoughts. The terms "cognitive defect" and "cognitive impairment" are intended to refer to the same and are used interchangeably.

Diseases classified as cognitive impairment include, but are not limited to, cognitive deficits associated with schizophrenia, cognitive deficiencies associated with age-induced memory impairment or psychosis, cognitive deficiencies associated with diabetes, cognitive deficiencies associated with post-stroke, hypoxia and Associated memory defects, cognitive and attention deficits associated with senile dementia, memory problems associated with mild cognitive impairment, impaired cognitive function associated with dementia, impaired cognitive function associated with Alzheimer's disease, impaired cognitive function associated with Parkinson's disease, impaired cognitive function associated with vascular dementia, Cognitive problems associated with brain tumors, Pick's disease, cognitive deficiencies due to autism, cognitive deficiencies after electroconvulsion therapy, cognitive deficiencies associated with traumatic brain injury, memory loss disorders and delirium.

In one embodiment of the present invention, it was confirmed that the composition of the present invention exhibits excellent cognitive function in the underwater maze and manual avoidance test in the animal model of scopolamine induced memory impairment administered the composition of the present invention (execution) See example 5).

The pharmaceutical composition according to the present invention may contain Lactobacillus helvetticus or its culture alone or may further contain one or more pharmaceutically acceptable carriers, excipients or diluents.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, it may include a variety of drug delivery materials used for oral administration to the peptide formulation. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycols, and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent and the like in addition to the above components. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The composition of the present invention may be administered to any mammal, including humans. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Can be.

The pharmaceutical composition of the present invention may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can be obtained by tablets or dragees by combining the active ingredients with solid excipients, milling them, adding suitable auxiliaries and then processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Formulations for parenteral administration may be formulated by methods known in the art in the form of injections, creams, lotions, external ointments, oils, humectants, gels, aerosols and nasal inhalants. These formulations are described in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995, a prescription generally known in all pharmaceutical chemistries.

The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol which is administered in multiple doses for a long time. . The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the extent of the disease. Preferably the preferred total dose of the composition of the present invention may be about 0.01 μg to 500 mg, most preferably 0.1 μg to 100 mg per kg of patient body weight per day. However, the dosage of the composition is determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the pharmaceutical composition is determined In view of this, one of ordinary skill in the art will be able to determine the appropriate effective dosage for the particular use of the composition as a prophylactic or therapeutic agent for dementia. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is shown.

In another aspect, the present invention provides a food composition for improving cognitive function containing Lactobacillus helvetticus or a culture thereof as an active ingredient.

The Lactobacillus helvetticus can be any of the same type of lactic acid bacteria that are effective in cognitive dysfunction symptoms, and the range includes all subspecies or variants of the Lactobacillus helveticus species. Preferably it may be Lactobacillus helveticus IDCC 3801 of accession number KCTC-11332BP.

The food composition includes all forms such as functional foods, nutritional supplements, health foods and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the lactic acid bacteria fermented product of the present invention may be prepared in the form of tea, juice, and drink for drinking, or may be consumed by granulation, encapsulation, and powdering. In addition, the lactic acid bacteria fermentation product of the present invention can be mixed with other active ingredients to prepare in the form of a composition.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef) Breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible oils, margarine, vegetable It can be prepared by adding the lactic acid bacteria fermentation product of the present invention to proteins, retort foods, frozen foods, various seasonings (for example, miso, soy sauce, sauce, etc.).

In addition, in order to use the lactic acid bacteria fermented product of the present invention in the form of food additives can be prepared in the form of powder or concentrate.

The preferred content of the lactic acid bacteria fermentation product of the present invention in the food composition of the present invention may include about 0.01 to 10% by weight relative to the total weight of the food.

Therefore, the pharmaceutical composition containing Lactobacillus helvetticus or its culture as an active ingredient of the present invention is effective in preventing or treating dementia or cognitive dysfunction. In particular, the composition of the present invention has an excellent effect of inhibiting the production of beta amyloid and promoting the production of APPα having a neuroprotective effect, and thus is excellent in preventing or treating dementia or cognitive dysfunction such as Alzheimer's disease. In addition, the culture production method of the present invention is effective in the production of a culture having excellent efficacy in the prevention or treatment of dementia or cognitive dysfunction of the present invention using the strain of the present invention. Therefore, the pharmaceutical composition or the method of preparing the culture containing Lactobacillus helvetticus or its culture as an active ingredient of the present invention is used for the prevention or treatment of a person of age at which dementia or cognitive dysfunction occurs or is likely to occur. It can be used in various fields such as pharmaceutical manufacturing.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Selection of Lactobacillus Suppresses β-Secretase Activity

<1-1> lactic acid bacteria fermentation product sample preparation method

Collect feces from breastfeeding infants within 2 weeks of birth at a postpartum cooking center in Seoul, Gyeonggi-do, and smear them on LBS flat medium (pH4.0, containing 0.3% bile, LBS agar, Becton, Dickson and Company, USA). Lactic acid bacteria were isolated and the fermentation products cultured with these strains were examined for APPβ inhibition activity.

<1-2> Preparation of lactic acid bacteria fermentation product sample

The lactic acid strain isolated in Example <1-1> was subcultured or precultured in MRS medium (de Man-Rogosa-Sharpe media). The culture medium was obtained by inoculating 3% (v / v) of SG (10% skim milk powder, 0.5% glucose, pH 6.8) medium or milk containing skim milk powder as a main ingredient, and incubating at 37 ° C for 24 hours. After cultivation in SG medium or milk, the culture state showed curd formation like lactic acid bacteria fermented milk and separated from whey. The medium to which 3% MRS medium was added was maintained under the same conditions and used as a control. Each culture was centrifuged at 15,000 rpm for 10 minutes to recover the supernatant, which was first filtered with a 0.22 μm syringe filer. Next, ultrafiltration was performed with a filter membrane for filtering a molecular weight of 5,000 or less. Then, acetone or alcohol was added to the ultrafiltrate first, and then mixed 1 times, and then centrifuged at 15,000 rpm for 10 minutes to obtain a 1x precipitate. A double precipitate was obtained by the method. A four-fold precipitate was also secured in the same way. Each precipitate was recovered and dried, weighed, and adjusted to a concentration suitable for 0.5% trifluoroacetic acid (TFA). Suspension was used for the test.

<1-3> Screening of lactic acid bacteria inhibiting β-secretase activity

Lactobacillus strains having β-secretase activity inhibitory activity were selected using the acetone precipitates (precipitation obtained by adding twice the acetone to the ultrafiltrate) of the nine strain cultures obtained in Example <1-2>. As a control, only a precipitate of acetone precipitated in the same manner as the SG medium culture medium not inoculated with lactic acid bacteria was used.

To investigate the effects of lactobacillus fermentation product samples on the metabolism of amyloid precursor protein (hereinafter called APP), HEK293 cell lines (hereinafter referred to as "BA-") were injected with β-secretase and wild-type APP695 genes. 3 ', Yeon et al, Peptides, 2007, 28: 838-844).

BA-3 is useful for the detection of active ingredients that directly or indirectly affect β-secretase activity by generating a lot of APPβ by overexpression of β-secretase. BA-3 was incubated for 1 day by dispensing in a 6-well plate using DMEM containing 10% FBS at a density of 80% or more. After removing the medium and adding 0.9 ml of fetal bovine serum (FBS) free of fetal bovine serum (FBS) warmed to 37 ℃ and suspended in 0.1ml free DMEM of the acetone or alcohol precipitate of Example <1-2> at a concentration of 100ug / ml Treated and incubated for 24 hours. 0.2 ml of each sample-treated medium was electrophoresed on 8% SDS-PAGE, followed by Western blot using MAB1560 (monoclonal, CHEMICON, USA) and Rb53 (polyclonal, Cephalon, USA) antibodies, antibodies of APPα and APPβ. APPα and β in the culture were measured.

To identify APPα and APPβ in the cells, the cells were collected, suspended in phosphate buffered saline (PBS), centrifuged to remove supernatant, and lysis buffer containing protease inhibitors (lysis buffer; 50 mM Tris-HCl, pH7.6, 180 mM NaCl, 2 mM EDTA, 1% Triton X-100) was added and left on ice for 30 minutes. Sonication was briefly performed, and the supernatant was recovered by centrifugation at 15,000 rpm for 10 minutes at 4 ° C., protein was quantified, and Western blot was performed using MAB1560 and Rb53 antibodies in the same manner as described above.

As a result, by examining whether there is a lactic acid strain that inhibits the production of APPβ than the control group to secure the IDCC 3801 showing the effect of inhibiting the production of APPβ.

In addition to lactic acid bacteria, similar results were observed in Bacillus subtilis and E. coli cultures, but they did not inhibit the production of APPβ.

Inhibitory Effects of APPβ Production on SG Cultured Lactic Acid Bacteria Samples. Strain APPβ inhibition rate (%) Strain APPβ inhibition rate (%) IDCC2053 0.0 IDCC2581 0.0 IDCC2097 0.0 IDCC3301 0.0 IDCC2135 3.1 IDCC3801 100.0 IDCC2145 0.0 Bacillus subtilis 0.0 IDCC2510 29.6 E. coli 0.0 IDCC2581 32.4

<1-4> Identification of Selected Lactic Acid Strains

Phylogenetic analysis was performed by comparing 16S rRNA gene sequences to identify IDCC 3801 strain, the lactic acid bacterium having the highest inhibitory effect on APPβ production.

The base sequence of 16S rRNA of IDCC 3801 strain and 16S rRNA of ATCC 15009, Lactobacillus helveticus standard strain, was compared using a Blast similarity search program (National Institute of Biotechnology Information). As a result, it was confirmed that the homology of 98.9%.

In addition, multisequence alignment was performed with 16S rRNA gene sequences of 9 Genus, 84 Lactobacillus Species, and 87 Ashdophilus strains registered in GenBank. The location in the phylogenic tree was determined. Multiple sequence alignment program was used for ClustalX. On the phylogenetic tree, the 16S rRNA gene of the IDCC 3801 strain was located closest to Lactobacillus helveticus. In addition, homology with 10 strains of 16S rRNA genes registered with Lactobacillus helveticus, such as AY369116, AM113779, DQ123580, AB362629, EF536362, EU377824, FJ172346, EU420177, FJ459815, GQ131286, showed a high identity of 99.1%.

As a result of molecular identification of the IDCC 3801 strain as described above, the strain was classified as Lactobacillus helveticus. Therefore, the strain with excellent probiotic characteristics such as acid resistance and bile acid resistance and high inhibitory effect on APPβ production was named as "Lactobacillus helvetticus IDCC 3801" and the Korea Microorganism Conservation Center (www.kccm.or.kr) Deposited on 8 May 2008 (accession number: KCTC-11332BP).

<Example 2>

Inhibitory Effect of Lactobacillus Helveticus IDCC 3801 on β-Secretase Activity and α-Secrease Activity

The inhibitory effect of APPβ production of Lactobacillus helvetticus IDCC 3801 strain culture was tested in acetone precipitated samples. It was confirmed that the acetone precipitated sample of the ultrafiltrate of the Lactobacillus helvetticus IDCC 3801 culture filtrate obtained by culturing in SG showed an inhibitory effect on APPβ production even at 25 ug / ml (see FIG. 1).

Acetone precipitates in SG culture filtrates were difficult to be applied as pharmaceuticals due to the solvent, and tested for Lactobacillus helvetticus IDCC 3801 strain to replace the solvent with alcohol.

Treatment of the alcoholic precipitate of the Lactobacillus helvetticus IDCC3801 SG culture filtrate obtained in Example <1-2 at a concentration of 100 ug / ml inhibited the production of APPβ or the production of APPα by the method of Example <1-3>. It was investigated whether or not it promoted. As a control, a precipitate precipitated by alcohol in the same manner was used only in the SG medium culture medium not inoculated with lactic acid bacteria.

As a result, when the ethanol sediment of Lactobacillus Helveticus IDCC 3801 strain culture solution was treated, the sediment precipitated at 4 times the alcoholic filtrate had the effect of inhibiting APPβ production, but unlike the acetone precipitate at the 2 sediment precipitated 100 It was confirmed that the percent inhibition effect. This double ethanol sediment showing high inhibitory activity means that the active ingredient in the culture product of Lactobacillus helvetticus IDCC 3801 strain is specifically precipitated and concentrated by double ethanol treatment. In addition, it was confirmed that the alcohol sediment samples of the Lactobacillus helvetticus IDCC 3801 strain increased the accumulation of APPα in the cells (see FIG. 2).

In conclusion, it was confirmed that the active ingredient of Lactobacillus helveticus IDCC 3801 alcoholic precipitates selectively inhibits the production of APPβ but has the property of inducing the accumulation of APPα in cells.

<Example 3>

Toxicity Test of Alcoholic Sediments in Lactobacillus Helveticus IDCC 3801 SG Culture

Single-dose toxicity studies were performed to determine the toxicity induced by oral administration of Lactobacillus helvetticus IDCC 3801 SG culture. Six-week-old, male ICR mice were observed for 7 days after a single oral dose of 3,000 mg / kg.

As a result, the toxicity test mice did not die and no abnormalities were found, indicating no toxicity (results not shown).

<Example 4>

In vivo  Dementia Improvement Efficacy Test of Alcohol Sediment in Lactobacillus Helveticus IDCC 3801 SG Culture

<4-1> Y-maze test

Controlling the Simple Effect of Lactobacillus Helveticus IDCC 3801 SG Cultured Ultrafiltrate Alcohol Sediment Using Tg2576 (Swedish mutant APP Overexpressing Mouse, 4, 9 Months Female), Alzheimer's Disease Animal Model Evaluated using. For administration, each sample was suspended in 1% carboxymethylcellulose (CMC), and the method of oral administration was performed three times a week for 8 weeks at 500 mg / kg.

The test was carried out using a Y-shaped labyrinth measuring device with three arms extending to form the letter Y and each branch having a length of 25 cm, a height of 14 cm, and a width of 5 cm. At the end of a passage in the Y-shaped maze, the head of the experiment animal was left facing the path freely for 8 minutes. The movement was recorded as an alteration in the way of admitting the entry into the passage to the hind paw of dementia rats, and the actual alteration of the passage through three passages in succession. One point was given and the spontaneous alteration behavior (%) was calculated by calculating the actual number of crossings as a percentage of the maximum possible number of crossings (ie, the total number of crossings minus two).

The Y-maze was performed once before sample processing and once after sample processing to compare the results before and after the test. As a result of the Y-maze test, 44% of the memory improvement effect after the sample treatment was compared with before the sample treatment. (See FIG. 3). The medium treated group, which was a control group, showed no significant difference from before the test.

<4-2> Aβ Inhibitory Effect of Alcohol Sediment in IDCC 3801 SG Culture in Alzheimer's Model Rats

After the test of Example <3-1>, the brain tissues of the mice were extracted and quantitatively compared with Aβ40 to determine the Aβ inhibitory effect of the alcoholic sediment in the IDCC 3801 culture.

 After completion of the test, the blood in the brain tissue was removed by perfusion with PBS, and the brain was extracted and weighed. Solution B (PBS, 5% BSA, 0.03% Tween-20, Protease), which was kept cool after 3 hours at room temperature by adding 8 times solution A (5M guanidine HCl, 50 mM Tris HCl, pH 8.0) by weight Inhibitor solution) was added 20 times by volume. The supernatant was recovered by centrifugation at 15,000 rpm for 20 minutes at 4 ° C. to quantify the protein and quantitatively compare Aβ40 in the extract using an ELISA kit (the GENETICS Company, Switzerland).

As a result, the Aβ40 value compared to the volume of brain tissue was 302.1 ± 2.1pg / ml in the control group, whereas the alcoholic sediment treated group of Lactobacillus helvetticus IDCC 3801 culture was 245.2 ± 3.2pg / ml in the group treated with Aβ40. Was found to decrease (see FIG. 4).

As a result of the above Y-maze test and Aβ40 quantitative test, it was confirmed that the alcohol sediment of Lactobacillus helvetticus IDCC 3801 SG culture filtrate ultrafiltration showed improvement effect of dementia in animal model of Alzheimer's disease.

Example 5

In vivo  Cognitive Improving Efficacy of Alcohol Sediment in IDCC 3801 SG Culture

Nine-week-old dominant ICR mice were used in each treatment group for cognitive improvement tests. The culture of Lactobacillus helvetus IDCC 3801 of the present invention and the ethanol sediment were suspended in 1% carboxymethylcellulose to prepare a sample for administration, and the control drug donepezil was used in physiological saline. 500 mg / kg of Lactobacillus Helveticus IDCC 3801 culture and medium spirit sediment, 2 mg / kg of donepezil were orally administered twice a week for 2 weeks and 60 mg of scopolamine 60 minutes before the start of the test. After oral administration of kg caused memory impairment, water maze and passive avoidance tests were performed. As a vehicle, the scopolamine-treated group and the physiological saline-treated group were used after administration of saline for two weeks instead of drugs.

<5-1> Underwater Maze Test

During the underwater maze test, the reference memory test was performed after adapting by allowing the swimmer to swim freely for 60 seconds in a platformless tank one day before the start of the test. The test was performed by measuring the time to find the platform submerged 4-5 times daily for 5 days (escape latency, in seconds), and the maximum allowable time was limited to 60 seconds. By the second day of the test, if the platform could not be located, it was guided to find the platform within the 60-second time limit.

24 hours after the end of the reference memory test, the probe test was performed by removing the platform in the tank and allowing the user to swim freely for 60 seconds to measure the time to stay at the platform.

As a result, in the reference memory test, the physiological saline treated group treated with scopolamine did not improve the time to find the platform during the test period, and the media alcohol sediment treated group had a weak learning effect. In contrast, the donepezil treated group used as a control drug significantly reduced the time to find the platform from the second day of the test showed similar improvement effect as the scopolamine non-administered group. The ethanol sediment treatment group of Lactobacillus helvetticus IDCC 3801 cultures decreased the time to find the platform as the test period elapsed, and the significance was 30.5 ± 6.2 seconds compared to the 50.0 ± 3.3 seconds of the scopolamine treatment group after the administration of saline on the 5th day of the test. (p <0.05) with the largest difference. 3 days (medium alcohol sediment treated group 51.0 ± 4.0 seconds, Lactobacillus helvetticus IDCC 3801 culture alcoholic sediment 35.3 ± 7.1 seconds. P <0.05) and 4 days (medium alcohol sediment treated group 49.0 ±) Lactobacillus Helveticus IDCC 3801 cultured alcoholic sediment showed significant difference in 4.2 sec, Lactobacillus Helveticus IDCC 3801 cultured alcoholic sediment 36.9 ± 4.6 seconds, p <0.05). 5). In the probe, Lactobacillus Helveticus IDCC 3801 cultured liquor sediment treatment group recovered 1.6 ± 0.6 (36.5%) from the place where the platform was located in the total swimming recovery, compared to 0.8 ± 0.3 (17.3%) of the saline treatment group. It was statistically significant higher than 2.1 times (p <0.05).

<5-2> Manual Evasion Test

The passive evasion test was conducted using a shuttle box divided into two compartments as a guillotine door. One compartment is illuminated by light and the other is unlit and wrapped in black cloth and darkened. On the first day, place the dementia rat in a bright room, give it 30 seconds of search time, open the compartment door to enter the dark room, measure the acquisition latency time into the dark room, and enter the compartment door when entering the dark room. The rat was closed and subjected to a 0.5 mA electric shock for 3 seconds through the bottom of the grid to allow the rat to remember the electrical stimulation. After 24 hours, the dementia rats were placed in a bright room, and after 30 seconds of searching time, the partition door was opened, and the retention latency time for all the feet of the rats into the dark room was measured up to 300 seconds. The larger the retention latency time, the better the memory of passive avoidance through learning.

 Retention latency time of each treatment group was measured after the study of electrical stimulation. After physiological saline administration, the scopolamine treatment group was 123.7 ± 11.0 seconds, and the media alcohol sediment treatment group was 80.3 ± 35.9 seconds. The ethanol sediment treatment group of Bacillus helvetus IDCC 3801 cultures was 276.1 ± 21.5 seconds, and the ethanol sediment treatment group of Lactobacillus helvetticus IDCC 3801 cultures was significantly more than 232% (p <0.01), compared to the physiological saline treatment group. Compared with the sediment treatment group, more than 343% (p <0.01) showed improvement of memory. In addition, the ethanol sediment treatment group of the Lactobacillus helvetticus IDCC 3801 culture showed better effects than the donepezil treatment group as a control drug under the present test conditions (see FIG. 6).

As a result, the composition of the present invention was confirmed to have excellent efficacy in improving memory and cognitive function by dementia.

Although a formulation example of a pharmaceutical composition comprising a compound of the present invention will be described, the present invention is not intended to be limiting but merely illustrative.

Preparation Example 1 Preparation of Powder

Alcohol sediment 20 mg of Lactobacillus helveticus culture

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

&Lt; Formulation Example 2 > Preparation of tablet

Alcohol sediment 10 mg of Lactobacillus helvetticus culture

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Preparation Example 3 Preparation of Capsule

Alcohol sediment 10 mg of Lactobacillus helvetticus culture

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 4 Preparation of Injection

Alcohol sediment 1 mg of Lactobacillus helvetticus culture

Mannitol 180 mg

Sterile distilled water for injection 2793 mg

Na2HPO412H2O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Preparation Example 5 Preparation of Liquid

Alcohol sediment 2 mg of Lactobacillus helvetus culture

10 g per isomer

5 g mannitol

Purified water quantity

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Preparation Example 6 Preparation of Health Food

Alcohol sediment 500 mg of Lactobacillus helvetticus culture

Vitamin mixture quantity

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Preparation Example 7 Preparation of Healthy Drinks

Alcohol sediment in Lactobacillus helvetica culture 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantity

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

As described above, the pharmaceutical composition containing Lactobacillus helvetticus or its culture as an active ingredient of the present invention is effective in preventing or treating dementia or cognitive dysfunction. In particular, the composition of the present invention has an excellent effect of inhibiting the production of beta amyloid and promoting the production of APPα having a neuroprotective effect, and thus has an excellent efficacy in preventing or treating dementia or cognitive dysfunction such as Alzheimer's disease. In addition, the culture production method of the present invention is effective in the production of a culture having excellent efficacy in the prevention or treatment of dementia or cognitive dysfunction of the present invention using the strain of the present invention. Therefore, the pharmaceutical composition or the method of preparing the culture containing Lactobacillus helvetticus or its culture as an active ingredient of the present invention is used for the prevention or treatment of a person of age at which dementia or cognitive dysfunction occurs or is likely to occur. It can be used in various fields such as pharmaceutical manufacturing, so it is highly applicable to the industry.

1 is a result of confirming the β-secretase inhibitory effect of IDCC 3801 lactic acid bacteria culture by Western blot (Media: band of APPα / β in the culture cultured BA-3 cell line using only the culture medium without administration of precipitate; IDCC 3801: band of APPα / β in the culture in which the BA-3 cell line was cultured by administering the acetone precipitate of the culture of IDCC 3801 to the culture medium).

Figure 2 is a result of comparing the active ingredient precipitation according to the volume ratio of the culture and the precipitation organic solvent in Western blot (Media: culture / cell lysate cultured BA-3 cell line using only the culture medium without the administration of the precipitate) Band of APPα / β in the IDCC 3801: band of APPα / β in the culture / cell lysate in which BA-3 cell line was cultured by administering a ethanol sediment of the culture of IDCC 3801 to the culture medium.

Figure 3 is a graph of the results of comparing the spontaneous alternating behavior (%) to determine the effect of memory improvement in the dementia model mice of the IDCC 3801 lactic acid bacteria culture through the Y maze test of dementia mice (medium: IDCC 3801 sediment) Mice administered SG medium instead; IDCC 3801: mice administered IDCC 3801 sediment; initial: measured values when IDCC 3801 cultures were administered to dementia model mice by oral administration; final: 8-week oral administration period Measured value at the end of time).

Figure 4 is a graph of the change in the amount of beta amyloid in the brain in accordance with the administration of IDCC 3801 lactic acid bacteria culture in the dementia model mice using a ELISA kit (medium: mice administered SG medium instead of IDCC 3801 precipitate; IDCC 3801: Mice administered IDCC 3801 precipitate).

FIG. 5 is a graph showing the results of comparing the time to find the platform for the memory improvement effect of the IDCC3801 lactic acid bacteria culture in the underwater maze test of scopolamine induced memory damage model mice (escape latency time (s): Time taken to get to the platform (seconds); Saline + Saline: Two weeks of physiological saline instead of drug, followed by saline instead of scopolamine; Saline + Scopolamine: Two weeks of saline instead of drug Donepezil + scopolamine: Scopolamine treated group after 2 weeks of donepezil administration; Medium + scopolamine: Scopolamine treated group after 2 weeks of medium alcohol sediment; Lactobacillus + scopolamine: Lactobacillus alcoholic precipitate for 2 weeks Scopolamine treated group after administration).

FIG. 6 is a graph showing the result of measuring the time taken to enter a dark room to examine the memory improvement effect of the IDCC3801 lactic acid bacteria culture in a passive avoidance test of a scopolamine induced memory injury model mouse (Retention Latency time (s) : Time taken to enter dark room (seconds); Sal + Sal: Physiological saline instead of scopolamine after 2 weeks of saline instead of drug; Sal + Sco: Scopolamine treated after 2 weeks of saline instead of drug Do + Sco: Scopolamine treated group after 2 weeks of donepezil; M + Sco: Scopolamine treated group after 2 weeks of media alcohol sediment; 3801+ Sco: Scopolamine treated group after 2 weeks of lactic acid bacteria alcohol sediment ).

Claims (12)

Lactobacillus helveticus (Accession No .: KCTC-11332BP), which produces a product that inhibits the production of beta amyloid protein. A pharmaceutical composition for preventing or treating dementia, comprising Lactobacillus helvetus or a culture thereof as an active ingredient. A pharmaceutical composition for preventing or treating cognitive dysfunction, comprising the Lactobacillus helvetus or a culture thereof as an active ingredient. A food composition for improving cognitive function, comprising Lactobacillus helvetus or a culture thereof as an active ingredient. delete The composition according to any one of claims 2 to 4, wherein the culture is incubated with a culture solution containing dairy material. The composition of claim 6, wherein the dairy material is one or a mixture thereof selected from the group consisting of skim milk, milk, goat oil, camel oil and goat milk. The method of claim 2, wherein the dementia is Alzheimer's disease dementia, Vascular dementia, Parkinson's disease dementia, Lewy body dementia, Huntington's disease dementia. , Dementia selected from the group consisting of Creutzfeldt-Jacob disease type dementia and Pick's disease type dementia. 4. The cognitive impairment of claim 3, wherein the cognitive impairment is cognitive deficit associated with schizophrenia, cognitive deficit associated with age-induced memory impairment or mental disorder, cognitive deficit associated with diabetes, cognitive deficit associated with post-stroke, hypoxia associated memory Defects, cognitive and attention deficits associated with senile dementia, memory problems associated with mild cognitive impairment, impaired cognitive function associated with dementia, impaired cognitive function associated with Alzheimer's disease, impaired cognitive function associated with Parkinson's disease, impaired cognitive function associated with vascular dementia, brain Cognitive impairment selected from the group consisting of tumor-related cognitive problems, Pick's disease, cognitive deficits due to autism, cognitive deficiencies after electroconvulsion therapy, cognitive deficiencies associated with traumatic brain injury, memory loss disorder, and delirium Characterized in that the composition. (a) inoculating the medium containing lactose into the culture medium by inoculating the Lactobacillus helveticus of claim 1; (b) removing Lactobacillus helvetica from the culture cultured in step (a); (c) adding a solvent selected from the group consisting of acetone or an alcohol having 1 to 6 carbon atoms to the culture from which the lactobacillus helvetticus has been removed in step (b) to prevent the dementia, comprising the steps of: A method for producing a Lactobacillus helvetticus culture having a therapeutic effect. (a) inoculating the medium containing lactose into the culture medium by inoculating the Lactobacillus helveticus of claim 1; (b) centrifuging in the culture cultured in step (a) to remove Lactobacillus helveticus; (c) obtaining a substance having a molecular weight of 5,000 or less in the culture from which the Lactobacillus helvetikus of step (b) is removed; And (d) adding a solvent selected from the group consisting of acetone and an alcohol having 1 to 6 carbon atoms to the obtained material to precipitate and separate the active ingredient, Lactobacillus helbetiti having an effect of preventing and treating dementia. Method of Making Cous Cultures. delete

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