KR102713042B1 - Fermented composition for antioxidant containing Lactobacillus plantarum 200655 strain (KCCM 12204P) and fructooligosaccharide and its use for preventing or treating neurodegenerative diseases - Google Patents

Abstract

The present invention relates to an antioxidant fermented composition comprising Lactobacillus plantarum 200655 strain and fructooligosaccharides, and specifically, a nerve cell protective effect was confirmed using the antioxidant fermented composition. The fermented composition of the present invention has increased storage stability and excellent antioxidant activity and nerve cell protective effect by including fructooligosaccharides, and therefore can be used as a pharmaceutical composition for preventing or treating degenerative neurological diseases and as a health functional food.

Description

{Fermented composition for antioxidant containing Lactobacillus plantarum 200655 strain (KCCM 12204P) and fructooligosaccharide and its use for preventing or treating neurodegenerative diseases}

The present invention relates to an antioxidant fermented composition comprising Lactobacillus plantarum 200655 (KCCM 12204P), a strain isolated from kimchi, and fructooligosaccharides, and more specifically, to a composition using the present fermented composition for antioxidant purposes and a protective effect against nerve cell death, which can be used for the prevention or treatment of degenerative neurological diseases.

Probiotics is a term referring to living microorganisms or their components that have a beneficial effect on the health of the host, such as humans or animals, and are known to provide benefits to the host that ingests them, such as by balancing intestinal bacteria. In general, probiotics include beneficial bacteria such as lactic acid bacteria and bifidobacteria, and yeast, and among them, lactic acid bacteria belonging to the genera such as Lactobacillus, Lactococcus, and Streptococcus are being widely studied and used.

Lactic acid bacteria (LAB) have been used to make various fermented foods. Lactic acid bacteria use various sugars, including lactose, as substrates and convert them into lactic acid. Through this process, they give food a sour taste and lower the pH, thereby inhibiting the growth of harmful microorganisms. Lactic acid bacteria have beneficial effects on humans in many aspects, such as suppressing various intestinal diseases and enhancing immunity by regulating the host's intestinal microflora in addition to antibacterial effects. Therefore, there is a lot of interest in developing lactic acid bacteria as various food ingredients.

Synbiotics are those that simultaneously exhibit the beneficial effects of probiotics and prebiotics, and the advantages of synbiotics include improved survival rate of probiotics when passing through the upper digestive tract, continued effects of probiotics in the host digestive tract ecosystem, and health effects from surviving microorganisms that cannot be secured with prebiotics.

Meanwhile, reactive oxygen species (ROS) can cause oxidative stress, which can cause various neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease (Tian et al., 2020). Hydrogen peroxide (H ₂ O- ₂ ) is a representative ROS that can promote apoptosis of nerve cells by activating apoptosis-related factors.

As interest in health increases, there is a demand for research on functional food materials that use probiotics to achieve neuroprotective effects through synbiotic foods or functional foods using compositions.

Korean Patent Publication No. 10-2019-0103755 reported that Lactobacillus plantarum 200655 strain has an antioxidant effect. However, there has been no report on directly confirming the antioxidant effect or neuroprotective effect of Lactobacillus plantarum 200655 strain by fermenting it together with prebiotics.

Accordingly, the inventors of the present invention completed the present invention by producing a fermented composition that increases antioxidant activity through fermentation and has a neuronal cell death protection effect.

The present invention confirmed the antioxidant activity and verified the neuroprotective effect by combining the optimal prebiotics of Lactobacillus plantarum 200655 strain, thereby confirming the possibility as a pharmaceutical composition and functional food material.

Accordingly, the purpose of the present invention is to provide an antioxidant fermentation composition comprising Lactobacillus plantarum strain 200655 (KCCM 12204P) and fructooligosaccharides.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a degenerative neurological disease comprising the fermented composition.

Another object of the present invention is to provide a food composition comprising the fermented composition.

The terminology used in this specification is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprise" or "have" should be understood to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but not to exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof. Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the art to which the embodiment belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an ideal or excessively formal sense unless explicitly defined in this application.

The present invention provides an antioxidant fermented composition comprising Lactobacillus plantarum strain 200655 (KCCM 12204P) and fructooligosaccharides.

In one embodiment of the present invention, the fructooligosaccharide may be present at a concentration of 3 to 5% (w/v).

In one embodiment of the present invention, the fermented composition may have a protective effect against neuronal cell death.

In one embodiment of the present invention, the fermented composition may be characterized in that the total phenol content increases by 150% or more compared to a case in which fructooligosaccharide is not added.

The present invention also provides a pharmaceutical composition for preventing or treating a degenerative neurological disease comprising the fermented composition.

In one embodiment of the present invention, the degenerative neurological disease may be characterized by at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, brain dysfunction due to immune system abnormality, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, and dementia due to cerebral ischemia and cerebral hemorrhage.

The present invention provides a food composition comprising the fermented composition.

In one embodiment of the present invention, the food composition may be at least one selected from the group consisting of soy milk, fermented milk, milk, and fermented soybean food.

In one embodiment of the present invention, the food composition can maintain 95% or more of the initial viable bacterial count for a storage period of 16 to 30 days.

In one embodiment of the present invention, the food composition may have a β-glycosidase activity of 7.0 to 8.6 mU/mL and a total phenol content of 15.0 to 20.0 mg GAE/mL.

The present invention relates to a fermented composition using a probiotic Lactobacillus plantarum strain and fructooligosaccharides, which has an excellent neuronal cell protection effect by improving antioxidant capacity through fermentation, and can be used as a pharmaceutical composition for preventing or treating degenerative neurological diseases and as a functional food material.

Figure 1 shows the growth index of Lactobacillus plantarum 200655 strain.
Figure 2 shows changes in the viable cell count (A), pH (B), and titratable acidity (C) of Lactobacillus plantarum 200655 strain during fermentation of fructooligosaccharide-containing soymilk.
Figure 3 shows changes in the number of viable cells (A), pH (B), and titratable acidity (C) of Lactobacillus plantarum 200655 strain in fermented soymilk containing fructooligosaccharides according to the storage period.
Figure 4 shows the DPPH radical scavenging ability of fermented soymilk supernatant.
Figure 5 shows the ABTS radical scavenging ability of the fermented soymilk supernatant.
Figure 6 shows the superoxide anion scavenging ability of fermented soymilk supernatant.
Figure 7 shows the hydroxyl radical scavenging ability of the fermented soymilk supernatant.
Figure 8 shows the reducing power of the fermented soymilk supernatant through FRAP assay.
Figure 9 shows the reducing power of fermented soymilk supernatant.
Figure 10 shows the copper ion reducing power of the fermented soymilk supernatant.
Figure 11 shows the iron ion chelating activity of fermented soymilk supernatant.
Figure 12 shows the cell viability of fermented soymilk supernatant using the MTT assay.
Figure 13 shows the neuroprotective effect of fermented _soymilk supernatant against _H2O2 using MTT assay.
Figure 14 shows the neuroprotective effect of fermented _soymilk supernatant on _H2O2 using LDH assay.
Figure 15 shows the expression of BDNF and TH genes in neurons induced with oxidative stress by H ₂ O ₂ .
Figure 16 shows the Bax/Bcl-2 gene expression ratio in neurons induced with oxidative stress by H ₂ O ₂ .
Figure 17 shows caspase-9 gene expression in neurons induced with oxidative stress by H ₂ O ₂ .
Figure 18 shows caspase-3 gene expression in neurons induced with oxidative stress by H ₂ O ₂ .

Hereinafter, the present invention will be described in more detail.

As described above, the present invention relates to a fermented food manufactured by adding prebiotics to Lactobacillus plantarum 200655 strain isolated from kimchi, and the β-glucosidase enzyme activity and total polyphenol content of soymilk containing the manufactured fermented product were measured, and in order to compare the antioxidant activity, DPPH, ABTS, superoxide anion, hydroxyl radical scavenging activity measurement, FRAP, reducing power, reducing power measurement using CUPRAC method, and Fe2 ⁺ chelating activity evaluation were performed. As a result, it was confirmed that the enzyme activity, total polyphenol content, and antioxidant activity increased when fructooligosaccharide was added compared to the control group. Accordingly, the fermented product of the present invention has been verified to have an antioxidant activity and a neuronal protective effect, and can be used as an excellent food material because it has an effect of alleviating degenerative brain diseases.

Accordingly, the present invention provides an antioxidant fermented composition comprising Lactobacillus plantarum strain 200655 (KCCM 12204P) and fructooligosaccharides.

In the present invention, the term "composition" means a mixture comprising a fermentation product of the present invention and a pharmaceutically acceptable excipient such as a diluent or carrier. The composition includes a composition for therapeutic use as well as a cosmetic composition. In some embodiments, a method of administering a composition of the present invention to a subject in need thereof is provided. In some embodiments, the composition of the present invention can be administered to a human.

The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in the compositions of the present invention will vary depending upon the identity, size, and/or disorder of the subject being treated and upon the route by which the composition is administered. For example, the composition may comprise from 0.1% to 100% (w/w) active ingredient.

The composition of the present invention may further contain, in addition to the above components, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). The composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, and a generally skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-1000 mg/kg.

The term "pharmaceutically acceptable carrier" as used herein refers to one commonly used in the formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

In one embodiment of the present invention, the fructooligosaccharide may be present at a concentration of 3 to 5% (w/v).

Specifically, in one embodiment of the present invention, Lactobacillus plantarum 200655 strain was fermented by adding fructooligosaccharide at concentrations of 1%, 3%, and 5% (w/v), and the stability and antioxidant capacity for the storage period were evaluated. In Example 5, the antioxidant capacity of the fermented composition was measured by DPPH, ABTS, superoxide anion, hydroxyl radical scavenging activity measurement, FRAP, reducing power, and CUPRAC methods. As a result, as shown in FIGS. 4 to 11, it was confirmed that the antioxidant effect was the highest when 3 to 5% of fructooligosaccharide was added.

In addition, in Example 6, which is a specific embodiment of the present invention, in order to measure the effect of protecting against nerve cell death, cytotoxicity was confirmed and the expression level of genes that can protect against nerve cell death was measured. When the expression levels of BDNF, a nerve cell growth promoting factor, TH, an enzyme related to dopamine synthesis, Bax , a factor that induces apoptosis, and Bcl-2 , caspase-9 , and caspase-3 , which are factors that inhibit apoptosis, were measured, it was confirmed that when 3 to 5% (w/v) of fructooligosaccharide was included, the expression of genes that promote nerve cell growth or inhibit apoptosis increased, and the expression level of genes that can induce nerve cell death decreased.

Therefore, in one embodiment of the present invention, the fermented composition may have a protective effect against neuronal cell death.

The term "neuron" refers to an animal cell comprising a cell body, an axon (i.e., axon or neurite) which is a projection of the cell body, and multiple dendrites (i.e., dendrites), and for example, the nerve cell may be a sensory nerve cell, a motor nerve cell, or an association nerve cell. In addition, the nerve cell may include neurons, nerve support cells, glia, Scheumann cells, etc. which form structures of the central nervous system, the brain, the brainstem, the spinal cord, the junction of the central nervous system and the peripheral nervous system, etc.

The term "death of neurons" is interpreted to mean the death of said neurons by apoptosis. Also, the term "neurodegeneration" means the progressive damage to the structure or function of neurons, including the death of said neurons.

It is widely known to those skilled in the art that death of nerve cells, or neurodegeneration, causes various brain diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease, and research on the mechanism of nerve cell death is being conducted to prevent or treat the above diseases. Nature Reviews Molecular Cell Biology 1:120-130 (2000) and Journal of Cellular and Molecular Medicine, 12:2263-2280 (2008) disclose that nerve cell death causes various symptoms such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemia, and sclerosis, and that research on the effects of oxidative stress and mitochondrial dysfunction on nerve cell death can suggest methods for preventing or treating neurodegenerative diseases. Accordingly, it will be clear to a person skilled in the art of medicine that a pharmaceutical composition comprising a compound having an effect of inhibiting neuronal death or neurodegeneration is meant for the prevention or treatment of the diseases disclosed above.

The composition according to one specific example is for neuroprotection through inhibition of neuronal cell death. The term "neuroprotection" means an action in the nervous system that protects nerve cells from neuronal death or neurodegeneration, and specifically, an effect of reducing, suppressing or improving nerve damage, and an effect of protecting, reviving or regenerating nerve cells in nerve tissue damaged by nerve damage. In addition, the term neuroprotection is a standard term in the art known to those skilled in the art with the above meaning and generally widely used (NeuroReport, 9:3955-3959(1998); Chen, J-F., J. Neurosci., 21:RC143(2001), etc.). The term "protection of neuron cells" means an action of alleviating or ameliorating a neuronal insult, or an action of protecting or recovering a nerve cell damaged by a neuronal insult. Therefore, the pharmaceutical composition can be used to prevent or treat various diseases by inhibiting oxidative stress by inducing a decrease in active acid species, thereby preventing the death of nerve cells.

Meanwhile, β-glucosidase plays a role in converting the β-glycosidic bond form of isoflavone, which is abundant in soybeans, into a non-glycosidic form with high bioavailability. When utilizing a β-glucosidase enzyme derived from natural products, the selectivity of the reaction product can be increased and the use of harmful organic solvents can be reduced. Therefore, when the activity of β-glucosidase is high, a functional fermented composition with high bioavailability can be produced.

Accordingly, in a specific embodiment of the present invention, the activity of β-glucosidase contained in fermented soymilk was measured. As a result, it was confirmed that when fermentation was performed by adding fructooligosaccharide, the enzyme activity significantly increased ( p <0.05) compared to fermentation using Lactobacillus plantarum 200655 strain in fermented soymilk without fructooligosaccharide addition. In particular, the highest enzyme activity was observed when 3% FOS was added.

In addition, phenolic compounds contained in plants such as soybeans are known to have antioxidant physiological activity as one of the metabolites, especially phenolic hydroxyl groups. The total phenol content also significantly increased ( p <0.05) when fructooligosaccharides were added according to β-glucosidase enzyme activity. It showed a very similar tendency to β-glucosidase enzyme activity, and also showed the highest value at 3%. This shows that fructooligosaccharides can increase the total phenol content in fermented soymilk by increasing the β-glucosidase enzyme activity of Lactobacillus plantarum 200655 strain.

That is, compared to using only the Lactobacillus plantarum 200655 strain and not adding fructooligosaccharides, adding fructooligosaccharides had a superior effect in increasing β-glucosidase enzyme activity and total phenol content. The increase in total phenol content increases the efficacy related to the antioxidant effect.

In one embodiment of the present invention, the fermented composition may be characterized in that the total phenol content increases by 150% or more compared to the case where fructooligosaccharide is not added. In a specific embodiment of the present invention, as shown in [Table 1], it can be confirmed that the total phenol content increases by about 150% or more when fructooligosaccharide is added (18.00 mg GAE/mL or more) compared to the case where fructooligosaccharide is not added (FS0, 11.81 ± 0.10 mg GAE/mL).

In addition, the present invention provides a pharmaceutical composition for preventing or treating a degenerative neurological disease comprising the fermented composition.

The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology or as developed hereinafter. Typically, such methods for tableting comprise the steps of bringing into association the active ingredient with excipients and/or one or more other auxiliary ingredients, followed by, if necessary or desired, shaping and/or packaging the product into desired single- or multi-dose units.

The pharmaceutical compositions of the invention described herein are typically prepared in dosage unit form for easy administration and uniform dosing. However, it will be understood that the total daily dosage of the compositions of the invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the disease, disorder, or disorder being treated and its severity; the activity of the specific active ingredient employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of treatment; drugs used in combination or concurrently with the specific active ingredient employed; and other factors well known in the medical arts.

The antioxidant fermentation composition comprising the Lactobacillus plantarum 200655 strain and fructooligosaccharides, a salt thereof, or a pharmaceutical composition comprising the same of the present invention may be administered by any route. In some embodiments, the antioxidant fermentation composition comprising the Lactobacillus plantarum 200655 strain and fructooligosaccharides, a salt thereof, or a pharmaceutical composition comprising the same is administered by various routes, including orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecal, subcutaneously, intracerebroventricularly, transdermally, intradermally, rectally, intravaginally, intraperitoneally, topically (by powders, ointments, creams, and/or drops), mucosally, nasally, buccally, enterally, sublingually; by intratracheal instillation, by bronchial instillation, and/or by inhalation; and/or by oral spray, nasal spray, and/or by aerosol. Routes specifically contemplated are intravenous injection by occlusive means, local administration via the blood and/or lymphatic supply, and/or direct administration to the affected area. In general, the most suitable route of administration will depend on a variety of factors, including the properties of the agent (e.g., stability in the gastrointestinal environment), and the impairment of the subject (e.g., whether the subject can tolerate oral administration). Currently, oral and/or nasal spray and/or aerosol routes are most commonly used to deliver therapeutic agents directly to the lungs and/or respiratory system. However, the present invention encompasses the delivery of a pharmaceutical composition comprising the Lactobacillus plantarum 200655 strain according to the present invention and an antioxidant fermentation composition comprising fructooligosaccharides by any suitable route that takes into account advances in the field of drug delivery.

In certain embodiments, the antioxidant fermentation composition comprising the Lactobacillus plantarum 200655 strain of the present invention and a fructooligosaccharide, a salt thereof, or a pharmaceutical composition comprising the same may be administered at a dosage level sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg of body weight of the subject once or more per day to achieve the desired therapeutic effect. The target dosage may be delivered three times daily, twice daily, daily, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the target dosage may be delivered via multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

In some embodiments, the pharmaceutical compositions of the present invention may be administered in combination with any therapeutically active agent or procedure (e.g., surgery, radiation therapy) useful for treating, alleviating, ameliorating, alleviating, delaying the onset, inhibiting the progression, reducing the severity, and/or reducing the incidence of one or more symptoms or characteristics of a neurological disorder.

In one embodiment of the present invention, the degenerative neurological disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, brain dysfunction due to immune system abnormalities, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, and dementia due to cerebral ischemia and cerebral hemorrhage.

Furthermore, the present invention provides a food composition comprising the fermented composition.

The food composition of the present invention can be added to health foods for the purpose of preventing or improving degenerative neurological diseases. When the fermentation composition containing the Lactobacillus plantarum 200655 strain of the present invention and fructooligosaccharides is used as an additive, the fermentation composition can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The mixing amount of the effective ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverage, the fermentation composition of the present invention is added in an amount of 1 to 20 wt%, preferably 5 to 10 wt%, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the effective ingredient can also be used in an amount above the above range.

There is no special limitation on the type of the above food. Examples of foods to which the above substance can be added include beverages, gum, vitamin complexes, or health supplements, and include all health foods in the conventional sense. Food compositions include beverage compositions and may also include health functional foods.

The beverage composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 ml of the composition of the present invention.

In addition, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention can provide a health functional food composition for preventing or improving degenerative neurological diseases, which contains a fermented composition as an effective ingredient. The term "health functional food" as defined herein means a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Functional Foods, and "function" means ingestion for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. When the fermented composition according to the present invention is used as a food additive, the fermented composition may be added as is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. Examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

In one embodiment of the present invention, the food composition may be at least one selected from the group consisting of soy milk, fermented milk, milk, and fermented soybean food. The fermented soybean food may be, but is not limited to, soy milk, tofu, soybean meat, soybean snack, soybean confectionery, fermented beverage, pills, granules, and powder.

In one embodiment of the present invention, the food composition can maintain 95% or more of the initial viable bacterial count for a storage period of 16 to 30 days.

In a specific embodiment, in Example 3 of the present invention, fermentation was carried out by including fructooligosaccharide, and the stability of soymilk was evaluated according to the storage period. When the storage period is long, there is a tendency for the initial viable cell count to decrease, whereas when fructooligosaccharide is included, the fermentation composition using the strain has improved storage stability and the viable cell count can be maintained compared to the case where fructooligosaccharide is not included, so that the convenience of distribution and stability against quality changes are excellent. The initial viable cell count can be maintained at 95% or more for 16 to 30 days, preferably for 18 to 25 days, and more preferably for 19 to 22 days.

In one embodiment of the present invention, the food composition may have a β-glycosidase activity of 7.0 to 8.6 mU/mL and a total phenol content of 15.0 to 20.0 mg GAE/mL.

In a specific embodiment, Example 4 shows that the total phenol content in fermented soymilk increased by increasing β-glucosidase enzyme activity by adding fructooligosaccharide. Compared to the case where only Lactobacillus plantarum 200655 strain was used and fructooligosaccharide was not added, when fructooligosaccharide was added, there was an excellent effect of increasing β-glucosidase enzyme activity and total phenol content. Therefore, the food composition of the present invention may have β-glycosidase activity of 7.0 to 8.6 mU/mL and a total phenol content of 15.0 to 20.0 mg GAE/mL, and preferably, the β-glycosidase activity may be 7.1 to 8.5 mU/mL and a total phenol content may be 16.0 to 19.0 mg GAE/mL.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[Preparation]

Strains used

Lactobacillus plantarum strain 200655 (strain deposit number: KCCM 12204P) was isolated from kimchi using MRS medium.

Production of fermented soy milk supernatant

Fermented soymilk 10 g was homogenized by adding 2.5 mL of sterilized distilled water. To obtain the supernatant, centrifugation was performed at 14,240Хg , 4℃ for 5 minutes. The supernatant was adjusted to pH 7.0 with 1 N NaOH, and then filtered using a 0.45 μm filter to produce a fermented soymilk supernatant. The fermented soymilk supernatant was stored at -20℃ and used as a sample for subsequent antioxidant and cell experiments.

Evaluation of Prebiotic Availability

Prior to the production of fermented soymilk, the prebiotic availability of Lactobacillus plantarum 200655 strain was evaluated. Glucose (positive control) and four kinds of prebiotics, fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, and xylitol, were added at 2% (w/v) each to the MRS medium that did not contain glucose to produce the medium. The MRS medium without added sugar was used as a negative control, and the MRS medium with added glucose was used as a positive control. Lactobacillus plantarum 200655 strain was inoculated into each medium at a concentration of 10 ⁷ CFU/mL at 1% (v/v), and then cultured at 37°C for 24 h. The culture medium was harvested, washed twice with phosphate buffered saline (PBS), suspended in PBS, and measured for absorbance at 600 nm. The equation for the growth index (GI) is as follows.

In the negative control, GI was 11.11±0.56%, and xylitol showed a lower GI of 9.96±0.18%. Therefore, it was confirmed that xylitol was not utilized at all as a carbon source. Inulin, GOS, and FOS showed GI of 29.31±0.67%, 88.70±2.92%, and 94.67±1.23%, respectively. Therefore, FOS, which showed the highest growth index, was selected as a prebiotic and fermentation was performed.

Stability evaluation of soymilk containing fructooligosaccharides (FOS) according to fermentation and storage period

Soymilk was sterilized by autoclaving at 121℃ for 15 minutes after adding FOS at concentrations of 0%, 1%, 3%, and 5% (w/v). The sterilized soymilk was cooled to 37℃ and then inoculated with 2% (v/v) of Lactobacillus plantarum 200655 strain. Fermentation was conducted at 37℃ until the pH reached 4.5, and samples were collected at each hour to measure the viable cell count, pH, and titratable acidity (FS0, fermented soymilk without FOS; FS1, fermented soymilk with 1% FOS; FS3, fermented soymilk with 3% FOS; FS5, fermented soymilk with 5% FOS).

The viable cell count was determined by diluting the collected fermented soymilk with 0.1% (w/v) peptone water and culturing it on MRS agar at 37℃ for 24 hours. The pH was measured using a pH meter. The titratable acidity (TA) was determined by taking 10 g of fermented soymilk, mixing it with 10 mL of distilled water, and titrating it with 0.1 N NaOH until the pH reached 8.3. The equation for titratable acidity is as follows.

FOS was added to soymilk at various concentrations and fermented until the pH reached 4.5. Regardless of the concentration of FOS in the soymilk, the fermentation time was the same at 20 hours.

As a result, as shown in Fig. 2, the initial viable cell count of each sample increased from 7.42 to 7.55 log CFU/mL until 10 hours of fermentation, and then was maintained between 8.74 and 9.04 log CFU/mL. In addition, as fermentation progressed, the pH decreased and the titratable acidity increased. The initial pH of each soymilk was 6.9, and decreased during fermentation to reach pH 4.5 after 20 hours of fermentation. The titratable acidity corresponding to lactic acid increased from 0.06 to 0.08% initially to 0.6% at the end. During the fermentation process, fructooligosaccharides did not significantly affect the growth and acid production of the Lactobacillus plantarum 200655 strain. This indicated that fructooligosaccharides did not have a negative effect on the growth of the strain.

Stability evaluation of fermented soy milk according to storage period

The stability of fermented soy milk was evaluated according to the storage period by measuring the number of viable cells, pH, and titratable acidity at 7-day intervals while storing the soy milk at 4℃.

As a result, as shown in Fig. 3(A), the viable cell count was initially 8.88 to 9.02 log CFU/mL, but the viable cell count tended to decrease over the storage period. In particular, the decrease was very large in FS0 that did not contain fructooligosaccharides, and the viable cell count was 7.77 log CFU/mL on the 21st day of storage. The fermented soymilk including FOS showed a slight decrease in the viable cell count during the storage period, but still showed a high viable cell count of 8.73 to 8.98 log CFU/mL on the 21st day. The decrease in the viable cell count of the fermented soymilk with FOS was less than 0.2 log CFU/mL, indicating that the addition of FOS helped maintain the viable cell count during the storage period.

As shown in Fig. 3(B), all fermented soymilk showed a change in pH of 4.40~4.51 on the first day of storage. However, after 21 days, the pH significantly decreased with storage period to pH 4.07~4.43 ( p <0.05). FS0 had pH 4.43 on the 21st day, but FOS-added fermented soymilk had pH 4.07~4.18, showing that the degree of decrease was greater in FOS-added fermented soymilk. Since the pH of commercially available liquid fermented milk is reported to be at the level of 3.25-4.27 (Koh Seok-ju et al., 2013), the pH of the fermented soymilk of the present invention was found to be above 4.0, indicating that there will be no problem in commercial storage when manufactured.

As shown in Fig. 3(C), the titratable acidity of fermented soymilk according to the storage period showed a trend opposite to that of pH and significantly increased ( p <0.05). Similar to the trend of pH, it was confirmed that the titratable acidity of fermented soymilk containing FOS was higher. After 21 days of storage, the titratable acidity of FOS-containing fermented soymilk increased to 1.36~1.54%, but the increase in FS0 was not large, increasing from 0.82% to 0.95%. It is known that a decrease in pH is an increase in titratable acidity, and this change, like pH, indicated that the addition of FOS was at a commercially available level.

Measurement of β-glucosidase enzyme activity

β-Glucosidase enzyme activity in fermented soymilk was measured. 200 μL of fermented soymilk and 400 μL of ρ NPG (4-Nitrophenyl β-D-glucopyranoside) dissolved in 0.2 M sodium phosphate buffer (pH 7.0) were reacted at 37°C for 30 minutes. 800 μL of 1 M sodium carbonate was mixed to terminate the reaction. The supernatant obtained by centrifugation at 14,240Х g for 10 minutes was measured for absorbance at 405 nm. β-Glucosidase enzyme activity was defined as the amount at which 1 μM ρ -nitrophenol is produced from the substrate per minute, 1 unit (U).

Measurement of total phenolic contents (TPC)

Total phenolic contents (TPC) were measured using the Folin-Ciocalteu assay. 50 μL of fermented soymilk and 1 mL of 2% sodium carbonate were mixed and reacted for 5 minutes. 50 μL of 50% Folin-Ciocalteu's reagent was added and reacted for 30 minutes. The supernatant obtained by centrifugation at 14,240Хg for 10 minutes was measured for absorbance at 750 nm. TPC was expressed as mg gallic acid equivalent (GAE)/mL by substituting it into a standard curve using gallic acid.

As a result, Lactobacillus plantarum 200655 strain showed enzyme activity of 6.66±0.12 mU/mL in fermented soymilk without FOS addition. However, when fermentation was performed with the addition of FOS, it was confirmed that the enzyme activity significantly increased ( p <0.05). When FOS was added at concentrations of 1%, 3%, and 5%, the enzyme activities were 6.95±0.18 mU/mL, 8.04±0.31 mU/mL, and 7.72±0.18 mU/mL, respectively. In particular, the highest enzyme activity was observed when 3% FOS was added.

According to β-glucosidase enzyme activity, TPC also significantly increased when FOS was added ( p <0.05). [Table 1] shows the β-glucosidase activity and total phenolic contents of fermented soymilk. FS0 showed 11.81±0.10 mg GAE/mL of TPC, whereas FS1, FS3, and FS5 showed 18.07±0.15 mg GAE/mL, 18.42±0.13 mg GAE/mL, and 18.21±0.16 mg GAE/mL of TPC, respectively. It showed a very similar tendency to β-glucosidase enzyme activity, and again showed the highest value at 3%. This suggests that FOS can increase the TPC content in fermented soymilk by increasing the β-glucosidase enzyme activity of Lactobacillus plantarum 200655 strain. With this increase in TPC content, an increase in antioxidant effect can be expected.

Sample β-Glucosidase activity
(mU/mL) Total phenolic contents
(mg GAE/mL) FS0 6.66 ± 0.12 ^d 11.81 ± 0.10 ^c FS1 6.95 ± 0.18 ^c 18.07 ± 0.15 ^b FS3 8.04 ± 0.31 ^a 18.42 ± 0.13 ^a FS5 7.72 ± 0.18 ^b 18.21 ± 0.16 ^b

5-1) 1,1-Diphenyl-2-picryl-hydrazyl(DPPH) radical scavenging assay

150 μL of fermented soymilk supernatant and 750 μL of 0.1 M DPPH solution dissolved in ethanol were mixed, and then reacted in the dark at room temperature for 30 minutes. The solution was centrifuged at 14,240Хg for 1 minute, and only the supernatant was collected, and the absorbance was measured at 517 nm. The equation for DPPH radical scavenging activity is as follows.

As a result, as shown in Fig. 4, the DPPH radical scavenging activity of the FOS-added group significantly increased compared to FS0 ( p <0.05). FS0, FS1, FS3, and FS5 showed radical scavenging activities of 47.26±1.74%, 57.16±1.44%, 60.53±1.38%, and 56.54±0.79%, respectively. When FOS was 1% and 5%, similar DPPH scavenging activities were shown, while the highest scavenging activity was shown at a concentration of 3%.

5-2) 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging assay

ABTS solution was prepared by mixing 14 mM ABTS stock and 5 mM potassium persulfate in a 1:1 ratio and incubating at room temperature for 16-18 hours in the dark. The ABTS solution was diluted with distilled water to an absorbance of 0.7±0.02 at 734 nm before use. 20 μL of fermented soymilk supernatant and 980 μL of ABTS solution were mixed and incubated at room temperature for 5 minutes in the dark. After the reaction, the absorbance was measured at 734 nm, and the equation for ABTS radical scavenging activity is as follows.

As a result, as shown in Fig. 5, although similar ABTS scavenging activities were observed among the groups, the scavenging activity of the FOS-added group significantly increased compared to FS0 ( p <0.05). FS0 exhibited a radical scavenging activity of 70.96±1.79%, and FS1, FS3, and FS5 exhibited ABTS radical scavenging activities of 75.02±1.31%, 76.82±1.45%, and 77.81±0.95%, respectively.

5-3) Superoxide anion radical scavenging assay

The superoxide anion scavenging activity of fermented soymilk was evaluated. Superoxide anion radical scavenging activity was measured using an assay based on the PMS (phenazine methosulfate), NADH (β-nicotinamide adenine dinucleotide), and NBT (nitrotetrazolium blue chloride) system. The fermented soymilk supernatant, 468 μM NADH solution in 20 mM potassium phosphate buffer (pH 7.4), 300 μM NBT solution in buffer, and 60 μM PMS were mixed (200 μL each) and reacted in the dark for 5 minutes. After the reaction, the absorbance was measured at 560 nm, and the equation for the superoxide anion radical scavenging activity is as follows.

As a result, as shown in Fig. 6, FS0 showed a scavenging activity of 47.84±1.88%, and only FS3 showed a significantly increased scavenging activity (54.72±6.71%) ( p <0.05). The superoxide anion scavenging activities of FS1 and FS5 were 50.69±4.08% and 50.12±5.97%, respectively.

5-4) Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity of fermented soymilk was evaluated. A solution containing 2.8 mM deoxyribose, 0.1 mM EDTA (ethylenediaminetetraacetic acid), and 0.1 mM ferric chloride was prepared using potassium phosphate buffer (20 mM, pH 7.4). 1 mL of the fermented soymilk supernatant and 1 mL of the previous solution were mixed, and 100 μL of 1 mM ascorbic acid and 400 μL of 20 mM hydrogen peroxide were added. After reaction at 37°C for 1 hour, 1 mL of 3% trichloroacetic acid and 300 μL of 1% thiobarbituric acid were added, and then boiled at 100°C for 15 minutes. The absorbance was measured at 532 nm after the reaction, and the equation for hydroxyl radical scavenging activity is as follows.

As a result, as shown in Fig. 7, unlike the previous results, FS0 showed the highest value at 70.49±0.58%, and the scavenging activity significantly decreased as the concentration of FOS in the fermented soymilk increased. FS1, FS3, and FS5 showed hydroxyl radical scavenging activities of 61.91±0.18%, 54.10±0.56%, and 47.02±0.45%, respectively. This is thought to be related to the iron ion chelating activity of fermented soymilk. In the following assay, hydroxyl radicals are generated by the Fenton reaction of Fe2 ⁺ _{and H2O2} . However, if the evaluated substance can chelate metal ions, it can interfere with or terminate the Fenton reaction. According to Fig. 11, it can be confirmed that the iron ion chelating activity increases depending on the concentration of FOS. Therefore, the following results are thought to have been derived.

5-5) Ferric reducing antioxidant power (FRAP) assay

The reducing power of the fermented soymilk supernatant was evaluated using the FRAP assay. The FRAP assay measures the degree of reduction of Fe ³⁺ -TPTZ complex to Fe ²⁺ -TPTZ. The FRAP solution was prepared by mixing 10 mM TPTZ (2,4,6-tris(2-pyridyl)- s -triazine (in 40 mM HCl), 20 mM ferric chloride, and 0.3 M sodium acetate buffer (pH 3.6) in a ratio of 1:1:10 and incubating at 37°C for 15 minutes. 50 μL of the sample and 950 μL of the FRAP solution were mixed, and the mixture was reacted in the dark at room temperature for 30 minutes. The absorbance was measured at 593 nm, and the FRAP activity was expressed by substituting it into a standard curve using FeSO ₄ (ferrous sulfate).

As a result, as shown in Fig. 8, the FRAP assay measured the degree of reduction of Fe ³⁺ -TPTZ complex to Fe ²⁺ -TPTZ. The reducing power of FOS-added fermented soymilk significantly increased compared to FS0 ( p <0.05). FS0 showed a reducing power corresponding to 365.88±12.55μM FeSO ₄ equivalent. In contrast, FS1, FS3, and FS5 showed reducing powers that increased by 13.8%, 20.3%, and 11.8%, respectively. It was confirmed that the highest reducing power was shown when the concentration of added FOS was 3%.

5-6) Reducing power assay

The reducing power of the fermented soymilk supernatant was evaluated using a reducing power assay. The following assay measures the degree to which a substance donates electrons. 50 μL of the fermented soymilk supernatant, 250 μL of 1% potassium ferricyanide, and 250 μL of 0.2 M sodium phosphate buffer (pH 6.6) were mixed and reacted at 50°C for 20 minutes. After the reaction, 250 μL of 10% trichloroacetic acid was mixed. 500 μL of the reaction solution was transferred to a new tube and reacted with 400 μL of distilled water and 100 μL of 0.1% ferric chloride at room temperature for 30 minutes. The absorbance was measured at 700 nm, and the reducing power was expressed by substituting it into a standard curve using L-cysteine.

As a result, as shown in Fig. 9, the reducing power of FOS-added fermented soymilk significantly increased compared to FS0 ( p <0.05). FS0 showed a reducing power corresponding to 1.25±0.04 mM L-Cysteine equivalent. In comparison, FS1, FS3, and FS5 showed increased values of 14.6%, 27.5%, and 27.7%. It was confirmed that the highest reducing power was shown when the concentration of FOS was 3% and 5%.

5-7) Cupric ion reducing antioxidant capacity (CUPRAC) assay

The copper ion reducing power of the fermented soymilk supernatant was evaluated using the CUPRAC assay. Fermented soymilk supernatant, 10 mM copper chloride (in 1 M ammonium acetate buffer pH 7.0), and 7.5 mM neocuprione (in ethanol) were mixed (200 μL each) with three solutions. The solution volume was adjusted to 2.0 mL with ammonium acetate buffer, and the solution was reacted in the dark for 30 minutes. After the reaction, the absorbance was measured at 450 nm, and the cupric ion reducing antioxidant capacity was expressed by substituting it into a standard curve using Trolox.

As a result, as shown in Fig. 10, the copper ion reducing power of FOS-added fermented soymilk significantly increased compared to FS0 ( p <0.05). FS0 showed a reducing power corresponding to 1.27±0.02 mM Trolox equivalent, and FS1, FS3, and FS5 showed increased values of 5.6%, 17.6%, and 16.9%, respectively.

5-8) Ferrous ion chelating (FIC) activity

The iron ion chelating activity of the fermented soymilk supernatant was evaluated. 125 μL of the fermented soymilk supernatant, 125 μL of 2 mM ferrous sulfate, and 500 μL of 0.1 M sodium phosphate buffer (pH 4.9) were mixed and incubated in the dark at room temperature for 30 minutes. 50 μL of 5 mM ferrozine was added to the solution, and the absorbance was measured at 562 nm. The equation for the chelating ability is as follows.

As a result, as shown in Fig. 11, all fermented soymilk supernatants showed high chelating activity of over 85%, and the activity of FOS-added fermented soymilk significantly increased compared to FS0 ( p <0.05). FS5 showed the highest value at 93.25±0.93%, followed by FS3 (91.59±1.06%), FS1 (87.64±1.03%), and FS0 (85.29±0.42%).

Measurement of the protective effect against neuronal cell death

To determine whether the fermented soymilk supernatant induces cytotoxicity in SH-SY5Y neurons and to confirm the cytoprotective effect against _{H2O2 ,} an oxidative stress inducer, an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was performed. SH-SY5Y neurons were cultured in a 96-well plate at ^1Х105 cells/well for 24 hours. To measure the cytotoxicity of the fermented soymilk _supernatant , the fermented soymilk supernatant diluted twice was treated for 24 hours. To measure the protective effect against _H2O2 , the fermented soymilk supernatant diluted twice was pretreated for 4 hours, and then _H2O2 (250 μM) was treated and cultured for 20 hours. After that, the existing medium was removed, MTT solution (0.5 mg/mL) was treated and cultured for ₄ hours. The MTT solution was removed, 100 μL DMSO (Dimethyl sulfoxide) solution was added, and the resulting formazan crystals were dissolved after reaction for 15 minutes. The absorbance was measured at 570 nm, and the equation for cell viability is as follows.

To confirm the cytoprotective effect on _H2O2 , an oxidative stress-inducing _substance for SH-SY5Y neurons, an LDH (lactate dehydrogenase) assay was performed. The following assay was performed according to the method described in the EZ-LDH assay kit (DoGenBio Co. Ltd., Seoul, Korea). SH-SY5Y cells were cultured in a 96-well plate at ^1Х105 cells/well for 24 hours. After pretreatment with a 2-fold diluted fermented soymilk supernatant for 4 hours, _H2O2 (250 μM) was treated and cultured for 20 hours. 10 μL of the culture supernatant was transferred to a new 96-well plate _, and 100 μL of LDH reaction mixture containing WST (water soluble tetrazolium salt) was added. After 30 minutes of dark reaction, 10 μL of stop solution was added to terminate the reaction, and the absorbance was measured at 450 nm.

Before evaluating the neuroprotective effect of fermented soymilk supernatant against _{H2O2 -} induced cell death, the cytotoxicity of the fermented soymilk supernatant diluted to various concentrations was evaluated. The fermented soymilk supernatant was treated to neurons at three concentrations: 1/4, 1/2, and 1 times. First, when the cell viability of the control group was set to 100%, all fermented soymilk supernatants showed toxicity, with cell viability ranging from 78.03 to 87.16% at 1 times the concentration. At 1/4 and 1/2 times the concentration, all showed cell viability of 99.26 to 101.86%, indicating no toxicity to neurons. Therefore, the 1 times concentration was excluded in subsequent experiments (Fig. 12).

After oxidative stress was induced in neurons by treating them with 250 μM H ₂ O ₂ , the protective effect of the fermented soymilk supernatant was evaluated. As a result, as shown in Fig. 13, when the cell viability of the control group was set as 100%, the H ₂ O- ₂ treatment group showed a cell viability of 65.19 ± 1.02%. At 1/2-fold concentration, all fermented soymilk supernatants showed cell viability similar to or lower than that of the H ₂ O ₂ treatment group. At 1/4-fold concentration, FS0 and FS1 showed cell viability of 55.62 ± 8.30% and 63.29 ± 5.53%, respectively, which were lower than that of the H ₂ O ₂ treatment group, but FS3 and FS5 showed cell viability of 95.74 ± 2.09% and 93.00 ± 1.39%, respectively. Therefore, the 1/2-fold concentration was excluded in subsequent experiments.

After oxidative stress was induced in neurons by treating them with 250 μM H ₂ O ₂ , the amount of lactate dehydrogenase (LHD) enzyme released from the cytoplasm of the damaged cells into the medium was measured. As a result, as shown in Fig. 14, 4.36 ± 1.00% LDH was measured in the control group, and 60.77 ± 1.47% LDH was measured in the H ₂ O- ₂ treated group. At 1/2 times the concentration, all fermented soymilk supernatants showed LDH amounts similar to or higher than those of the H ₂ O ₂ treated group. At 1/4 times the concentration, FS0 and FS1 showed LDH levels of 65.14 ± 3.58% and 62.09 ± 0.28%, respectively, which were similar to those of the H ₂ O ₂ treated group. In contrast, FS3 and FS5 measured LDH of 31.27±1.14% and 22.36±5.68%, respectively. That is, since an increase in LDH indicates cytotoxicity of nerve cells, a decrease in LDH in FS3 and FS5 indicates a protective effect on nerve cells.

Gene expression studies in nerve cells

The increase or decrease in BDNF (brain-derived neurotrophic factor), a nerve growth promoting factor, TH (tyrosine hydroxylase), an enzyme involved in dopamine synthesis, and Bax , Bcl-2 , caspase-9 , and caspase-3 , factors related to apoptosis, were measured by real-time PCR for SH-SY5Y neurons. SH-SY5Y neurons were cultured at 2Х10 ⁶ cells/well in a 6-well plate for 24 hours, pretreated with fermented soymilk supernatant for 4 hours, and treated with H ₂ O ₂ (250 μM) for 3 hours. Afterwards, each cell was collected, RNA was extracted, converted to cDNA, and changes in apoptosis-related factors were measured by real-time PCR. The following [Table 2] shows the related gene base sequences used in the present invention.

Gene Primer sequence Sequence number BDNF Forward 5'-TGACCATCCTTTTCCTTACT-3' 1 Reverse 5'-GCCACCTTGTCCTCGGAT-3' 2 TH Forward 5'-GAGGAGAAGGAGGGGAAG-3' 3 Reverse 5'-ACTCAAACACCTTCACAGCT-3' 4 Bax Forward 5'-GTGGTTGCCCTCTTCTACTTTGC-3' 5 Reverse 5'-GAGGACTCCAGCCACAAAGATG-3' 6 Bcl-2 Forward 5'-CGGCTGAAGTCTCCATTAGC-3' 7 Reverse 5'-CCAGGGAAGTTCTGGTGTGT-3' 8 Caspase-9 Forward 5'-CACTTCCCCTGAAGACGAGTC-3' 9 Reverse 5'-GTGGGCAAACTAGATATGGCG-3' 10 Caspase 3 Forward 5'-CATGGAAGCGAATCAATGGACT-3' 11 Reverse 5'-CTGTACCAGACCGAGATGTCA-3' 12 GAPDH Forward 5'-GAGTCAACGGATTTGGTCGT-3' 13 Reverse 5'-GACAAGCTTCCCGTTCTCAG-3' 14

BDNF, brain-derived neurotrophic factor, TH , tyrosine hydroxylase, Bax, Bcl-2 associated X protein; Bcl-2 , apoptosis regulator; caspase-9 , initiator caspase, critical to the apoptotic pathway; caspase-3 , interactor with caspase-8 and caspase-9; GAPDH , housekeeping gene.

To measure the neuroprotective effect of fermented soymilk, SH-SY5Y was pretreated with 1/4-fold fermented soymilk supernatant, and then oxidative stress was induced with _{H2O2 .} The expression levels of BDNF, a neuronal growth promoting factor , TH, an enzyme related to dopamine synthesis, Bax , a factor inducing apoptosis, and Bcl-2 , caspase-9 , and caspase-3, which are factors that inhibit apoptosis, were measured by RT-PCR.

As a result, as shown in Fig. 15, when oxidative stress was induced in neurons with H ₂ O- ₂ , the expression levels of BDNF and TH decreased by 0.82 and 0.81 times compared to the control group, respectively. The expression levels of BDNF in FS0 and FS1 were 0.85 and 0.84 times, which were similar to those in H ₂ O ₂ , but FS3 and FS5 showed increases of 1.09 and 1.19 times. The expression levels of TH in FS0, FS1, FS3, and FS5 were 0.96, 0.94, 1.23, and 1.66 times, respectively, compared to the control group, showing an increase in all sample groups compared to the H ₂ O ₂ treatment group. Through this, it was confirmed that the expression levels of BDNF, a neuronal growth promoting factor, and TH, an enzyme related to dopamine synthesis, increased, thereby promoting neuronal growth and protecting against death.

Also, as shown in Fig. 16, when only H ₂ O- ₂ was treated in neurons, the expression ratio of Bax and Bcl-2 increased 2.96-fold compared to the control group, and 3.00, 3.12, 0.42, and 0.27-fold for FS0, FS1, FS3, and FS5, respectively. FS0 and FS1 showed a significant increase in the expression ratio compared to the H ₂ O ₂ treatment group, but FS3 and FS5 showed a lower expression ratio than the control group that did not induce toxicity. Therefore, it was confirmed that in the case of FS3 and FS5, the expression ratio of Bcl-2, which inhibits apoptosis, was higher than that of Bax, which induces neuronal apoptosis, and thus neuronal apoptosis was protected.

Caspase-9 acts as an initiator that activates caspase-3 in the early stage of apoptosis. As shown in Fig. 17, when neurons were treated with only H ₂ O- _{2 ,} the expression level of caspase-9 increased 1.45-fold compared to the control group, and FS0, FS1, FS3, and FS5 increased 1.49-, 1.44-, 1.31-, and 1.29-fold, respectively. FS0 and FS1 were similar to the H ₂ O ₂ treatment group, but FS3 and FS5 showed significantly lower expression levels.

Caspase-3 is a protein hydrolase that performs cell death in the final stage of apoptosis. As shown in Fig. 18, when neurons were treated with only H ₂ O- ₂ , the expression level of caspase-3 increased 1.21-fold compared to the control group, and FS0, FS1, FS3, and FS5 showed 1.19-, 1.23-, 1.00-, and 0.90-fold increases, respectively. FS0 and FS1 showed similar expression levels to the H ₂ O ₂ treatment group, but FS3 and FS5 showed expression levels similar to or lower than the control group.

The expression levels of Caspase-9 and Caspase-3 were significantly lower in FS3 and FS5, which confirmed that the fermented soymilk supernatant containing 3% and 5% FOS alleviated the apoptosis-inducing action of _{H2O2 .}

SEQUENCE LISTING <110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Fermented composition for antioxidant containing Lactobacillus plantarum 200655 strain (KCCM 12204P) and fructooligosaccharide and its use for preventing or treating neurodegenerative diseases <130> 1070203 <160> 14 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial <220> <223> BDNF forward primer <400> 1 tgaccatcct tttccttact 20 <210> 2 <211> 18 <212> DNA <213> Artificial <220> <223> BDNF reverse primer <400> 2 gccaccttgt cctcggat 18 <210> 3 <211> 18 <212> DNA <213> Artificial <220> <223> TH forward primer <400> 3 gaggagaagg aggggaag 18 <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> TH reverse primer <400> 4 actcaaacac cttcacagct 20 <210> 5 <211> 23 <212> DNA <213> Artificial <220> <223> Bax forward primer <400> 5 gtggttgccc tcttctactt tgc 23 <210> 6 <211> 22 <212> DNA <213> Artificial <220> <223> Bax reverse primer <400> 6 gaggactcca gccacaaaga tg 22 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> Bcl-2 forward primer <400> 7 cggctgaagt ctccattagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial <220> <223> Bcl- 2 reverse primer <400> 8 ccagggaagt tctggtgtgt 20 <210> 9 <211> 21 <212> DNA <213> Artificial <220> <223> Caspase-9 forward primer <400> 9 cacttcccct gaagacgagt c 21 <210> 10 <211> 21 <212> DNA <213> Artificial <220> <223> Caspase-9 reverse primer <400> 10 gtgggcaaac tagatatggc g 21 <210> 11 <211> 22 <212> DNA <213> Artificial <220> <223> Caspase3 forward primer < 400>11 catggaagcg aatcaatgga ct 22 <210> 12 <211> 21 <212> DNA <213> Artificial <220> <223> Caspase 3 reverse primer <400> 12 ctgtaccaga ccgagatgtc a 21 <210> 13 <211> 20 <212> DNA < 213> Artificial <220> <223> GAPDH forward primer <400> 13 gagtcaacgg atttggtcgt 20 <210> 14 <211> 20 <212> DNA <213> Artificial <220> <223> GAPDH reverse primer <400> 14 gacaagcttc ccgttctcag 20

Claims (10)

An antioxidant fermented composition comprising Lactobacillus plantarum strain 200655 (KCCM 12204P) and fructooligosaccharide at a concentration of 3 to 5% (w/v). delete In the first paragraph,
The above fermented composition is an antioxidant fermented composition having a protective effect against nerve cell death. In the first paragraph,
The above fermented composition is an antioxidant fermented composition characterized in that the total phenol content increases by 150% or more compared to a case in which fructooligosaccharide is not added. A pharmaceutical composition for preventing or treating a degenerative neurological disease, comprising a fermented composition according to any one of claims 1, 3 and 4. In paragraph 5,
A pharmaceutical composition for preventing or treating a degenerative neurological disease, characterized in that the above-mentioned degenerative neurological disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, brain dysfunction due to immune system abnormalities, progressive neurodegenerative disease, metabolic brain disease, Niemann-Pick disease, and dementia due to cerebral ischemia and cerebral hemorrhage. A food composition comprising a fermented composition according to any one of claims 1, 3 and 4. In Article 7,
A food composition characterized in that the food composition comprises at least one selected from the group consisting of soy milk, fermented milk, milk, and fermented soybean food. In Article 7,
The above food composition is characterized in that it maintains 95% or more of the initial viable bacterial count for a storage period of 16 to 30 days. In Article 7,
The above food composition is a food composition characterized in that the β-glycosidase activity is 7.0 to 8.6 mU/mL and the total phenol content is 15.0 to 20.0 mg GAE/mL.