KR20230142931A - Composition for preventing, treating, or improving degenerative brain disease comprising enzyme reactant of citron pomace - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or ameliorating degenerative brain disease containing an enzyme reaction product of citron marmalade. The composition exhibits a protective and anti-stress effect on brain cells and an effect of improving cognitive function, thereby effectively treating degenerative brain disease. It can be used for prevention, treatment or improvement.

Description

Composition for preventing, treating, or improving degenerative brain disease comprising enzyme reactant of citron pomace}

The present invention relates to a composition for preventing, treating or improving degenerative brain diseases comprising a citron marmalade enzyme reaction product, and more specifically, to a culture medium of Leuconostoc mesenteroides subsp. dextranicum NY203 strain and It relates to a composition for preventing, treating or improving degenerative brain diseases containing an enzyme reaction product prepared by reacting an enzyme solution derived from Aspergillus niger strain with citron peel as a substrate.

Citron is the only specialty product of Korea's southern coast, and is estimated to be produced annually around 20,000 tons, mainly on Geoje Island. Currently, citron is overproduced and farmers have almost given up on growing it, and the citron syrup (for citron tea) that has been processed and produced so far is only consumed seasonally in the winter and cannot be stored during the summer, so a huge amount is being discarded. This is the situation.

Yuzu is not only rich in vitamins C, P, and E, but also contains limonin and citron, a compound of the limonoid family that is currently found in grapefruits and oranges in the United States and Japan and has been shown to be a powerful anti-cancer substance. It was confirmed that nomilin was contained in a significant amount. In particular, it was found that it contains a large amount of flavonoid compounds such as limonene, carvone, coumarin, and terpinene, which have high anti-cancer properties. Citron is a fruit that must be eaten with its peel because the peel contains a large amount of these bioactive substances.

Until now, citrons were mainly used as citron syrup, and the only way to do this was to pickle finely chopped citrons in about 60% sugar, add a certain amount of water, and use it as tea. Recently, citron juice sold by some companies is made by adding water to citron extract or diluting citron juice squeezed by pressing citron with water, so the effective bioactive substances in the peel are hardly utilized. Additionally, if you look at the patent technologies registered for citron, you can see that they are not very diverse compared to other fruits.

The main components of yuzu's bitter taste are naringin and hesperidin, which are contained in large amounts compared to other citrus fruits. Even when the fruit is ripe, yuzu retains its bitter taste because, unlike other citrus fruits, it does not have the naringinase enzyme that breaks down these bitter substances. Additionally, it contains more than three times the pectin content of other citrus fruits, so it can be enjoyed raw. It was impossible to juice it with the skin. When making yujacheong, it is made by pickling it with more than 60% of the total amount of sugar to remove the bitter taste. Therefore, the excessive amount of sugar sweetness masks the bitter taste of naringin, making it less bitter. Currently, most of the citron produced is consumed as citron syrup, which results in excessive amounts of sugar being consumed, and most of the citron peels in the citron syrup are discarded without being eaten.

The bitter taste present in some citrus fruits, especially yuzu, is present in excessive amounts and limits the production of yuzu juice. Also, due to the strong bitter taste of ginseng, ginseng drinks are not becoming a favorite beverage for the public, including children. Therefore, making drinks by removing the bitter taste of citrus fruits and ginseng is very commercially useful in popularizing citrus drinks and ginseng drinks.

Methods for producing fungal cultures have been studied to produce naringinase, which removes the bitter taste of citron. Naringinase-producing strains are known to produce beta-glucosidase enzymes in addition to naringinase during culture. It is known that the mechanism by which the bitter taste of yuzu is removed is because beta-glucosidase and naringinase act in stages to decompose naringin, which produces a bitter taste, into prunin and naringenin, which do not have a bitter taste.

Accordingly, the present inventors reacted the culture medium of the Leuconostoc mesenteroides subsp. dextranicum NY203 strain and the enzyme solution derived from the Aspergillus niger strain with citron peel as a substrate, producing a product. It was confirmed that the enzyme reaction product had excellent protection and anti-stress effects on brain cells and improved cognitive function.

Accordingly, the object of the present invention is Leuconostoc genus ( Leuconostoc sp .) microorganism or its culture medium; and Aspergillus ( Aspergillus sp .) For preventing or treating degenerative brain disease, comprising an enzyme reaction product prepared by reacting at least one selected from the group consisting of microorganisms of the genus Aspergillus, a culture medium thereof, or a fraction of a culture medium thereof in the presence of vegetable fiber. To provide a pharmaceutical composition.

Another object of the present invention is a microorganism of the genus Leuconostoc or its culture medium; and an enzyme reaction product prepared by reacting at least one selected from the group consisting of Aspergillus microorganisms, their cultures, or fractions of their cultures in the presence of vegetable fiber, providing a health functional food composition for improving degenerative brain diseases. will be.

Another object of the present invention is a microorganism of the genus Leuconostoc or its culture medium; It relates to the use of an enzyme reaction product prepared by reacting one or more species selected from the group consisting of Aspergillus microorganisms, their cultures, or fractions of their cultures in the presence of vegetable fiber to prevent, treat, or improve degenerative brain diseases.

The present invention relates to a composition for preventing, treating or improving degenerative brain diseases containing an enzyme reaction product of citron marigold. The composition according to the present invention exhibits protective and anti-stress effects on brain cells and an effect of improving cognitive function.

The present inventors have developed an enzyme prepared by reacting the culture medium of Leuconostoc mesenteroides subsp. dextranicum NY203 strain and the enzyme solution derived from Aspergillus niger strain with citron peel as a substrate. It was confirmed that the reactants were excellent in suppressing cell death of brain cells, reducing the content of cortisol, a stress hormone, and reducing the content of AChE (Acetylcholinesterase), which is considered to improve cognitive function.

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

One aspect of the present invention is a microorganism of the genus Leuconostoc ( Leuconostoc sp .) or a culture medium thereof; and Aspergillus ( Aspergillus sp .) For preventing or treating degenerative brain disease, comprising an enzyme reaction product prepared by reacting at least one selected from the group consisting of microorganisms of the genus Aspergillus, a culture medium thereof, or a fraction of a culture medium thereof in the presence of vegetable fiber. It is a pharmaceutical composition.

In the present invention, the microorganism of the Leuconostoc genus may be the Leuconostoc mesenteroid subspecies Dextranicum NY203 strain deposited under the accession number KCTC13679BP.

The term “culture medium” in this specification means that the strain is removed from the culture produced by culturing the strain, and the culture medium includes enzymes produced by the strain. Since the culture medium of the strain used in this specification contains an enzyme derived from the strain, it may be used as an enzyme used in the preparation of an enzyme reaction product.

The fraction of the culture medium is a result obtained by performing fractionation to separate a specific component or group of specific components from the culture medium containing various components. In this specification, the fraction of the culture medium refers to enzymes or enzymes isolated from the culture medium of the strain. It means a combination of some of the

For example, in the examples herein, NY enzyme refers to the supernatant of the culture medium of Leuconostoc mesenteroid subspecies Dextranicum NY203 strain.

The culture medium of the microorganism of the Leuconostoc genus may be 0.1 to 20.0% (v/v) relative to the total enzyme reaction, preferably 0.5 to 20.0% (v/v), 1.0 to 20.0% (v/v), or It may be 5.0 to 20.0% (v/v), for example, 10.0 to 20.0% (v/v), but is not limited thereto.

The reaction for preparing an enzyme reaction using the culture medium of the Leuconostoc microorganism may be carried out at 32 to 40 ° C. for 2 to 10 hours, and is preferably carried out at 35 to 38 ° C. for 5 to 7 hours. It may be, but is not limited to this.

The microorganism of the Aspergillus genus may be Aspergillus niger.

In the present invention, the fraction of the culture medium of the Aspergillus microorganisms includes a first enzyme solution containing at least one selected from the group consisting of pectinase, glucanase, and arabinase; And it may be at least one type selected from the group consisting of a second enzyme solution consisting of at least one type selected from the group consisting of cellulase, hemicellulase, and protease. Preferably, the fractions of the culture solution of the Aspergillus genus microorganism may be both the first enzyme solution and the second enzyme solution, and the first enzyme solution and the second enzyme solution may be used sequentially or mixed, but are limited thereto. That is not the case.

The concentration of the first enzyme solution may be 0.01 to 20.00% (v/v), preferably 0.05 to 2.00% (v/v), 0.10 to 2.00% (v/v), 0.50 to 20.00% (v/v), relative to the total enzyme reaction product. 2.00% (v/v), 1.00 to 2.00% (v/v), 0.01 to 1.00% (v/v), 0.05 to 1.00% (v/v), 0.10 to 1.00% (v/v) or 0.50 to 1.00% (v/v) It may be 1.00% (v/v), for example, 1.00% (v/v), but is not limited thereto.

The reaction for preparing the enzyme reaction using the first enzyme solution may be carried out at 45 to 55°C for 1 to 8 hours, and preferably at 48 to 52°C for 2 to 4 hours. It is not limited to this.

For example, the first enzyme solution herein may be a UF enzyme, which contains pectinase, glucanase and arabinase derived from Aspergillus niger strain.

According to one embodiment of the present invention, the UF enzyme exhibits an excellent dissolution rate for both soluble and insoluble dietary fiber.

The concentration of the second enzyme solution may be 0.01 to 20.00% (v/v), preferably 0.05 to 2.00% (v/v), 0.10 to 2.00% (v/v), or 0.50 to 20.00% (v/v) of the total enzyme reaction product. 2.00% (v/v), 1.00 to 2.00% (v/v), 0.01 to 1.00% (v/v), 0.05 to 1.00% (v/v), 0.10 to 1.00% (v/v) or 0.50 to 1.00% (v/v) It may be 1.00% (v/v), for example, 1.00% (v/v), but is not limited thereto.

The reaction for preparing an enzyme reaction using the second enzyme solution may be carried out at 45 to 55°C for 2 to 10 hours, and preferably at 48 to 52°C for 5 to 7 hours. It is not limited to this.

For example, the second enzyme solution herein may be KN enzyme, which contains cellulase, hemicellulase and protease derived from Aspergillus niger strain.

According to one embodiment of the present invention, when the KN enzyme reacts with naringenin, glucose, and rhamnose, which constitute bitter-tasting naringin, it can decompose them into prunine and rhamnose, which do not have a bitter taste, thereby reducing the bitter taste. It can be used for reduction purposes.

According to one embodiment of the present invention, 1) Leuconostoc mesenteroid subspecies Dextranicum NY203 strain or a culture medium thereof; and 2) Enzyme reactants prepared using Aspergillus niger strain, its culture medium, or fractions of its culture medium have excellent protective and anti-stress effects on brain cells and cognitive function improvement effects, thereby preventing and treating degenerative brain diseases. Or it can be used for improvement purposes.

In the present invention, the vegetable fiber may be derived from one or more species selected from the group consisting of fruits, leaves, stems, and roots of plants.

The plant may be one or more species selected from the group consisting of citron, tangerine, orange, and tangerine.

In the present invention, the concentration of the vegetable fiber may be 1.0 to 20.0% (w/v), preferably 2.5 to 20.0% (w/v), 5.0 to 20.0% (w/v), relative to the total enzyme reaction product. , 10.0 to 20.0% (w/v), 1.0 to 10.0% (w/v), 2.5 to 10.0% (w/v), or 5.0 to 10.0% (w/v), for example, 10.0 It may be % (w/v), but is not limited thereto.

The reaction may be carried out for 1 to 15 hours, preferably 1 to 12 hours, 1 to 9 hours, 2 to 15 hours, 2 to 12 hours, 2 to 9 hours, 3 to 15 hours, 3 to 12 hours. It may be performed for 3 to 9 hours, 6 to 15 hours, or 6 to 12 hours, for example, 6 to 9 hours, but is not limited thereto.

In the present invention, the degenerative brain disease may be one or more types selected from the group consisting of dementia, Alzheimer's disease, stroke, paralysis, Parkinson's disease, Huntington's disease, Pick's disease, and Creutzfeldt-Jakob disease.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition containing a pharmaceutically effective amount of an enzyme reactant and/or a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to achieve the efficacy or activity of the above-described enzyme reaction product.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

The pharmaceutical composition according to the present invention can be administered to mammals, including humans, through various routes. The administration method may be any commonly used method, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc. administration, and is preferably administered orally.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, body weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (e.g., hydrogel), and may additionally contain a dispersant or stabilizer. .

Another aspect of the present invention is a microorganism of the genus Leuconostoc or a culture medium thereof; and an enzyme reaction product prepared by reacting at least one selected from the group consisting of Aspergillus microorganisms, their cultures, or fractions of their cultures in the presence of vegetable fiber. It is a health functional food composition for improving degenerative brain disease.

In the present invention, the microorganism of the Leuconostoc genus may be the Leuconostoc mesenteroid subspecies Dextranicum NY203 strain deposited under the accession number KCTC13679BP.

The culture medium of the microorganism of the Leuconostoc genus may be 0.1 to 20.0% (v/v) relative to the total enzyme reaction, preferably 0.5 to 20.0% (v/v), 1.0 to 20.0% (v/v), or It may be 5.0 to 20.0% (v/v), for example, 10.0 to 20.0% (v/v), but is not limited thereto.

The microorganism of the Aspergillus genus may be Aspergillus niger.

In the present invention, the fraction of the culture medium of the Aspergillus microorganisms includes a first enzyme solution containing at least one selected from the group consisting of pectinase, glucanase, and arabinase; and a second enzyme solution containing at least one type selected from the group consisting of cellulase, hemicellulase, and protease. Preferably, the fraction of the culture solution of the Aspergillus genus microorganism may be the first enzyme solution, but is not limited thereto.

The concentration of the first enzyme solution may be 0.01 to 20.00% (v/v), preferably 0.05 to 2.00% (v/v), 0.10 to 2.00% (v/v), 0.50 to 20.00% (v/v), relative to the total enzyme reaction product. 2.00% (v/v), 1.00 to 2.00% (v/v), 0.01 to 1.00% (v/v), 0.05 to 1.00% (v/v), 0.10 to 1.00% (v/v) or 0.50 to 1.00% (v/v) It may be 1.00% (v/v), for example, 1.00% (v/v), but is not limited thereto.

The concentration of the second enzyme solution may be 0.01 to 20.00% (v/v), preferably 0.05 to 2.00% (v/v), 0.10 to 2.00% (v/v), or 0.50 to 20.00% (v/v) of the total enzyme reaction product. 2.00% (v/v), 1.00 to 2.00% (v/v), 0.01 to 1.00% (v/v), 0.05 to 1.00% (v/v), 0.10 to 1.00% (v/v) or 0.50 to 1.00% (v/v) It may be 1.00% (v/v), for example, 1.00% (v/v), but is not limited thereto.

In the present invention, the vegetable fiber may be derived from one or more species selected from the group consisting of fruits, leaves, stems, and roots of plants.

The plant may be one or more species selected from the group consisting of citron, tangerine, orange, and tangerine.

In the present invention, the concentration of the vegetable fiber may be 1.0 to 20.0% (w/v), preferably 2.5 to 20.0% (w/v), 5.0 to 20.0% (w/v), relative to the total enzyme reaction product. , 10.0 to 20.0% (w/v), 1.0 to 10.0% (w/v), 2.5 to 10.0% (w/v), or 5.0 to 10.0% (w/v), for example, 10.0 It may be % (w/v), but is not limited thereto.

The reaction may be carried out for 1 to 15 hours, preferably 1 to 12 hours, 1 to 9 hours, 2 to 15 hours, 2 to 12 hours, 2 to 9 hours, 3 to 15 hours, 3 to 12 hours. It may be performed for 3 to 9 hours, 6 to 15 hours, or 6 to 12 hours, for example, 6 to 9 hours, but is not limited thereto.

In the present invention, the degenerative brain disease may be one or more types selected from the group consisting of dementia, Alzheimer's disease, stroke, paralysis, Parkinson's disease, Huntington's disease, Pick's disease, and Creutzfeldt-Jakob disease.

When using the health functional food composition of the present invention as a food additive, the health functional food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. In general, when manufacturing a food or beverage, the health functional food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. These include alcoholic beverages and vitamin complexes, and include all foods in the conventional sense.

The beverage may contain various flavors or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, and glycerin. , alcohol, and carbonating agents used in carbonated beverages. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

The present invention relates to a composition for preventing, treating or ameliorating degenerative brain disease containing an enzyme reaction product of citron marmalade. The composition exhibits a protective and anti-stress effect on brain cells and an effect of improving cognitive function, thereby effectively treating degenerative brain disease. It can be used for prevention, treatment or improvement.

Figure 1 is a graph comparing the degree of dietary fiber solubilization of selected strains according to an embodiment of the present invention.
Figure 2a is a photograph comparing the dietary fiber dissolution rates of 11 types of commercial enzymes according to an embodiment of the present invention.
Figure 2b is a graph comparing the dietary fiber dissolution rates of 11 types of commercial enzymes according to an embodiment of the present invention.
Figure 3a is a photograph comparing the bitter taste removal effect of 11 types of commercial enzymes according to an embodiment of the present invention.
Figure 3b is a graph comparing the bitter taste removal effect of 11 types of commercial enzymes according to an embodiment of the present invention.
Figure 4a is a photograph showing the substrate content, enzyme content, and dietary fiber dissolution rate by reaction time of the culture medium of Leuconostoc mesenteroides subsp. dextranicum NY203 strain according to an embodiment of the present invention.
Figure 4b is a graph showing the dissolution rate of dietary fiber by substrate content by the culture medium and UF enzyme of the NY203 strain according to an embodiment of the present invention.
Figure 4c is a graph showing the dissolution rate of dietary fiber by enzyme content in the culture medium of the NY203 strain according to an embodiment of the present invention.
Figure 4d is a graph showing the dissolution rate of dietary fiber by enzyme content by UF enzyme according to an embodiment of the present invention.
Figure 4e is a graph showing the dissolution rate of dietary fiber by reaction time in the culture medium of the NY203 strain according to an embodiment of the present invention.
Figure 4f is a graph showing the dissolution rate of dietary fiber by reaction time by UF enzyme according to an embodiment of the present invention.
Figure 5a is a graph showing the change in rhamnose content by substrate content by KN enzyme according to an embodiment of the present invention.
Figure 5b is a graph showing the change in rhamnose and naringin content by enzyme content by KN enzyme according to an embodiment of the present invention.
Figure 5c is a graph showing the change in rhamnose and naringin content over reaction time by KN enzyme according to an embodiment of the present invention.
Figure 6a is a photograph showing the upper and lower layers and powder of the citron peel enzyme reaction product following enzyme treatment according to an embodiment of the present invention.
Figure 6b is a graph showing the dissolution rate of the citron pumpkin enzyme reaction product according to enzyme treatment according to an embodiment of the present invention.
Figure 6c is a photograph showing the dissolution rate of the citron peel enzyme reaction product according to enzyme treatment according to an embodiment of the present invention.
Figure 6d is a graph showing the results of HPLC analysis of free sugars in the citron peel enzyme reaction product according to enzyme treatment according to an embodiment of the present invention.
Figure 7 is a graph showing the distribution of dietary fiber in the enzyme reaction product of citron peel according to enzyme treatment according to an embodiment of the present invention.
Figure 8 is a scanning electron microscope (SEM) photograph of an enzyme-treated citron peel enzyme reaction product according to an embodiment of the present invention.
Figure 9 is a graph showing the results of FTIR (Fourier transform infra-red spectroscopy) analysis of the enzyme-treated citron marmalade enzyme reaction product according to an embodiment of the present invention.
Figure 10 is an HPLC analysis graph of changes in functional components of the enzyme-treated citron pumpkin enzyme reaction product according to an embodiment of the present invention.
Figure 11a is a graph showing the results of measuring cytotoxicity by Scopolamine, Donepezil, and Theanine according to an embodiment of the present invention.
Figure 11b is a graph of the cytotoxicity measurement results of the enzyme-treated citron pumpkin enzyme reaction product at different concentrations according to an embodiment of the present invention.
Figure 12a is a photograph showing the change in cell viability by treatment with citron marmalade enzyme reaction under scopolamine treatment conditions according to an embodiment of the present invention.
Figure 12b is a graph showing the change in cell viability by treatment with citron marmalade enzyme reaction under scopolamine treatment conditions according to an embodiment of the present invention.
Figure 13 is a graph showing the change in cortisol (Cprtisol) content due to treatment of citron peel enzyme reaction under scopolamine treatment conditions according to an embodiment of the present invention.
Figure 14 is a graph showing the change in AChE (Acetylcolinesterase) content by treatment of citron peel enzyme reaction under scopolamine treatment conditions according to an embodiment of the present invention.
Figure 15 is a graph showing the antioxidant activity of the citron pumpkin enzyme reaction product according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/volume)% for solid/solid, (weight/volume)% for solid/liquid, and Liquid/liquid is (volume/volume)%.

Example 1: Enzyme selection

1-1. Securing the optimal strain for solubilization of insoluble dietary fiber

The citron peel used as a substrate is a traditional citron variety harvested in November, and is a powder obtained by freeze-drying and grinding citron peel with the juice and seeds removed. The reaction conditions were 0.05 M sodium acetate buffer (pH 5.2), 10% citron peel as the substrate, and Leuconostoc citreum NY126, a superior strain of our own selection, as the enzyme, and Leuconostoc mesen. 10% of the culture medium of Leuconostoc mesenteroides subsp. dextranicum NY174 and NY203 strains was added and reacted at 37°C for 6 hours, and then confirmed by thin layer chromatography (TLC). The TLC development conditions were Nitromethane:1-Propanol:H ₂ O = 2:5:1.5, followed by development twice, followed by sulfuric acid development.

As can be seen in Figure 1, the dissolution rates of cellulose, etc. by the strain culture medium were 47.4%, 39.4%, and 62.0%, respectively, and the NY203 strain was observed to be the most efficient. Among these, strain NY203 is a strain of Leuconostoc mesenteroid subgenus Dextranicum NY203 deposited with accession number KCTC13679BP.

1-2. Securing optimal enzyme solution for dissolving soluble dietary fiber

The optimal conditions were searched using the following 11 commercial enzymes.

Strains and derived enzymes for dissolving commercial soluble dietary fiber and removing bitter ingredients turn product Derived bacteria activation One P.T. Aspergillus aculeatus Polygalactronase 2 VS Aspergillus aureatus β-Glucanase, Arabanase, Cellulase, Hemicellulase, Xylanase 3 CL Trichoderma reesei β-glucanase, cellulase 4 CP Aspergillus niger Pectinase, polygalactronase, pectinmethylesterase 5 PR Aspergillus niger Pectinase, pectin methyl esterase 6 UF Aspergillus niger Pectinase, glucanase, arabinase 7 TA Aspergillus oryzae Tannin acylhydrolase 8 B.G. Trichoderma Ressey β-glucanase, cellulase 9 PL Aspergillus niger β-Glucosidase, Cellobiase 10 AC Aspergillus niger β-glucosidase, cellulase, hemicellulase 11 KN Aspergillus niger Cellulase, hemicellulase, protease

The reaction conditions were 0.05 M sodium acetate buffer (pH 5.2), adding 10% of citron foil as a substrate and 1% of each of the 11 enzyme solutions in Table 1 above as enzymes, and reacting at 50°C for 6 hours to produce a citron foil enzyme reaction product. was manufactured.

As can be seen in Figures 2a and 2b, all enzymes showed a dissolution rate of 13.2 to 51.9%, which was lower than the 62.0% dissolution rate shown in NY203. The one with the highest dissolution rate is UF enzyme No. 6 (PECLYVE UF CLEANER, Glucanase 50 U/g, Arabinase 450 U/g, Lyven Zac Normmandial), which is Aspergillus niger . An enzyme solution containing derived pectinase, polygalactronase, and pectinmethylesterase was selected.

1-3. Securing the optimal enzyme solution to remove bitter taste

Bitterness is mainly caused by glycosides (glucose, rahmnose) bound to aglycon. During the fermentation process, when the sugar component of this glycoside is broken down and changed to aglycone, most of it becomes bitter. It is known to lose its taste. To remove the bitter taste, naringin, which produces the unique bitter taste of yuzu, is made into a non-glycoside by decomposing sugars such as rhamnose or glucose bound to it using beta-glucosidase and nariniginase. did.

The reaction conditions were 0.05 M sodium acetate buffer (pH 5.2), adding 10% of citron foil as a substrate and 1% of each of the 11 enzyme solutions in Table 1 above as enzymes, and reacting at 50°C for 6 hours to produce a citron foil enzyme reaction product. was manufactured. As standard substances, FS (0.1% fructose, sucrose), GR (0.1% glucose, rhamnose), IMO (1%, Isomaltose series), and MO (1% Maltose series) were used.

As can be seen in Figures 3a and 3b, the sugar content of rhamnose after the enzyme reaction varied from 0.48 to 3.1 mg/mL. Aspergillus niger with No. 11 KN enzyme (Celluase KN, B-glucosidase 20 U/g Vision Biocam) The decomposition rate of naringin was highest when an enzyme solution containing cellulase, hemicellulase, and protease was used, and the sugar content of rhamnose derived from it was 3.1 mg/mL. This was selected because it was the highest.

Example 2: Optimization of manufacturing conditions for citron marmalade enzyme reaction product

Leuconostoc mesenteroid subgenus Dextranicum in Example 1-1 Cellulose and hemicellulose, which are insoluble dietary fibers, were decomposed using 10% of the NY203 strain culture medium, and soluble dietary fiber was produced using 1% of UF, an enzyme derived from Aspergillus niger in Example 1-2. Optimization conditions were investigated for dissolving pectin and removing bitter taste components using KN 1%, an enzyme derived from Aspergillus niger in Examples 1-3.

The substrate, citron peel, is 0%, 1%, 2.5%, 5%, 10%, 20%, the enzyme is NY (NY203 strain culture medium) 10%, and the UF and KN enzymes are 0%, 0.01%, 0.1%, and 0.5%, respectively. , 1%, 2%, 5%, 10%, 20%, reaction time is 0, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, 15h. After reacting for a while and analyzing by TLC, the weight difference between the samples before and after enzyme treatment was compared and the solubilization rate was expressed as a %. The solubilization rate was expressed in % by comparing the weight difference of the sample before and after enzyme treatment. (GP(0.1% glucose, pectin), FS(0.1% fructose, sucrose), Isomalto series(IMO), Maltose series(MO))

As can be seen in Figures 4a and 4b, the solubility rate was highest at 10% for NY enzyme and 20% for UF with respect to the content of citron peel used as a substrate, but the content of 10% was optimal in terms of solubilization efficiency of enzyme and substrate. It was confirmed that it was.

As can be seen in Figures 4a, 4c, and 4d, the optimal dissolution rate of dietary fiber by enzyme concentration was 10% for NY and 1% for UF.

As can be seen in Figures 4a, 4e, and 4f, the optimal reaction time was 6 hours for NY and 3 hours for UF, and in general, the NY enzyme had a higher dietary fiber solubilization rate than the commercial UF.

As can be seen in Figures 5a to 5c, the result of enzyme treatment to remove the bitter taste is that when the citron peel content was 10%, the KN concentration was 1%, and the reaction time was 6 hours, rhamnose sugar was removed from naringin and the bitter taste was reduced. Since a tendency appeared, this was established as the optimal reaction condition.

Example 3: Quantitative and qualitative analysis of citron peel enzyme reaction

3-1. Analysis of functional components and free sugar content of citron peel enzyme reaction

Yujapak enzyme reaction product was prepared based on the optimal reaction conditions determined in Example 2.

As a single enzyme treatment group, the NY treatment group (NY) was treated with 10% citron peel and 10% NY203 strain culture medium and reacted at 37°C for 6 hours. In the UF treatment group (UF), 10% citron peel and 1% UF enzyme were applied and reacted at 50°C for 3 hours. In the KN treatment group (KN), 10% citron peel and 1% KN enzyme were applied and reacted at 50°C for 6 hours.

As a complex enzyme treatment group, the NY and UF complex treatment group (NY+UF) was treated with 10% citron peel and 10% NY203 strain culture medium, reacted at 37°C for 6 hours, and enzyme deactivated at 100°C for 10 minutes, followed by UF enzyme treatment. 1% was applied and reacted at 50°C for 3 hours. For the NY, UF and KN combination treatment group (NY+UF+KN), 1% of KN enzyme was applied to the deactivated NY and UF combination treatment group and reacted at 50°C for 6 hours.

As can be seen in Figures 6a to 6c, the dissolution rate of the citron peel enzyme reaction was 48% for NY, 42% for UF, 52% for NY+UF, and 68% for NY+UF+KN, all of which increased compared to the untreated group (2%). In particular, the dissolution rate was found to be highest in the case of combined treatment of NY, UF, and KN.

Afterwards, the functional components and free sugar content were investigated from the obtained citron peel enzyme reaction using HPLC. Free sugar analysis was performed by adding distilled water to the pulp, homogenizing it, filtering it, diluting it 10 to 40 times, passing it through a 0.45 ㎛ membrane filter, and analyzing it using HPLC. Since cellulose is released into glucose, hemicellulose into arabinose, and pectin into galactronic acid, the degree of increase in their contents was investigated.

Specifically, HPLC was performed using the Agilent 1216 infinity LC series system (Agilent Technologies, Palo Alto, CA, USA). The analysis column was ZORBAX eclipse plus C18 (4.6x250mm, 5-Micron, Agilent Technologies, Palo Alto, CA, USA), and the solvent composition was A: 0.1% formic acid, B: methanol-acetonitrile. did.

The solvent gradient starts with A:80, B:20, A:60, B:40 from 5 to 10 minutes, A:50, B:50 from 10.1 to 15 minutes, and A:30, B from 15.1 to 20 minutes. :70, A:0 and B:100 were used for 20.1-25 minutes, and A:80 and B:20 were analyzed for 25.1-30 minutes. The solvent flow rate was set at 0.5 mL/min and the column temperature was fixed at 35°C. 10 uL of sample was injected and detected at 280 nm.

As can be seen in Figure 6d, glucose, fructose, and arabinose increased when treated with NY compared to untreated, and when treated with UF, there was little change in glucose and fructose, and the content of galactronic acid increased significantly. The contents of glucose and galactronic acid increased during NY+UF treatment, but were lower than those of the single enzyme treatment groups. When NY+UF+KN combined treatment, galactronic acid, glucose, fructose, and arabinose were all the highest.

3-2. Analysis of dietary fiber composition of citron peel enzyme reaction

The dietary fiber composition was investigated from the citron peel powder remaining after enzyme treatment.

The insoluble and soluble dietary fiber content of the dietary fiber source isolated from citron peel was measured using the enzymatic gravimetric method of Prosky et al., and the sum of the insoluble and soluble was taken as the total dietary fiber. Using citron dietary fiber, accurately weigh 1 g of each dried sample, add 50 mL of phosphate buffer (pH 6.0), and then use amylase, protease, and amyloglucosidase. and were sequentially hydrolyzed. Afterwards, the dietary fiber content was calculated through the difference between each protein and ash content measurement value.

To derive the insoluble dietary fiber (IDF) content, the washed residue after enzyme treatment was washed with 95% and 78% ethanol and acetone, and then the difference in protein and ash content measurements was used in the oven-dried residue. The calculation was performed. In addition, after enzyme treatment, 95% ethanol was added to the filtrate at 60°C, the precipitated solid was dried and measured, and the soluble dietary fiber (SDF) content was calculated using the difference between the protein and ash content measurements, and the insoluble dietary fiber (SDF) content was calculated. and total dietary fiber (TDF) content was calculated using the sum of the soluble dietary fiber content.

TDF (%) IDF (%) SDF (%) Untreated 34.52a 17.85a 16.67a NY processing 18.06b 7.22c 10.84b UF treatment 18.04b 11.68b 6.36c NY+UF processing 9.51c 7.03c 2.48d NY+UF+KN processing 8.76c 6.09c 2.67d

As can be seen in Table 2 and Figure 7, the total dietary fiber (TDF) content decreased in all groups. In the NY treatment group, insoluble dietary fiber (IDF) was significantly reduced due to the decomposition of cellulose and hemicellulose. In particular, cellulose decreased by 2 times and hemicellulose by 3 times. In the UF-treated group, soluble dietary fiber (SDF) decreased due to the dissolution of pectin, and in particular, cellulose decreased by 2-fold and pectin decreased by 5-fold. In the NY+UF+KN treatment group, both soluble and insoluble dietary fiber compositions were dissolved, and 60-65% of the total was dissolved, which was consistent with the dissolution rate results.

Example 4: Confirmation of structural changes in citron marmalade enzyme reactants

Untreated, NY, and UF treated samples were analyzed using scanning electron microscopy (ZEMINI SEM, FEI Co., The Netherlands) and Fourier transform infra-red spectroscopy (FTIR) chromatogram analysis. Structural changes in citron peel powder were confirmed. For the citron peel enzyme reaction product, images of particles were observed under a scanning electron microscope at 400x magnification.

As can be seen in Figure 8, it can be observed that the structure is collapsed in the NY+UF+KN treated group compared to the untreated group, many pores are created in the tissue, and the structure swells like a balloon and is easily dissolved.

To confirm changes in the chemical properties of enzyme-treated dietary fiber, FTIR analysis (Fourier transform infra-red spectroscopy, perkin Elmer Spectrum 400) was performed.

As a result of the analysis, the dietary fiber treated with the citron mixed enzyme had three peak ranges in the FTIR spectrum: 1) 3500 to 2900 cm ^-1 , 2) 1700 to 1300 cm ^-1 , 3) 1200 to 900 cm. It was displayed as ^-1 . According to existing research results, 1) in each range refers to the generation of OH groups, and mainly water or ethanol characteristics appear. 2) mainly refers to the presence, increase or production of phenolic compounds and represents the main characteristics of flavonoids and phenols. 3) indicates the expansion or increase of the carbonyl group, and an increase in the curve height indicates an increase in the C=O bond.

As can be seen in Figure 9, the peak patterns of the untreated group, NY-treated group, and UF-treated group are similar, and most of the peaks are concentrated at 1700 to 1300 cm ^-1 , which indicates the presence of phenol compounds as flavonoids. In particular, the size of the peak in the NY+UF+KN complex treatment group increased compared to the untreated group or the single and complex enzyme treatment groups, and the creation of aromatic rings increased through the presence of OH groups and the combination of phenolic compounds. showed. The complex enzyme treatment groups appeared similar to each other in terms of chemical structure.

Example 5: Change in functional ingredient composition by complex enzyme treatment

In order to specify changes in the functional components of the enzyme reaction product, HPLC analysis was performed by comparing the untreated sample and the NY+UF+KN mixed enzyme-treated supernatant powder.

mg/g DW Nari routine Naringin hesperidin Neohesperidin naringenin Hesperetin Total Untreated 22.30d 3.36d 10.73d 3.84d 0.68b 0.11b 41.03d NY 23.43c 3.67c 11.39c 4.16c 0.62c 0.08c 43.36c UF 27.52b 6.58b 12.70b 5.41b 0.00d 0.00d 52.21b NY+UF 31.88a 7.91a 15.58a 6.78a 0.00d 0.00d 62.15a NY+UF+KN 0.62e 2.16e 0.80e 0.00e 2.20a 0.39a 6.16e

As can be seen in Table 3 and Figure 10, the functional ingredients (Total) increased by 4.8%, 27.3%, and 51.2% in the NY, UF, and NY+UF treated groups, respectively, compared to the untreated group, but in the NY+UF+KN combined treatment group, In fact, functional ingredients decreased by 88.0%.

Compared to the untreated group, in the NY+UF+KN combination treatment group, the diet efficacy ingredients Narirutin and Hesperidin decreased slightly, while Naringin and Neohesperidin, which are known to produce a bitter taste, decreased by 50~65%. It appears to have decreased by more than %, showing that the bitter taste has been greatly reduced.

Overall, the content of functional ingredients increased as more enzymes were added to react with the citron powder, but in the UF treatment group, the functional ingredients increased by 51%, while more than 90% of the functional ingredients were destroyed when KN was added, so the combined treatment group had NY+ It was judged that it would be better to use it only up to UF. Yujapak enzyme reaction product is a powder form obtained by freeze-drying the supernatant produced after each enzyme reaction and was used to evaluate the brain cell protection effect and other efficacy.

Example 6: Confirmation of brain cell protection effect by citron pumpkin enzyme reaction product

6-1. Toxicity Assessment

SH-SY5Y brain nerve cell damage was induced by treating scopolamine, which is known to accelerate the decomposition of acetylcholine by increasing the expression of AChE (Acetylcholinesterase), and then used as a positive control to treat dementia. The test was conducted using donepezil or theanine, a natural ingredient known to improve cognitive function and relieve mental stress.

SH-SY5Y, epithelial cells, were purchased from the American Cell Line Bank. SH-SY5Y stored in freezing media was removed from the nitrogen tank and quickly placed in a 37°C water bath for 1 minute. After dissolving, it was slowly transferred to 10 ml RFP (media + 10% FBS + 1% Streptomycin-penicillin) medium prepared in advance and subjected to centrifugation.

After removing (suction) the RFP medium, leaving only the pellet, add 1 ml of RFP and gently suspend it, then transfer to a T-25 flask containing 20 ml of RFP and place at 37°C in the presence of 5% CO ₂ Incubation was performed under these conditions, and subculture was performed when the amount of cells grew to about 70-80%.

As can be seen in Figure 11a, as a result of measuring cytotoxicity to SH-SY5Y brain nerve cells, scopolamine was found to be optimal at 5 mM, donecortex at 10 uM, and theanine at 200 uM.

As can be seen in Figure 11b, it was confirmed that there was no toxicity to cells in the citron peel enzyme-treated experimental group at a treatment level of 1.00 to 0.01% of the sample.

6-2. Brain cell protection effect

To determine the protective effect on SH-SY5Y brain nerve cells, cells were treated with 5 mM scopolamine 24 hours after treatment at concentrations of 0.01%, 0.10%, and 1.00%, respectively, and MTT analysis was performed 24 hours later. Cell viability was confirmed using (assay).

To perform MTT analysis, 20 μl of MTT (3-(4,5-Dimethylthiazol-2-yl) (5 mg/ml) diluted in PBS was treated with cells in each well of a 96-well plate. Cultured in an incubator at 37°C with 5% CO ₂ , blocked from light, for 3 hours. Afterwards, 150 μl of DMSO was added and absorbance was measured at a wavelength of 570 nm. Cell viability relative to the control was expressed as a percentage (%). .

As can be seen in FIGS. 12a and 12b, cell photos observed under an optical microscope showed that the scopolamine-treated group was reduced to less than 40% compared to the untreated group, and the positive control group, theanine-treated group and Donepezil. , Aricept, 5 mg Pfizer) treatment group, the cell survival rate increased to over 70%. As enzyme treatment at a concentration of 1% was observed to be effective in the survival rate of brain nerve cells, an overall protective effect against apoptosis was observed in the enzyme treatment group. In particular, it was confirmed that the survival rate of the NY+UF enzyme-treated group increased to 85% compared to the survival rate of 65% in the non-enzyme treated group.

6-3. anti-stress effect

To determine the anti-stress effect on SH-SY5Y brain nerve cells, the cells were treated with 5 mM scopolamine 24 hours after the sample was treated, and analyzed 24 hours later.

To analyze the anti-stress effect, Cortisol for Serum ELISA (COS6134, CALBIOTECH, U.S.A.) was used, and the citron peel enzyme reaction was diluted 100, 200, and 500 times as a sample, and the dilution ratio was set to fall within the range of the standard material calibration curve. Put 25 μl of the sample and standard into a well of a 24-well plate, add 50 μl of Biotin reagent and 100 μl of Cortisol Enzyme Conjugate in turn, mix for 10 seconds, and mix for 10 seconds. The reaction was carried out in an incubator at 37°C for an hour.

The liquid (sample, biotin reagent, enzyme conjugate) in the well was removed and then washed once with cortisol wash buffer. After treating with 100 μl of TMB substrate and culturing for 15 minutes in an incubator at 37°C, 50 μl of stop solution was added and the reaction was stopped. Absorbance was measured at a wavelength of 450 nm using a spectrophotometer (BioTex, Epoch, USA). Conversion of cortisol content according to absorbance was converted into a formula according to the standard material value.

As can be seen in Figure 13, cortisol, a stress hormone, When treated with scopolamine, the cortisol content increased more than 4 times compared to untreated, and in the theanine treated group, the cortisol content decreased by 2 times to 45 ng/mL. The NY, UF, NY+UF, and NY+UF+KN treatment groups showed 49.4, 48.6, 45.1, and 51.8 ng/mL, respectively, showing that cortisol secretion decreased by about 10% compared to the untreated group (56.1 ng/ml). .

6-4. Cognitive function improvement effect

To determine the cognitive function improvement effect confirmed from SH-SY5Y brain nerve cells, the cells were treated with 5 mM scopolamine 24 hours after the sample was treated, and analyzed 24 hours later.

SH-SY5Y brain nerve cells were treated with 200 μl of RIPA Lysis extraction buffer, and then cell lysate was obtained at -20°C and used in the experiment. Acetylcholinesterase ELISA Kit (CSB-E0967H, CUSABIO, USA) was used, and the cell lysate was diluted 200-fold and 400-fold to set a dilution ratio within the range of the standard material calibration curve. 100 μl of sample and standard material were added to the well and then reacted in an incubator at 37°C for 2 hours. The liquid (sample, standard material) in the well was removed, treated with 100 μl of biotin-antibody, and incubated at 37°C for 1 hour. To remove unbound antibodies, the liquid (Biotin-antibody) in the well was removed and then washed three times with AchE washing buffer.

100 μl of HRP-avidin was added and reacted in an incubator at 37°C for 1 hour, then washed 5 times in the same manner as above. 90 μl of TMB substrate was added and reacted in an incubator at 37°C for 20 minutes, then 50 μl of termination reagent was added and the reaction was stopped. Absorbance was measured at a wavelength of 450 nm using a spectrophotometer (BioTex, Epoch, USA). Conversion of AchE content according to absorbance was converted to a formula according to the standard material value.

Scopolamine
0mM Scopolamine
5mM theanine donee cortex Untreated NY UF NY+UF NY+UF+KN AChE (uM/mL) 375+
0.25e 1,365+
0.33a 1,092+
0.06b 725+
1.06d 1,112+
0.08b 1,050+
0.48b 877 +
0.05c 777+
0.05d 852+
0.09c

As can be seen in Figure 14, the AChE content in the scopolamine-treated group increased by more than three times compared to the untreated group. The donecortex-treated group showed an AchE content of 53%, which was 47% of the scopolamine-treated group. The non-treatment, NY, UF, NY+UF, and NY+UF+KN treatment groups were each 19% to 43%, and the cognitive function improvement effect of the NY+UF treatment group was observed.

Example 7: Analysis of antioxidant activity of citron peel enzyme reaction

The antioxidant activity of the citron peel enzyme reaction was investigated.

Specifically, antioxidant activity was analyzed by measuring the radical scavenging effect using DPPH (1,1-diphenyl-2-picrylhydrazyl). In a 96-well plate, the citron peel enzyme reaction was added to 100 μM DPPH ethanol solution, reacted at 37°C for 30 minutes, and then analyzed at 515 nm using a VERSAmax microplate reader (Molecular Devices, USA). Absorbance was measured. Antioxidant activity was expressed as the radical scavenging ability (IC50) of the citron peel enzyme reaction, which appears when the absorbance decreases by 50%, and data were obtained by repeating the experiment three times.

As can be seen in Figure 15, the antioxidant effect was improved by 60% in the NY+UF treated group compared to the untreated group.

Korea Research Institute of Bioscience and Biotechnology KCTC13679BP 20181024

Claims (16)

Leuconostoc sp . Microorganisms or cultures thereof; and Aspergillus ( Aspergillus sp .) For preventing or treating degenerative brain disease, comprising an enzyme reaction product prepared by reacting at least one selected from the group consisting of microorganisms of the genus Aspergillus, a culture medium thereof, or a fraction of a culture medium thereof in the presence of vegetable fiber. Pharmaceutical composition. The method of claim 1, wherein the microorganism of the Leuconostoc genus is a strain of Leuconostoc mesenteroides subsp. dextranicum NY203 deposited under the accession number KCTC 13679BP, for preventing or treating degenerative brain disease. Pharmaceutical composition. The method of claim 1, wherein the fraction of the culture medium of the Aspergillus genus microorganism is a first enzyme solution comprising at least one selected from the group consisting of pectinase, glucanase, and arabinase; and a second enzyme solution that is at least one type selected from the group consisting of cellulase, hemicellulase, and protease, preventing degenerative brain disease, or Pharmaceutical composition for therapeutic use. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 3, wherein the concentration of the first enzyme solution is 0.01 to 20.00% (v/v) based on the total enzyme reaction product. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 3, wherein the concentration of the second enzyme solution is 0.01 to 20.00% (v/v) based on the total enzyme reaction product. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 1, wherein the vegetable fiber is derived from one or more species selected from the group consisting of fruits, leaves, stems, and roots of plants. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 1, wherein the concentration of the vegetable fiber is 1 to 20% (w/v) based on the total enzyme reaction product. The method of claim 1, wherein the degenerative brain disease is at least one selected from the group consisting of dementia, Alzheimer's disease, stroke, stroke, Parkinson's disease, Huntington's disease, Pick's disease, and Creutzfeldt-Jakob disease. Pharmaceutical composition. Leuconostoc sp . Microorganisms or cultures thereof; and Aspergillus ( Aspergillus sp .) Microorganisms, cultures thereof, or fractions of cultures thereof. A health function for improving degenerative brain disease containing an enzyme reaction product prepared by reacting one or more species selected from the group consisting of vegetable fibers in the presence of vegetable fiber. Food composition. The method of claim 9, wherein the microorganism of the Leuconostoc genus is a strain of Leuconostoc mesenteroides subsp. dextranicum NY203 deposited under the accession number KCTC 13679BP. Health function for improving degenerative brain disease. Food composition. The method of claim 9, wherein the fraction of the culture medium of the Aspergillus genus microorganism is a first enzyme solution containing at least one selected from the group consisting of pectinase, glucanase, and arabinase; and a second enzyme solution that is at least one type selected from the group consisting of Cellulase, Hemicellulase, and Protease, for improving degenerative brain disease. Health functional food composition. The health functional food composition for improving degenerative brain disease according to claim 11, wherein the concentration of the first enzyme solution is 0.01 to 20.00% (v/v) based on the total enzyme reaction product. The health functional food composition for improving degenerative brain disease according to claim 11, wherein the concentration of the second enzyme solution is 0.01 to 20.00% (v/v) based on the total enzyme reaction product. The health functional food composition for improving degenerative brain disease according to claim 9, wherein the vegetable fiber is derived from one or more species selected from the group consisting of fruits, leaves, stems, and roots of plants. The health functional food composition for improving degenerative brain disease according to claim 9, wherein the concentration of the vegetable fiber is 1 to 20% (w/v) relative to the total enzyme reaction product. The health function for improving degenerative brain disease according to claim 9, wherein the degenerative brain disease is at least one selected from the group consisting of dementia, Alzheimer's disease, stroke, stroke, Parkinson's disease, Huntington's disease, Pick's disease, and Creutzfeldt-Jakob disease. Food composition.