KR102222846B1 - Fermented black rice powder having antioxidant ability or nerve cell protective ability and the method for manufacturing the same - Google Patents

Abstract

The present invention relates to a fermented black rice powder capable of expressing an excellent antioxidant effect and neuronal protective effect by fermenting black rice with specific lactic acid bacteria, and a method for producing the same.

Description

Fermented black rice powder having antioxidant or nerve cell protection and its manufacturing method {FERMENTED BLACK RICE POWDER HAVING ANTIOXIDANT ABILITY OR NERVE CELL PROTECTIVE ABILITY AND THE METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a fermented black rice powder having antioxidant activity or neuronal cell protection, and a method for producing the same.

The oxidative stress of human tissue cells not only plays an important role in the pathogenesis of cardiovascular disease, but also the oxidative stress induced by free radicals breaks the harmony between the expansion and contraction of blood vessels and reduces the lipid peroxidation of the biological membrane. It is known to cause various diseases such as arteriosclerosis, diabetes and hypertension by causing oxidative damage to tissue cells.

On the other hand, rice, a staple food of Koreans, is known to contain various physiologically active ingredients such as dietary fiber, as well as antioxidants such as tocopherol, tocotrienol, gamma-oryzanol, and phytic acid. Black rice, a kind of black rice, is known to have various physiological activities, such as anti-inflammatory, antioxidant, antibacterial, and anti-cancer, as it is rich in anthocyanin, a natural pigment component. As peonidin, malvidin, pelargonidin flavylium and their glycosides, it has been reported that they have an antioxidant activity similar to that of α-tocopherol (Cho MH et, al. 1996).

On the other hand, the present inventors confirmed through an experiment that the fermented powder obtained by fermenting black rice with a specific lactic acid bacteria can express excellent antioxidant effects and neuronal protective effects, and completed the present invention.

An object of the present invention is to provide a fermented black rice powder capable of expressing excellent antioxidant effect and neuronal protective effect by fermenting black rice with specific lactic acid bacteria and a method for producing the same.

In order to achieve the above object, in the present specification, a step of mixing black rice powder, protein powder, purified water, and freeze-dried Leuconostoc mesenteroides lactic acid bacteria powder, and fermenting for 20 to 40 days It provides a method for producing fermented black rice powder containing.

In addition, in the present specification, as a fermented black rice powder prepared according to the above method, the powder provides a fermented black rice powder having a lactic acid bacteria content of 1,000,000 cfu/g or more.

Hereinafter, a fermented black rice powder and a manufacturing method thereof according to a specific embodiment of the present invention will be described in more detail.

As described above, according to an embodiment of the present invention, mixing black rice powder, protein powder, purified water, and freeze-dried Leuconostoc mesenteroides lactic acid bacteria powder, and fermenting for 20 to 40 days Including, a method for producing fermented black rice powder may be provided.

The inventors of the present invention confirmed through an experiment that black rice can be fermented with a specific lactic acid bacteria, but when fermented for a specific temperature and a specific time, an excellent antioxidant effect and a neuroprotective effect can be expressed through experiments, and the present invention was completed.

According to an embodiment of the present invention, black rice is a kind of colored rice and is rich in anthocyanin, a natural pigment component, and has various physiological activities such as anti-inflammatory, antioxidant, antibacterial and anti-cancer. Specifically, the black rice used in the present invention may be used in the form of a powder.

On the other hand, the protein powder according to an embodiment of the present invention is to help the growth and growth of lactic acid bacteria, specifically, one or more raw materials selected from vegetable proteins such as rice protein, soy protein, pea protein, oat protein, pumpkin protein, etc. Using, it may be a powder prepared by various drying methods.

On the other hand, Leuconostoc mesenteroides lactic acid bacteria used in the above step are plant lactic acid bacteria discovered in "Kimchi," a representative traditional fermented food in Korea, and have effects such as intestinal function, inhibition of growth of harmful bacteria and smooth bowel movement. It is known to have.

Specifically, according to an embodiment of the present invention, based on the total weight of the powder when blended in the above step, black rice powder is 20 to 30wt%, protein powder is 20 to 30wt%, purified water is 45 to 55wt%, and freeze-dried Cono Stock mesenteroides (Leuconostoc mesenteroides ) lactic acid bacteria powder may be blended in an amount of 0.005 to 0.015 wt%.

Meanwhile, in the above step, the fermentation period may be within the range of 20 to 40 days, and more specifically, fermentation may be performed for 20 to 22 days, and in the case of fermentation during the period, intestinal action, harmful bacteria through the growth of lactic acid bacteria and enhancement of enzyme activity It can effectively express beneficial effects such as inhibition of proliferation, smooth bowel movement and promotion of digestion. In addition, in the above step, the fermentation temperature may be 35 to 37 °C, and when fermented within the above temperature range, beneficial effects such as intestinal action, inhibition of growth of harmful bacteria, smooth bowel movement and digestion promotion effect through increase of lactic acid bacteria growth and enzyme activity Can be effectively expressed.

In the manufacturing method of the fermented black rice powder according to an embodiment of the present invention, after the fermentation step, the fermented powder may be freeze-dried and then pulverized.

Specifically, the freeze drying and pulverization method may be performed by a method generally used in the art, and is not particularly limited. The diameter of the fermented black rice powder pulverized through the above step may be 250 μm or less, for example.

The fermented black rice powder prepared by the method according to an embodiment of the present invention as described above may have a lactic acid bacteria content of 1,000,000 cfu/g or more. In addition, the fermented black rice powder prepared by the method according to an embodiment of the present invention as described above has antioxidant activity or neuronal cell protection.

The fermented black rice powder prepared according to the present invention has antioxidant properties and has an effect of preventing oxidative damage to cells. In particular, the fermented black rice powder according to the present invention has the ability to protect nerve cells.

1 is a graph showing the result of measuring the pH according to the fermentation period of a 10% solution of fermented black rice powder prepared according to an embodiment of the present invention.
Figure 2 is a graph showing the ABTS radical scavenging activity by concentration of the black rice fermented powder water extract prepared according to an embodiment of the present invention.
3 is a graph showing ferric reducing antioixdant power by concentration of water extract of fermented black rice powder prepared according to an embodiment of the present invention.
Figure 4 is a graph showing the lipid peroxidation inhibitory activity by concentration of the black rice fermented powder water extract prepared according to an embodiment of the present invention.
FIG. 5 is a graph showing the survival rate of neurons in oxidative stress conditions according to concentrations of water extract of fermented black rice powder prepared according to an embodiment of the present invention.
6 is a graph showing the effect of inhibiting cell membrane damage by concentration of water extract of fermented black rice powder prepared according to an embodiment of the present invention.
7 is a graph showing intracellular ROS production inhibitory activity according to concentrations of water extract of fermented black rice powder prepared according to an embodiment of the present invention.

The present invention will be described in detail below and exemplify specific embodiments, as various modifications can be made and various forms can be obtained. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

Example 1. Method for producing fermented black rice powder

First, 250 g of black rice powder, 250 g of rice protein powder, 499.85 g of purified water, and 0.15 g of freeze-dried Leuconostoc mesenteroides lactic acid bacteria powder were mixed, and then fermented at a temperature of 37° C. for 20 days in a constant temperature and humidity environment. Next, the obtained fermented powder was freeze-dried using a cochlear dryer under vacuum conditions in which aerobic pathogenic microorganisms are difficult to grow, and then pulverized using a grinder, so that the diameter of the fermented powder was 250 μm or less.

Example 2. Preparation of water extract of fermented black rice powder

900 mL of distilled water was added to 100 g of the fermented black rice powder prepared according to Example 1, followed by immersion for 24 hours for extraction. The extracted solution was concentrated using a vacuum rotary concentrator and then freeze-dried and used in the following experiment.

Experiment 1: pH measurement

The black rice fermented powder water extract (10% solution) of Example 2 was measured using a pH meter (Orion star A211, Thermo scientific Co., USA) (see FIG. 1).

Referring to the results of FIG. 1, the pH was 5.81 on day 0 of the fermentation period, but the pH gradually decreased as the fermentation period elapsed, and on the 20th day of fermentation, the pH was 4.48, which is during fermentation. It is judged as a result of the proliferation of lactic acid bacteria.

Experiment 2: ABTS radical scavenging activity

Mix 5 mL of 7 mM ABTS and _{88 μL of 140 mM K 2} S ₂ O ₈ and leave for 14 to 16 hours in a dark place, mix this with absolute ethanol at a ratio of about 1: 88 (v/v), and absorb the absorbance of the control at 734 nm. The ABTS solution adjusted to a value of 0.7±0.02 was used. Mixing 20 μL of the black rice fermented powder water extract by concentration prepared using the black rice fermented powder of Example 1 and 980 μL of ABTS solution, shaking for 30 seconds, reacting for 2.5 minutes, and measuring the absorbance at 734 nm for radical scavenging activity Is shown (see Fig. 2).

Referring to the results of FIG. 2, as a result of measuring ABTS radical scavenging activity using the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1, the ABTS radicals gradually increased as the concentration of the extract increased. The scavenging activity also showed a tendency to increase in a concentration-dependent manner, and at a concentration of 1,000 μg/mL, it showed a similar tendency to vitamin C used as a positive control.

Experiment 3: Ferric reducing antioxidant power

Reagents used in the FRAP assay were 0.3 M sodium acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) solution dissolved in 40 mM HCl, and 20 mM FeCl ₃ solution. . Pre-prepared sodium acetate buffer, TPTZ solution, and FeCl ₃ solution were mixed in a ratio of 10:1:1 (v/v/v), respectively, and incubated at 37°C for 10-15 minutes to prepare a FRAP reagent. 1.5 mL of the FRAP reagent was mixed with 50 μL of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1, vortexed, and allowed to stand at room temperature for 30 minutes, and the absorbance was measured at 593 nm (see FIG. 3 ). ).

FRAP assay is a method of measuring the antioxidant power by measuring the absorbance at 593 nm of a colored ferrous tripyridyl triazine complex obtained by reducing ferric ions to ferrous ions by antioxidants present in the sample.Refer to the results of FIG. In the same manner as in the ABTS radical scavenging activity, the absorbance value tended to increase as the concentration of the water extract of the fermented black rice powder increased.

Experiment 4: lipid peroxidation inhibitory activity

The brain tissue was homogenized in 10 volumes of ice cold Tris-HCl buffer (20 mM, pH 7.4) and centrifuged at 12,000 × g for 15 minutes at 4°C. To 0.1 mL of the supernatant, 0.1 mL of 10 μM FeSO _4, 0.1 mL of 0.1 mM ascorbic acid, and 0.2 mL of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 were added and incubated at 37°C for 1 hour. I did. 0.1 mL of 28% trichloroacetic acid was added to the reaction solution to terminate the reaction, 0.3 mL of 1% thiobarbituric acid was added, heated at 80° C. for 30 minutes, and absorbance was measured at 532 nm (see FIG. 4).

In the oxidative damage of brain tissues caused by harmful free radicals, the inhibitory effect on lipid peroxidation by the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 was analyzed. In contrast, since the content of unsaturated fatty acids is particularly high, it is advantageous to investigate changes in oxidative damage, and this experiment was conducted because the oxidation of lipid components has a deep relationship with cell membrane damage and other protein damage. Referring to FIG. 4, as a result of measuring lipid peroxidation inhibitory activity using mouse brain homogenate, addition of water extract of fermented black rice powder prepared using the fermented black rice powder of Example 1 is similar to the above two antioxidant activity results. As the concentration increased, lipid peroxidation in brain tissues was very inhibited, and in particular, the sample to which 1,000 μg/mL was added showed a very high lipid peroxidation inhibitory activity of 76.25%.

Experiment 5: Measurement of neuronal cell viability and cell membrane damage inhibition effect

Cell and culture method

PC12 cells (KCLB 21721, Korean Cell Line Bank, Seoul, Korea) used in this experiment were cells that exhibit neuronal characteristics and were derived from murine pheochromocytoma. Of the PC12 cells, 25 mM HEPES, 25 mM sodium bicarbonate , 10% fetal bovine serum (GIBCO), 50 units / mL penicillin and 100 μg / mL and streptomycin inoculated in RPMI 1640 medium is contained 37 ℃, 5% CO ₂ conditions It was cultured in an incubator.

Cell viability measurement

The protective effect of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 on PC12 cells induced by H ₂ O _{2 was measured by MTT reduction assay.} The black rice fermented powder water extract according to concentration prepared using the black rice fermented powder of Example 1 was treated in PC12 cells and pre-incubated for 48 hours, followed by treatment with 200 μM H ₂ O ₂ for 3 hours, respectively. PC12 cells in this state were treated with MTT stock solution and incubated at 37°C for 3 hours, and then 150 μL of MTT solubilization solution was added to terminate the reaction. Finally, absorbance was measured at 570 nm and 690 nm in a microplate reader (680, Bio-rad, Tokyo, Japan). For the positive control, vitamin C (200 μM) was used, and the cell viability was expressed as a% concentration for the control group (see FIG. 5).

As a result of measuring the protective effect on PC12 neurons in a state of oxidative stress induced by _{H 2} O ₂ of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1, see FIG. 5 In the case of lower cell viability _{, treatment with H 2} O ₂ showed a survival rate of 58% compared to 100% in the control group, and in the treatment group treated with H ₂ O ₂ and vitamin C at the same time, the survival rate of 79% was about 21%. It showed a cell protective effect. The sample treated with the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 showed a protective effect similar to 200 μM of vitamin C used as a positive control at a concentration of 200 μg/mL, and the added concentration of the extract As was increased, the neuronal protective effect showed a tendency to increase significantly.

Cell membrane damage inhibitory effect

The black rice fermented powder water extract prepared using the black rice fermented powder of Example 1 was pre-incubated for 48 hours _{, treated with 200 μM H 2} O ₂ and cultured for 3 hours, and then centrifuged for 5 minutes (250 × g) After transferring 100 μL of the supernatant to a new well, the cell membrane damage effect was measured with an LDH assay kit (Sigma Chemical Co., St. Louis, MO, USA) (see FIG. 6).

Neurons contain relatively many lipid components and are very susceptible to oxidative stress, so the following study was conducted to investigate the relationship between the above neuronal protective effect and neuronal cell membrane damage using these structural properties. . 6, the cytoplasm contained in nerve cells to confirm the protective effect of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 against _{H 2} O _{2 induced nerve cell membrane damage.} As a result of measuring the LDH emission of the component, the emission of the control group was about 21%, whereas the H ₂ O ₂ treated sample showed a 64% emission, and the LDH emission increased by 43% due to oxidative stress such as _{H 2} O ₂ I did. The positive control vitamin C 200 μM treatment group showed a 32% LDH release, and the black rice fermented powder water extract prepared using the black rice fermented powder of Example 1 was treated at a concentration of 12 to 200 μg/mL. The LDH release tended to decrease in a concentration-dependent manner, ranging from 38 to 23%.

DCF-DA assay

DCF-DA assay was performed to measure the protective effect of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 against oxidative damage to PC12 cells. First, the cells were aliquoted into a 96 well plate at 1×10 ⁴ cells/well and cultured for 24 hours at 37°C and 5% CO _2. The black rice fermented powder extract was treated with PC12 cells and pre-incubated for 48 hours, followed by treatment with 200 μM H ₂ O ₂ for 3 hours each. After incubation, the medium was removed, and then 10 μM DCF-DA diluted with phosphate buffered saline (PBS) buffer was added, followed by incubation for 45 minutes. After washing twice with PBS buffer, 200 μM H ₂ O ₂ was added, and after 3 hours, fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a fluorescence microplate reader (see FIG. 7 ).

As a result of measuring the ROS production inhibitory activity in PC12 cells induced by _{H 2} O ₂ of the black rice fermented powder water extract prepared using the black rice fermented powder of Example 1 _{, DCF formation is H 2} O _The treatment group treated with 2 showed 142% of DCF formation compared to 100% of the control group, and 104% in the treatment group treated with H ₂ O ₂ and vitamin C at the same time showed an oxidative stress reduction effect of about 38%. The black rice fermented powder water extract prepared by using the black rice fermented powder of Example 1 showed a concentration-dependent tendency to reduce oxidative stress compared _{to the H 2} O _{2 treatment group.} At a concentration of 200 μg/mL, it was 105%, showing a similar oxidative stress reduction effect to vitamin C 200 μM (104%) used as a positive control. This is believed to be due to anthocyanin-based compounds such as cyanidin-3-O-glucoside, a powerful antioxidant contained in black rice.

Analysis of total phenolic compounds and cyanidin-3-O-glucoside content

For the analysis of the total phenolic compound content contained in the fermented black rice powder, 9 mL of tertiary distilled water was added to 1 mL of the water extract of the fermented black rice powder prepared using the fermented black rice powder of Example 1, and then Folin Ciocalteau phenol reagent. 1 mL was added, mixed, and reacted at room temperature for 5 minutes. _{10 mL of a 7% Na 2} CO ₃ solution was added to the reaction solution, mixed again, and then distilled to 25 mL with tertiary distilled water. The mixed solution was allowed to stand at 23°C for 2 hours, and the absorbance was measured at 760 nm, and the measured absorbance was calculated using a calibration curve prepared using gallic acid to calculate the total phenolic compound content. For the analysis of cyanidin-3-O-glucoside content in the fermented black rice powder, 2 mL of the water extract of the black rice fermented powder prepared using the black rice fermented powder of Example 1 was filtered through a 0.45 μm membrane filter, followed by HPLC (1100, Agillent Co. , Santa clara, CA, USA). Analysis condition column was used as shiseido C18 (4.6 mm × 250 mm, 5 μm), and the mobile phase was linear gradient analysis for 30 minutes with 0.01 M potassium phosphate monobasic (pH 3.0) and 80% methanol. The flow rate was 1.5 mL/min, the injection amount was 20 μL, the detector was a diode array detector, and the wavelength was analyzed at 530 nm.

As a result of specific experiments, the total phenolic compound content and cyanidin-3-O-glucoside content contained in 1 g of the water extract of the fermented black rice powder prepared using the fermented black rice powder of Example 1 were 44.27 mg/g GAE and respectively. It was 30.61 mg/g.

As described above, the fermented black rice powder prepared according to the present invention has antioxidant properties and has an effect of preventing oxidative damage to cells. In particular, it was confirmed that the fermented black rice powder according to the present invention has the ability to protect nerve cells.

In the above, although specific embodiments of the present invention have been described and illustrated, the present invention is not limited to the described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. It is self-evident to those who have. Accordingly, such modifications or variations should not be individually understood from the technical spirit or viewpoint of the present invention, and the modified embodiments should be said to belong to the claims of the present invention.

Claims (7)

Blending black rice powder, protein powder, purified water, and freeze-dried Leuconostoc mesenteroides lactic acid bacteria powder, and fermenting for 20 to 40 days,
When blended, based on the total weight of the powder, black rice powder is 20 to 30 wt%, protein powder is 20 to 30 wt%, purified water is 45 to 55 wt%, and freeze-dried Leuconostoc mesenteroides lactic acid bacteria powder is 0.005 to Blended at 0.015 wt%,
The fermentation temperature during the fermentation is 35 to 37 ℃,
After the fermentation step, freeze-drying the fermented powder, and then pulverizing the fermented powder as a method for producing a fermented black rice powder, further comprising,
The prepared black rice fermented powder has a lactic acid bacteria content of 1,000,000 cfu/g or more,
The fermented powder has antioxidant activity or nerve cell protection through lipid peroxidation inhibitory activity, and has a therapeutic or preventive use for at least one disease selected from arteriosclerosis, diabetes and hypertension.
delete delete delete The method of claim 1,
After the pulverizing step, the diameter of the obtained fermented powder is 250 μm or less, a method for producing black rice fermented powder.
delete delete

Images