JP2003292448A - Antioxidant using kefir, remedy for diabetes using the same, tissue-repairing agent and healthy food having dna reparative action, method of producing antioxidant using kefir and identification - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of producing an antioxidant substance from fermented milk kefir, a method for identification, and provide a remedy for diabetes including kefir, a tissue-repairing agent that has DNA-repairing action, and healthy food. <P>SOLUTION: The method of producing an antioxidant from kefir comprises the first step where kefir is centrifuged to precipitate the milk fat and obtain the supernatant, the second step where the supernatant is dialyzed with a dialysis membrane of a fraction molecular weight of 1,000 to collect the external fluid with a molecular weight of ≤1000, the third step where the external fluid is concentrated, the concentrate is neutralized with alkali to remove the precipitate and obtain separated fluid, the fourth step where the separated fluid is dialyzed with a dialysis membrane of a fraction molecule of 11 to obtain the inner fluid with a molecule of ≥100, and the fifth step where the antioxidant obtained from kefir is identified by the on-line HPLC process and the fraction is collected. In addition, a remedy for diabetes and a tissue-repairing agent having DNA-repairing action are provided. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antioxidant effect of kefir, which is a health food, and an antidiabetic agent utilizing the effect, a tissue repair agent having a DNA repair effect, and a health food.

[0002]

2. Description of the Related Art Kefir is a fermented milk that people in the Caucasus region of the Republic of Georgia love, and milk of cows, goats, and sheep is stored in goat leather bags and is accidentally mixed with lactic acid bacteria and yeast. It is produced by lactic acid and alcohol fermentation. Caucasus-type fermented milk kefir is a stable kefir grain obtained by researching the manufacturing method over many years by the Central Research Institute for Microorganisms of the Russian Academy of Sciences of the Independent State Community, and using it as a bacterial species. It is used in medical institutions as a diet for the treatment of kidney diseases and cardiovascular diseases by enhancing digestive function. Kefir grains are sponge-like masses of about 1 to 3 cm and contain about 20 kinds of lactic acid bacteria, yeasts and acetic acid bacteria, and are generally called yogurt mushrooms in Japan. Kefir grains (called kefir yogurt) can be obtained by adding milk to kefir grains and sealing at room temperature for a specified time. You can easily obtain kefir by filtering the kefir grains and removing the kefir grains. It is also used as a health food in homes.

[0003]

In recent years, active oxygen generated from the metabolic system of human beings has been widely noticed as a aging factor of human beings when they perform life activities. Oxidative stress due to this active oxygen increases in the body due to inappropriate dietary habits, stress, ultraviolet rays, etc., and this active oxygen is not only a problem of aging, but also a cause of deterioration or aggravation of cancer and other various diseases. Therefore, reducing this active oxygen has become a major research subject for maintaining human health. In addition, in Japan, insulin-independent type II diabetes has been increasing in recent years from Western diets, and many Japanese suffer. In this type II diabetes, inability to maintain cellular homeostasis due to inability to take up glucose, which is a physiological function at the core of health maintenance, results in various complications and serious symptoms. Is not uncommon. It is considered that active oxygen is largely involved in the decrease in glucose uptake by cells, which is a factor of this type II diabetes, and the decrease in active oxygen activates glucose uptake by cells, and is considered to be useful for the treatment of type II diabetes. Furthermore, there is a demand for the development of foods and drugs that have an action of reducing active oxygen. It is speculated that fermented milk kefir, which has long been a long-lived drink in the Caucasian region of Russia and has recently received attention as a health food in Japan, may also have this active oxygen-reducing effect. Isolation and identification of a substance having an oxygen reducing action (antioxidant action) to produce a substance having an antioxidant action will greatly contribute to the maintenance of human health and the treatment of diabetes, particularly the treatment of type II diabetes. Conceivable.

The present invention has been made in view of such circumstances, and a first object of the present invention is to produce a substance (antioxidant substance) having an active oxygen reducing action (antioxidant action) from fermented milk kefir. Secondly, the second purpose is to provide a method for isolating and identifying an antioxidant, and further, a therapeutic agent for diabetes that mainly uses the antioxidant, a tissue repair agent having a DNA repair action, and health. Third to provide food
The purpose of.

[0005]

The antioxidant using kefir according to the first aspect of the invention comprises an antioxidant in the kefir water-soluble fraction. The antioxidant using kefir according to the second invention is the antioxidant according to the first invention,
The antioxidant has a molecular weight of 100-700. Third
The antioxidant using kefir according to the invention of 1) is the antioxidant according to 2nd invention, wherein the antioxidant has a hydroxyl group. The antioxidant according to the fourth invention is the second and third antioxidants.
In the antioxidant according to the invention, the antioxidant substance has an aromatic group. An antioxidant according to a fifth aspect of the present invention is the antioxidant according to any one of the second to fourth aspects, wherein the antioxidant substance is acidic or neutral and stable, and is stable to autoclaving. The therapeutic agent for diabetes according to the sixth invention has the antioxidant according to the first to fifth inventions. A tissue repairing agent having a DNA repairing action according to the seventh invention comprises:
It has an antioxidant according to the fifth invention. The health food according to the eighth invention comprises the antioxidant according to the first to fifth inventions.

A method for producing an antioxidant according to the ninth invention is
The first step of centrifuging kefir to precipitate milk fat to obtain a supernatant, and the second step of dialyzing the supernatant with a dialysis membrane having a molecular weight cut off of 1000 to obtain an external solution having a molecular weight of 1000 or less; The third step of obtaining a separated liquid by removing the precipitate generated by neutralizing the concentrated liquid with alkali with a concentrated liquid, and dialyzing the separated liquid with a dialysis membrane having a molecular weight cutoff of 100 to obtain a liquid having a molecular weight of 100 or more. It has a fourth step of obtaining a liquid and a fifth step of identifying and fractionating an antioxidant substance from the inner liquid by using an online HPLC method. The method for producing an antioxidant according to the tenth invention is
In the method for producing an antioxidant according to the ninth invention, the online HPLC method is a DPPH-HPLC method, and the antioxidant substance of kefir collected in the fourth step is purified by gel filtration.

Here, the on-line HPLC method is a method capable of simultaneously performing separation and identification of antioxidants in a mixture, and activity evaluation. In particular, the on-line HPLC method using DPPH is applied to the HPLC analysis system using DPPH.
(Diphenyl 1,2-picrylhydrazyl) in combination, the components in the mixture are separated by ordinary HPLC (High Performance Liquid Chromatography) analysis, and then the DPPH solution is mixed in the line to cause reaction, Through a photodiode array detector capable of detecting a large number of wavelengths at the same time, and by simultaneously measuring the spectra of each component and the decrease in absorbance due to the reaction of DPPH with an antioxidant, the peaks with antioxidant ability can be identified. It is something to do.
The structure is shown in FIG. As shown in FIG.
By supplying a DPPH solution using methanol as a solvent to the eluent after C, the antioxidant and DPPH react with each other.
It is intended to identify the component having antioxidant activity in the mixture by monitoring the decrease in absorbance at 17 nm,
Analysis is possible even with low pH buffers (buffers).
The baseline absorbance of PPH is more stable than using luminol instead of DPPH. In addition, it is possible to separate and separate antioxidant substances without losing their antioxidant activity. DPPH has a maximum absorption at 517 nm in a methanol solvent and is visually recognized as purple. Figure 2
Fig. 2 shows a conceptual diagram of the detection of antioxidants by DPPH. As shown in Fig. 2, when DPPH causes a reaction that accepts hydrogen from antioxidants and is reduced, it becomes reduced DPPH, and the maximum absorption at 517 nm occurs. Remarkably reduced, the purple color is decolorized. The antioxidant is detected by measuring the peak of this purple decolorization by HPLC analysis.

The antidiabetic agent using kefir according to the eleventh invention is mainly composed of the antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention.
The tissue repairing agent having a DNA repairing action using kefir according to the twelfth invention is mainly composed of the antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention. A health food using kefir according to the thirteenth invention,
It is mainly composed of an antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention. The method for identifying an antioxidant using kefir according to the 14th aspect of the invention comprises the first step of centrifuging kefir to precipitate milk fat to obtain a supernatant,
The second step of dialyzing the supernatant with a dialysis membrane having a molecular weight cut off of 1000 to obtain an external solution having a molecular weight of 1000 or less, and a concentrated solution obtained by concentrating the external solution is neutralized with an alkali to remove precipitates formed. And the third step of obtaining a separated liquid by
Dialysis with a dialysis membrane to obtain an internal solution with a molecular weight of 100 or more
The method includes a step, a fifth step of identifying and fractionating an antioxidant substance from the inner solution using an online HPLC method, and a sixth step of identifying the fractionated antioxidant substance.

[0009]

EXAMPLES Hereinafter, the method for identifying and manufacturing the antioxidant of kefir according to the present invention will be described below with reference to specific examples. FIG. 3 is a process diagram for preparing a fractionated sample having a molecular weight of 1000 or less, FIG. 4 is an explanatory diagram of the antioxidant activity of ascorbic acid, and FIG.
0 or less), and FIG. 6 shows the ultraviolet of Kefir antioxidant.
Visible spectrum diagram, FIG. 7 is a UV-visible spectrum diagram of kefir antioxidant and representative flavonoids, FIG. 8 is an absorbance diagram when the active substance is purified by gel filtration, and FIG. 9 is a mass spectrum of kefir antioxidant (FAB -MS) diagram,
FIG. 10 is an infrared absorption spectrum diagram of kefir antioxidant, FIG. 11 is an explanatory diagram of the result of the antioxidant activity measurement in a test tube, FIG. 12 is an explanatory diagram of the action mechanism of DCFH-DA,
FIG. 13 is a process diagram of an intracellular reactive oxygen scavenging test using a human leukemia-derived cell line K562, FIG. 14 is a measurement diagram of intracellular active oxygen scavenging activity by FCM, and FIG. 15 is a kefir antioxidant for adipocytes. FIG. 16 is an explanatory view of the effect of promoting sugar uptake, FIG. 16 is an explanatory view of the effect of enhancing expression of hMLHI gene, which is a DNA repair enzyme of kefir antioxidant, and FIG. 17 is an effect of enhancing irregular DNA synthesis (UDS) of kefir antioxidant, and its effect. It is explanatory drawing of thermal stability.

Example 1 In this example, first, the water-soluble fraction of kefir was fractionated into several molecular weight ranges by a dialysis membrane to identify the fraction having major antioxidant activity. Furthermore, the constituent components in the fraction were separated by an anion exchange column, and online HPLC using DPPH was performed.
The method was adopted to identify the components having antioxidant activity. Further, this was fractionated and further purified. The molecular weight and infrared absorption spectrum of the obtained purified product were measured by fast atom bombardment mass spectrometry. The antioxidant activity of the purified product in vitro was measured using the DPPH reducing ability as an index.
First, the materials and devices used in this example will be briefly described. (1) Materials Kefir as a starting material is a Kefir fermented liquid (milk solid content 17%, internal milk fat content 2%, lactic acid 50% solution, pectin 0.6%, pH adjustment) provided by Nippon Kefir Co., Ltd. 90) refrigerated (4 ° C) is used. (2) In order to investigate the molecular weight of the dialysis membrane and the active ingredient for osmotic pressure measurement, the spectrum (SP
ECTRUM) dialysis membrane with a molecular weight cut off of 2,000 (Spectra / Por
Membrane MWCO: 2,000), molecular weight cutoff of 1,000 (Spectra /
Por Membrane MWCO: 1,000), molecular weight cutoff 500 (Spectr
a / Por Membrane MWCO: 500), molecular weight cutoff 100 (Spectra /
Por Membrane MWCO: 100) was used. For measuring the osmotic pressure of water-soluble fractions in each molecular weight range, VOGEL
An osmometer OM802-D manufactured by the same company was used. (3) In vitro antioxidative ability test using DPPH DPPH was obtained from Wako Pure Chemical Industries, Ltd. DPP
H was prepared by using 80% methanol as a solvent on the day of the experiment, protected from light and refrigerated. In addition, in the antioxidant capacity test, the spectrum reader of TECAN was used to measure the absorbance of multiple samples under the same conditions, and the absorbance of DPPH at the absorbance at 492 nm on a 96-well plate (perforated plate). The decrease was measured simultaneously.

(4) Online HPLC-DPPH System As shown in FIG. 1, the online HPLC-DPPH system 10 includes a system controller 12 (Waters 600E).
System Controller), Auto Sampler 13 (Waters 7
17 plus Autosampler) and a photodiode array detector 14 (Waters 996 Photodiode Array Detector) were used to perform system control and data analysis by a Chromatography Manager MILLENIUM 32 (Chromatography Manager 18) manufactured by Waters.
In addition, DPPH pump 1 as a pump for supplying DPPH solution
5 (Waters 501 HPLC pump) was used. As a column for separation, TSKgel DEAE-5PW (φ7.5 m from TOSOH)
m × 75 mm) was used as the anion exchange column 16. In addition, the fraction collector 17 (SF-212 of ADVANTEC) can be used to automate the collection of target substances.
0 SUPER FRACTION COLLECTOR) was used.

(5) Gel filtration Biogel / BIO-Gel P-2 resin (separated molecular weight range 100-1) manufactured by Bio-Rad (BIO-RAD) for gel filtration purification.
800) was used to produce a column (φ10 mm × 45
0 mm). The column was used by connecting to an online HPLC-DPPH system by an FPLC-HPLC conversion adapter. Furthermore, the molecular weight of the purified product was estimated by the gel filtration elution pattern. Vitamin B12 (MW 1
355.4), bromophenol blue (MW 669.97) and phenol red (MW 354.38) were obtained from Wako Pure Chemical Industries, Ltd. and used for the experiment.

(6) Reversed-phase HPLC analysis A split tube (Waters 2695 Se) for analysis of the purified product after gel filtration.
paration module) was controlled by a chromatography manager 18 manufactured by Waters Co., Ltd. to perform data analysis. As a separation column, TSKgel ODS-80Ts (φ 4.6 mm × 150 mm), which is a reverse phase column manufactured by Tosoh Corporation, was used.

(7) Fast atom bombardment mass spectrometry (FAB-MS)
Molecular weight measurement by Kyushu University Graduate School of Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Department of Medicinal Resource Control, 1 mg of purified product after gel filtration was used for Fast Atom Bombarment Mass Spectrometry,
FAB-MS) was requested for molecular weight measurement. (8) Infrared absorption spectrum measurement The Shimadzu General Analysis Center Co., Ltd. was requested to measure infrared absorption spectrum. After gel filtration, 0.5 mg of the purified product was provided, and a microscopic infrared absorption spectrum was measured with a Shimadzu Fourier transform infrared spectrophotometer FTIR-8200PC and an infrared microscope AIM-8000.

Experimental Method and Results (1) Preparation of Kefir Water-Soluble Fraction and Molecular Weight Fractionation FIG. 3 shows the sample weight, volume and osmotic pressure at each stage when preparing a kefir water-soluble fraction sample having a molecular weight of 1000 or less. Show. As shown in FIG. 3, the kefir fermentation liquid 27
Centrifuge 5g (17% milk solids content, 2% internal milk fat content, 50% lactic acid solution, 0.6% pectin, pH adjustment 3.90) to precipitate milk fat content on kefir. Kiyoshi (Kefir water-soluble fraction sample A) 115
g was obtained (first step). The resulting supernatant was cut to a molecular weight of 10
It was transferred to a dialysis membrane of No. 00 and dialyzed with 1000 ml of ultrapure water for 24 hours to obtain an external solution having a molecular weight of 1000 or less (second step). The obtained external solution was concentrated by an evaporator. The concentrated solution was neutralized with a 4N sodium hydroxide aqueous solution, and the resulting protein precipitate was centrifuged (550
g, 20 min) to remove (third step). The supernatant (separation liquid) obtained here has a molecular weight cut-off of 10 for the purpose of desalting.
It was transferred to a dialysis membrane of No. 0 and dialyzed with 1000 ml of ultrapure water for 24 hours to obtain an internal solution having a molecular weight of 100 or more (fourth step). The obtained internal solution is concentrated by an evaporator,
A sample (B) having a molecular weight of 1000 or less was prepared by adjusting the osmotic pressure to 0.300 osmol / kg.

[0016] Here, the dialysis was repeated twice while keeping the amount of the internal liquid remaining in the dialysis membrane having a molecular weight cut off of 1000 to remove substances having a molecular weight of 1000 or less. Next, the thus prepared internal solution containing a substance having a molecular weight of about 1000 or more was transferred to a dialysis membrane having a molecular weight cut off of 2000, and 1000 ml was added.
Dialysis was carried out for 24 hours with the above ultrapure water. The obtained external solution was concentrated by an evaporator and the osmotic pressure was adjusted to 0.300 os.
Molecular weight adjusted to mol / kg from 1000 to 20
00 sample (C). The obtained internal solution was concentrated by an evaporator and the osmotic pressure was adjusted to 0.300 osm.
The sample adjusted to ol / kg was used as a sample (D) having a molecular weight of 2000 or more.

(2) Qualitative Antioxidant Activity Test of Molecular Weight Fraction Sample DPPH was dissolved in 80% methanol solvent on the day of the experiment and stocked at a concentration of 0.1 mM. The prepared kefir water-soluble fraction sample (A), a sample (B) having a molecular weight of 1000 or less, a sample (C) of 1000 to 2000, and a sample (D) of 2000 or more were subjected to molecular weight fractionation. A qualitative antioxidant capacity test using DPPH was performed on 4 samples including the minute samples (B, C, D). First, all samples containing a 0.1 mM ascorbic acid aqueous solution used as a positive control were dispensed in 50 ml portions into 1.5 ml Eppendorf tubes. 50 ml of 100% methanol was dispensed into each well and mixed to precipitate a protein component. This is because the residual protein in the kefir water-soluble fraction is precipitated with methanol. An Eppendorf tube with a total volume of 100 ml was centrifuged to completely precipitate the protein component (4500 g, 10 mi).
n). Next, 50 μl of the supernatant was taken from each sample and dispensed into a new Eppendorf tube. As a negative control, 50% methanol 50μ
A sample dispensed in 1 was prepared. 6 prepared in this way
The above 0.1 mM DPPH solution 10 was added to all samples.
0 μl was quickly dispensed, left at 37 ° C. protected from light. Three
After standing for 0 minutes, place each sample in a 96-well plate.
Each μl was dispensed, and the absorbance at 492 nm was measured. The results are shown in Table 1. Here, the strongest decrease in absorbance was observed in the sample having a molecular weight of 1,000 or less, and the strongest antioxidant ability was confirmed.

[0018]

[Table 1]

(3) Identification of Antioxidants by Online HPLC-DPPH System Eluent 1 of Online HPLC-DPPH System 10
As 1 was used a 0.1 M aqueous solution of ammonium acetate.
As shown in FIG. 1, the eluent 11 having a single composition was used at a flow rate of 0.7.
It was supplied to the online HPLC-DPPH system 10 at ml / min. DPPH was dissolved in 80% methanol solvent on the day of the experiment and stocked at a concentration of 0.1 mM.
This was diluted 10 times with 80% methanol solvent to 0.01 m.
The DPPH solution 19 of M was mixed with the line of the online HPLC-DPPH system 10 after separation from the anion exchange column 16 by the DPPH pump 15 at a flow rate of 0.7 ml / min. Online HPLC from the day before the experiment
The anion exchange column 16 of the DPPH system 10 was equilibrated at a flow rate of 0.7 ml / min. The DPPH solution 19 was prepared on the day of the experiment, and the DPPH pump 15 was operated. 80% methanol solvent 2 simultaneously with pump operation
00 ml was injected and the system was run until the baseline DPPH absorbance at 517 nm was stable. After confirming the stability of the baseline, a 0.1 mM ascorbic acid aqueous solution prepared on the day to confirm that this system can detect water-soluble antioxidants 20
0 ml was injected from the auto sampler 13. The results are shown in Fig. 4. When ascorbic acid was monitored at a wavelength of 280 nm, a peak was detected at around 10 minutes, and a decrease in the absorbance of DPPH at 517 nm was confirmed in a form corresponding to the peak. Therefore, 200 ml of a sample of the kefir water-soluble fraction having a molecular weight of 1,000 or less was injected to search for antioxidants. Results are shown in FIG. When the component was monitored at a wavelength of 280 nm, multiple peaks were recognized.
Among them, only the peak (Q) confirmed around 38 minutes showed a corresponding decrease in the absorbance of DPPH, and it was proved that it has antioxidant activity. Further, FIG. 6 shows the results obtained by simultaneously acquiring the UV-visible spectrum of the target substance at the peak (Q) by the photodiode array detector 14, which is a device capable of detecting the absorbance at various wavelengths. It was found that the target substance has a maximum absorption at 236.0 nm and 291.4 nm (λ max). As shown in FIG. 7, flavonoids were found to have similar spectral patterns among natural antioxidants. In this example, an online HPLC method using an online HPLC-DPPH system was adopted as the online HPLC method, but the case of using luminol instead of DPPH is also included in the method of the present invention.

Next, the DPPH supply system was separated from the system to fractionate the antioxidant while maintaining its activity (fifth step). The injection amount of the kefir water-soluble fraction sample (B) having a molecular weight of 1000 or less was increased to 1000 ml, and 1
Large-scale collection of the target peak was performed using a program with a cycle of 60 minutes. When collecting a large amount, the fraction collector 17 was used and the range from 35 minutes to 50 minutes was divided into 5 fractions every 3 minutes for automatic fractionation. After continuous operation for 20 hours each, 200 ml of 80% methanol solvent, 200 m of 0.2M aqueous sodium hydroxide solution
The anion exchange column 16 was washed by injecting in the order of 1. Further, the anion exchange column was washed with a 1 M sodium chloride aqueous solution. From the elution pattern in the final sample injection, only the fraction containing the target substance was selected and concentrated by the evaporator. Further, here, the concentration by an evaporator and the reconcentration operation by adding ultrapure water again were repeated to remove the volatile ammonium acetate salt as much as possible. Finally, the sample (B) was concentrated to about 5 ml, frozen and stored, and freeze-dried.

(4) Purification by gel filtration and estimation of molecular weight The gel filtration column that was made by myself is an online HPLC-DPP.
It was connected to the H system 10 and equilibrated by supplying 10% acetic acid aqueous solution at a flow rate of 0.5 ml / min from the day before the experiment. The freeze-dried sample (B) was dissolved in the smallest possible amount of 10% acetic acid aqueous solution, and 100 ml was injected by an autosampler. From the result of the UV-visible spectrum in FIG. 6, the elution of the target substance was monitored at 290 nm by gel filtration. The results of purification / desalting by gel filtration are shown in FIG. As shown in FIG. 8, the target peak eluting from around 110 minutes was collected, and concentration by an evaporator and reconcentration by adding ultrapure water again were repeated to completely remove acetic acid, which is a volatile acid. Finally, the sample was concentrated to about 5 ml, frozen and stored, and freeze-dried. At the same time, various substances with known molecular weights were subjected to gel filtration under the same conditions, and the size of the target substance in terms of molecular structure was estimated from their elution time. First, 1 mg of purified product after gel filtration
When the amount was adjusted to / ml and 50 ml was supplied, the peak was eluted at 121.3 minutes. Vitamin B12 (MW 1355.4), Bromophenol Blue (MW 669.97) and Phenol Red (MW 354.38) were tested at the same concentration and supply amount. Vitamin B12 peaked at 32.2 minutes and phenol red peaked at 157.9 minutes. Was eluted as. Also, bromophenol blue could not be detected under these conditions. The results are shown in Table 2. On the other hand, the molecular weight of the kefir antioxidant estimated from the data of vitamin B12 and phenol red was 650. Results here and molecular weight cut-off 5
Since the target substance can be concentrated in the external liquid obtained using the dialysis membrane of No. 00, the molecular weight of the target substance was estimated to be in the range of about 500 or less. However, since the concentration by this dialysis membrane can change depending on the shape and charge of the substance, the value of 500 or less is not accurate. Similarly, since the molecular weight estimation by gel filtration also changes depending on the shape of the molecule, it is difficult to accurately estimate the molecular weight even with a calibration curve prepared by using a molecular weight marker as a position marker of the molecular weight.

[0022]

[Table 2]

(5) Reversed-phase HPLC analysis For purification after gel filtration, a general reverse-phase HPLC elution condition of 0.05% TFA (trifluoroacetic acid) water / methanol system at a flow rate of 1.0 ml / min. Tried to elute. After injecting the sample (B) after gel filtration into reverse phase HPLC, three peaks were confirmed at the stage where the composition of the eluent was 100% water. Further, no other peak was observed even when the concentration gradient was applied up to 80% of methanol. It was confirmed that the peak monitored by UV absorption at 230 nm around 2.4 minutes was ammonium acetate in the eluent used for large-scale fractionation on the anion exchange column. The peak at around 3.8 minutes was found from the UV-visible spectrum pattern obtained from the photodiode array detector 14 to be a contaminant of the peak before the target peak at the elution of the anion exchange column. Further, the target substance was detected from around 5 minutes, and it was found that it was a substance having extremely high polarity. Since the recovered amount of the sample after gel filtration was very small, purification / preparation by reverse phase HPLC was abandoned at this stage, and various instrumental analyzes were performed using the sample after gel filtration.

(6) Fast atom bombardment mass spectrometry (FAB-MS /
Molecular weight measurement by mass spectrum) Fig. 9 shows the results of molecular weight measurement requested to fast atom bombardment mass spectrometry (FAB-MS) from the Department of Medicinal Resource Control, Kyushu University Graduate School of Pharmaceutical Sciences. Result Maximum molecular weight 60
A peak at 7.8 m / z was detected. Also, 341.4
A strong peak was observed at m / z. From this, it was estimated that the molecular weight of this substance was 607.8, and the molecular weight of the metastable degradation product was 341.4.

(7) Infrared absorption spectrum measurement FIG. 10 shows the results of infrared (IR) absorption spectrum (microscopic IR) of the antioxidant in Kefir, which was commissioned to Shimadzu Integrated Analysis Center Co., Ltd. In summary of the report, infrared (I
(R) Absorption spectrum (microscopic IR) There is no spectrum similar to the requested kefir antioxidant in the library of Sudler, which stores analysis values, and it is a compound having an ethylene-like secondary bond as a substance having the closest structure. There is a possibility. From the partial structure analysis, it was reported that there is a benzene ring having a substituent at the ortho position. Also 1125, 1029, 991, 783, 744 cm ^-1
The absorption at 3 represents the out-of-plane bending vibration of a benzene ring having three adjacent substituents, and the absorption at 3300 to 2800 cm ^-1 suggests the presence of a hydroxyl group.

(8) Antioxidant activity measurement of the purified product in a test tube The specific activity of the purified kefir antioxidant and the water-soluble antioxidant ascorbic acid was examined. DPPH was dissolved in 80% methanol solvent on the day of the experiment and prepared at a concentration of 0.5 mM. Ascorbic acid aqueous solution 1 on the day of the experiment
Prepared at a concentration of 0 mM. First, dilute a 10 mM aqueous ascorbic acid solution to give 0, 50, 100, 150, 20
0, 250, 300, 350, 400, 600, 80
Samples of 0, 1000 μM were prepared. 1 mg /
Dilute ml of kefir antioxidant to 0, 0.2, 0.
Samples of 4, 0.6, 0.8, 1.0 mg / ml were prepared. Next, 1.5 ml of each sample, 50 ml
Dispense into an Eppendorf tube. 100 there
50 μl of% methanol was dispensed and mixed. Total amount 1
0.5 mM DPPH was added to all of the samples that reached 00 μl.
Quickly dispense 100 μl of the solution, protect it from light at 30
Let stand for a minute. Add each sample to a 96-well plate
Three points of 0 ml each were dispensed, and the absorbance at 492 nm was measured. The results are shown in Tables 3 and 4 and FIG.

[0027]

[Table 3]

[0028]

[Table 4]

As shown in Tables 3 and 4, and FIG. 11, the 50% DPPH reducing concentration of ascorbic acid was 300 μM, and that of kefir antioxidant was 0.4 mg / ml. Assuming the molecular weight of kefir is 608, 0.4m
The g / ml was equivalent to 660 μM, and it was found that the kefir antioxidant has an antioxidant activity that is about half that of ascorbic acid at a weight concentration using 50% DPPH reduction as an index.

(9) Discussion When the active substance was searched for from the water-soluble fraction with respect to the antioxidative effect of fermented milk kefir, it was found that the active substance mainly exists in the range of molecular weight of 1000 or less. Molecular weight 100 for confirmation
Online H for samples in the range 0, 2000
When the active ingredient was searched for using the PLC-DPPH method, no new ingredient was found. Further, the same search was conducted for the antioxidative action with respect to the molecular weight of 100 or less, which was previously separated, but no new component having the antioxidative action was found. Online HPLC method DPPH-HPLC
It can be said that the active substance whose activity is confirmed by the method is negatively charged because it can be separated by an anion exchange column. In addition, the results of analysis by reverse phase HPLC strongly suggested that this kefir antioxidant is a very hydrophilic (highly polar) substance. Furthermore, this activity is maintained even when a kefir water-soluble sample fractionated to a molecular weight of 1,000 or less, which is a starting material for purification, is subjected to high-pressure steam sterilization, and therefore it is considered to be a thermostable substance. Further, by separating the online-DPPH supply system from the system in the above-mentioned example and collecting the antioxidant substance in the state where the activity was maintained, it became possible to produce the antioxidant substance of kefir. Furthermore, a more purified antioxidant can be produced by a method such as gel filtration. In this example, the purification of the anti-acidic substance after the online HPLC-DPPH method was performed by gel filtration, but the case of using other purification methods is also included in the method of the present invention.

Next, in order to know the molecular characteristics of the Kefir antioxidant, the molecular weight was first examined. The molecular weight was estimated to be 500 or less from the molecular weight fraction sample in the early stage of the experiment and the experimental results of gel filtration. But this 500
The following numerical values are not accurate, and it is difficult to estimate the molecular weight accurately because the molecular weight estimation by gel filtration changes depending on the shape of the molecule. Next, the Kefir antioxidant substance purified by gel filtration was requested for molecular weight measurement by fast atom bombardment mass spectrometry (mass spectrum), and a peak showing a molecular weight of 607.8 at the maximum was detected. It is considered that the estimation of the molecular weight by this mass spectrum is the most accurate estimation of the molecular weight, and the molecular weight of this substance is 607.8,
It was estimated that the metastable degradation product would have a molecular weight of 341.4. Furthermore, in order to know the structural characteristics of this kefir antioxidant, we requested the measurement of infrared absorption spectrum. Results There is no spectrum similar to the requested kefir antioxidant in the library of Sadler, and it is highly likely that the compound has novelty. From the assignments corresponding to the spectra, the existence of a benzene ring having multiple substituents and an ethylene-like double bond was suggested as the molecular skeleton. The presence of hydroxyl groups, which are characteristic of natural antioxidants, was also suggested. This result is mainly due to the hydrogen acceptance from the hydroxyl group.
Consistent with the fact that PPH is reduced. Therefore, at this stage, the antioxidant substance contained in the kefir water-soluble fraction has a molecular weight of 100 to 700, a hydroxyl group, and an aromatic group, and is a substance that is stable in acidity and neutrality and stable even in high temperature steam sterilization. You can say that.

Example 2 (10) Physiological Activity of Kefir Antioxidant in Cultured Animal Cells Generally, even a compound showing antioxidant activity in vitro,
In fact, it does not always have an appropriate amount of antioxidant activity in cells. Then, the water-soluble kefir antioxidant produced by the above-mentioned embodiment of the method of the present invention, that is, purified from the water-soluble fraction of kefir and having a molecular weight of 100 to 700 m / z, has a proper intracellular action amount. It was examined below by examining the physiological activity in cultured animal cells to determine whether or not it has antioxidant activity. Here, the fluorescent dye DCFH-
The intracellular oxidation state was measured using DA (2,7-dichlorofluorescein diacetate).

FIG. 12 is an explanatory view of the action mechanism of DCFH-DA, and FIG. 13 is a process chart of an intracellular reactive oxygen scavenging test using a human leukemia-derived cell line K562. As shown in FIG. 12, DCFH-DA, which is a membrane-permeable probe, moves from the outside to the inside of the cell, and is hydrolyzed by esterase in the cytoplasm to become DCFH. This DCFH stays in the cell because it loses its membrane permeability, where it reacts with intracellular reactive oxygen species such as hydrogen peroxide and is oxidized to become DCF. This DCF emits fluorescence (λex = 502n
Therefore, the intracellular oxidation state can be measured by measuring the DCF fluorescence intensity of the DCFH-DA-administered cells. In the case of adherent cells, DCFH-DA is treated while the cells are adhered, and the DCF fluorescence intensity is measured by a confocal laser microscope, but it is difficult to determine the observation position and quantify the experimental results at this time. Therefore, in this study, we used the non-adherent cell line K562 derived from human leukemia to irradiate FCM (fluorescent dye-stained cells with laser light to measure the cell size, DNA content, and distribution of membrane antigen expression. Method) was used to measure the intracellular oxidation state.

(11) Experimental materials Cell culture RPMI1640 medium (basic nutrient medium) was obtained from Nissui Pharmaceutical Co., Ltd. As shown in FIG. 13, human leukemia-derived cells were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS).
K562 was cultured in a 90 mm dish, and the number of cells was diluted to 1/10 at each saturation density, and the cells were passaged. Reagent fluorescent dye DCFH-DA Hank's solution was obtained from Nissui Pharmaceutical Co., Ltd.

(12) Equipment used (FCM) DCF fluorescence intensity is measured by Beckman Coulter (BECKMAN CO
ULTER's EPICS XL / XL-MLL SystemII (aka FAC)
The measurement was performed using S). (13) Results and Discussion DCF Fluorescence Intensity Analysis by FCM (Flow Cytometry) FIG. 13 shows the procedure of an intracellular reactive oxygen scavenging test using the human leukemia-derived cell line K562. As shown in FIG. 13, human leukemia-derived cells K562 contained 10% fetal bovine serum (FBS).
Incubate in RPMI1640 medium and increase the number of cells to 1 at each saturation.
It was diluted to 1/0 and passaged. Transfer the cells with 1 × 10 ⁶ cells into a test tube and add 5 μM DCFH-DA.
A water-soluble kefir antioxidant having a molecular weight of 100 to 700 prepared according to the above-described example of the method of the present invention was added to the fluorescent dye and treated for 7 minutes. The one which was not treated with the kefir antioxidant was used as a control. FCM of human leukemia-derived cell line K562 treated with control and kefir antioxidant after 10 minutes
Fluorescence detection was performed. The result is shown in FIG. As can be seen from FIG. 14, the fluorescence intensity of the cells was moved to the left as compared with the control by the treatment with the kefir antioxidant. This indicates that the fluorescence intensity per cell was totally decreased, and it was speculated that the hydrogen peroxide in the cell was erased by the kefir antioxidant. When the fluorescence intensity of the control is 100%, 1 and 10 μg / m
It can be said that the kefir antioxidant has a strong intracellular active oxygen scavenging ability because 50 to 60% of the intracellular active oxygen was eliminated at the concentration of 1. These results indicate that oxidative stress causes various functional declines in animal cells and causes many diseases. Therefore, kefir antioxidants are expected to be effective in preventing and treating various diseases. It was

(Example 3) (14) Glucose uptake promoting activity of kefir-derived antioxidant into adipocytes The kefir antioxidant purified by the above-mentioned online HPLC-DPPH method promotes glucose uptake into adipocytes. The following experiment was carried out in order to determine whether or not to do so, and to confirm the effect on diabetes, which is said to be caused by insufficient glucose uptake, particularly type II diabetes.

Experimental Example 3T3 / L1 preadipocytes were
After culturing in 10% FBS-DMEM medium and reaching half saturation, 0.5 mM isobutylmethylxanthine (isobuty
l metylxanthine), 0.25 μM dexamethasone (dexa
methasone), and 10 μg / ml insulin added 7
The cells were cultured for 2 hours, cultured for 48 hours in a medium containing only 10 μg / ml insulin, and then returned to a medium containing only 10% FBS-DMEM to induce differentiation. Cells differentiated by culturing in a normal medium for 72 hours after the differentiation treatment were used for the glucose uptake assay. The differentiated cells were cultured for 4 hours in a medium containing Kefir-derived antioxidants, stimulated with insulin for 20 minutes, then rinsed twice with 300 μl of glucose-free HBS solution, and 500 μl per well.
l HB containing 1 μCi / ml 2-Deoxy-D- [1-3H] glucose
S was added and treatment was performed for 10 minutes. After 10 minutes, the radioactive solution was removed by suction and 300 μl of ice-cold 0.9% NaCl was added.
The solution was rinsed 3 times quickly. After lysing the cells by adding 500 μl of 0.5 N sodium hydroxide, 100 μl of 2
Neutralized with N 2 acetic acid. 2 ml of each sample 500 μl
Then, 800 μl of Aquasol liquid scintillation cocktail (fluorescent dye-containing solution for radiation measurement) was added to the Eppendorf, and the measurement was performed with a liquid scintillation counter (radiation dose measuring device). The measurement was performed for 5 minutes per sample.

Results FIG. 15 is a diagram showing the measurement of intracellular active oxygen scavenging activity by FCM. As shown in FIG. 15, the Kefir antioxidant has a stimulating effect about 2.0 times at 0.1 μg / ml concentration and about 2.5 times at 10 μg / ml concentration, accelerating glucose uptake in a concentration-dependent manner. It became clear to do. From these results,
It was expected that it would be possible to develop new antidiabetic therapeutic agents and functional foods mainly containing the antioxidants derived from kefir. In addition, health foods using kefir antioxidants may be added with other nutrients or substances in order to improve their nutritional value and to expect further effects in order to make them delicious.

Next, DN for Kefir's antioxidants
The action as a tissue repairing agent having A repairing action was also searched. (Example 4) Effect of mismatch repair enzyme hMLH1 on gene expression The mismatch repair gene repairs an error generated during DNA replication to maintain genome stability and suppresses carcinogenesis by suppressing the accumulation of error bases. is doing. Further, in the individual in which the mismatch repair gene does not function normally, dysfunction is also observed in the excision repair function, and therefore it is considered that the mismatch repair gene is also involved in the excision repair mechanism. However, the details are not clear.
Since the kefir antioxidant could be highly purified by the HPLC-DPPH method, the mismatch repair gene h
The gene expression enhancing effect of MLH1 was examined. Also, 100
The thermal stability by heating at 0 ° C for 10 minutes was also investigated.

Method Antioxidants were added to HMV-1 cells (melanoma cells / skin model cells) and cultured for 15 hours, and the expression of the mismatch repair gene hMLH1 was determined by RT-PCR (messenger RNA was extracted from the cells, A phase-corrected DNA (cDNA) is synthesized by reverse transcriptase, and then a cDNA partial sequence fragment (primer) of a specific gene is used.
The method was used to examine the degree of gene expression by amplifying DNA). Results and discussion results are shown in FIG. The numerical data are as follows. Control 1.0 Kefir antioxidant (0.4 μg / ml) 1.26 Kefir antioxidant (1 μg / ml) 1.33

It was revealed that the cells to which the purified kefir antioxidant was added increased the expression of the mismatch repair gene hMLH1. From this, the gene expression of the mismatch repair gene hMLH1 is redox regulated (a biological reaction in which gene expression is regulated by oxidative stress).
Was suspected to have been received. About 1/3 of Vitamin C
The addition of this kefir antioxidant having antioxidative properties has enabled the development of a new anticancer agent that repairs an error in DNA replication.

Example 5 Next, the promoting effect of kefir antioxidant and heat-treated kefir antioxidant on irregular DNA synthesis was examined. D, which is irrelevant to DNA replication when the target DNA is damaged
DNA synthesis for NA repair is performed. This is called irregular DNA synthesis (UDS). It can be inferred that when the irregular DNA synthesis was promoted, the DNA repair activity of the cell was promoted. Irradiation with ultraviolet light (UVA) damages the bases of DNA, and the damaged bases are repaired by the base excision repair mechanism. Method UVA was added to HMV-1 cells plated on a 96-well plate.
After irradiating with 400 J / m ^2, 0.40 Kefir antioxidant was added.
It was added at a concentration of μg / ml and cultured for 1 hour. Irregular D
NA synthesis (UDS) is based on bromodeoxyuridine (Br
It was measured by the incorporation of dU). In order to minimize DNA replication caused by cell division, the cell cycle was arrested by treatment with 50 mM hydroxylurea (Wako), which acts to arrest the cell cycle that causes cell division. Results As shown in FIG. 17, UDS was clearly increased in the cells to which the kefir antioxidant was added. Also, 100
UDS, a kefir antioxidant, even if heated at 10 ° C for 10 minutes
This kefir antioxidant was considered to be thermostable, as the promoting effect was not diminished. Discussion From the above results, it was considered that the kefir antioxidant activated the mismatch repair mechanism of cells and promoted DNA repair. The kefir antioxidant used here was presumed to be highly purified since it showed a single peak even when the solvent was changed in the HPLC purification. It is considered that the heat stability of this substance is also advantageous in terms of application to functional foods. Further, from these results, it has become possible to develop a new tissue repair agent and a functional food that promote the DNA repair activity of cells and have a DNA repair action. The kefir antioxidant can be widely used for the development of a method for preventing UV damage to skin cells or a method for preventing skin photoaging, and the prevention and treatment of cancer and genetic diseases caused by DNA mutations.

The present invention has been described above with reference to the embodiments.
The present invention is not limited to these examples. In the above examples, kefir used was kefir (kefir fermented liquid) donated by Japan Kefir Co., Ltd.
Kefir using the sold Kefir bacterium, those from other countries other than Russia, or those cultivated for Kefir grains by themselves may be used. Further, the device and the medicine used in the above-mentioned examples are not limited to these, and can be changed as long as they do not affect the result.

[0044]

The antioxidant using kefir according to any one of claims 1 to 5 has a function of reducing active oxygen in a living body, which has been regarded as a cause of not only aging but also cancer or other various diseases or aggravating factors. Is useful because it has Especially claim 5
The described antioxidant using kefir is easy to use because it is stable in acid or neutral and stable in high-pressure steam sterilization. Since the antidiabetic agent having the antioxidant of kefir according to claims 6 and 11 has an effect of enhancing glucose uptake, it is possible to develop an antidiabetic agent for diabetes, especially type II, and to use it as an antidiabetic agent. Became. DN with antioxidant of kefir according to claims 7 and 12
A tissue repair agent having an A-repairing action is caused by an error-repairing action of DNA replication of an antioxidant of kefir, a DNA repair-promoting action, which prevents UV damage to skin cells, photo-aging of skin, and DNA mutation. It has become possible to be widely used for prevention and treatment of cancer and genetic diseases that occur. The health foods containing the kefir antioxidants according to claims 8 and 13 have enabled the development of functional foods or health foods that reduce active oxygen in the living body. The method for producing an antioxidant using kefir according to claim 9 or 10, wherein a substance having an active oxygen reducing action (antioxidant action) is isolated and produced from kefir. It has become possible to contribute to the development of a diabetes functional food and a tissue repair agent having a DNA repair action. Particularly, in the method for producing an antioxidant using kefir according to claim 10, since the DPPH-HPLC method is used for the online HPLC method, the system construction is simple, and the antioxidant can be easily extracted from the kefir water-soluble fraction. The agent can be dispensed. Also, a low pH buffer solution
However, analysis is possible, the baseline of the absorbance of DPPH is more stable than that using luminol instead of DPPH, and the economy is good. Since the antioxidant substance can be separated and collected without losing the antioxidant activity, the antioxidant can be easily collected by separating the HPLC column. By the method for identifying an antioxidant using kefir according to claim 14, it is estimated that the antioxidant of kefir is a low molecular weight organic compound having a molecular weight of 100 to 700, and has a plurality of hydroxyl groups and aromatic groups, and Stable in acid or neutral,
And since it was found to be stable for autoclaving,
Further utilization of antioxidants can be expected.

[Brief description of drawings]

FIG. 1 is a configuration diagram of an online HPLC-DPPH system.

FIG. 2 is a conceptual diagram of detecting an antioxidant by DPPH.

FIG. 3 is a process diagram of preparing a fractionated sample having a molecular weight of 1000 or less.

FIG. 4 is an explanatory view of antioxidant activity of ascorbic acid.

FIG. 5 is an explanatory diagram of the results of searching for antioxidants in the kefir water-soluble fraction (molecular weight of 1000 or less).

FIG. 6 is an ultraviolet-visible spectrum diagram of Kefir antioxidant.

FIG. 7 is an ultraviolet-visible spectrum diagram of kefir antioxidants and representative flavonoids.

FIG. 8 is an absorbance diagram when purifying an active substance by gel filtration.

FIG. 9: Mass spectrum of Kefir antioxidant (FAB
-MS) is an explanatory diagram of FIG.

FIG. 10 is an infrared absorption spectrum diagram of Kefir antioxidant.

FIG. 11 is an explanatory diagram of the results of measurement of antioxidant activity in a test tube.

FIG. 12 is an explanatory diagram of a mechanism of action of DCFH-DA.

FIG. 13 is a process diagram of an intracellular reactive oxygen scavenging test using a human leukemia-derived cell line K562.

FIG. 14 is a measurement diagram of intracellular active oxygen scavenging activity by FCM (flow cytometry).

FIG. 15 is a graph showing the effect of promoting the uptake of sugar into adipocytes by the kefir antioxidant.

FIG. 16 is an explanatory diagram of the effect of enhancing the expression of repair enzyme hMLHI gene of kefir antioxidant.

FIG. 17: Irregular DNA synthesis of Kefir antioxidants (U
It is explanatory drawing of the DS) enhancement effect and its thermal stability.

[Explanation of symbols]

10: Online HPLC-DPPH system, 11:
Eluent, 12: System controller, 13: Autosampler, 14: Photodiode array detector, 1
5: DPPH pump, 16: anion exchange column, 1
7: Fraction collector, 18: Chromatography manager, 19: DPPH solution

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/10 A61P 3/10 35/00 35/00 39/06 39/06 43/00 105 43/00 105 (72) Inventor Minoru Shirahata 1-2-19-9 Maisato, Koga City, Fukuoka Prefecture (72) Inventor Teruichiro Teruya 1-3-4-406 Chidori, Koga City, Fukuoka Prefecture (72) Yoshinori Katakura Fukuoka Prefecture, Koga Prefecture Chidori, Ichi 1-3-4-205 (72) Inventor Yagaku Tsutomu 4-25-8-102 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka (72) Inventor Shin-ichi Kiyonari 11-15 Ohata-cho, Takatsuki-shi, Osaka Joyful Takatsuki Ohata 309 (72) Inventor Junko Naruzawa 17-14 Inaricho, Sasebo City, Nagasaki Prefecture (72) Inventor David Burns United States Virginia Virginia Cattlett Charity Lane 9049 (72) Inventor Koichiro Tokumaru Tsuji Fujisawa, Kanagawa Prefecture Taiheidai 1-10-3 (72) Inventor Chinosuke Tokumaru Fujisawa City, Kanagawa Tsujido Taiheidai 1-10-3 F Term (reference) 4B001 AC31 AC32 BC99 EC05 4B018 MD71 MD91 ME03 ME06 ME14 MF01 4C087 AA01 AA02 AA03 AA04 AA05 BB39 BC11 BC58 BC61 CA07 CA10 CA11 CA24 CA25 CA26 CA27 MA52 NA14 ZB26 ZC35 ZC37

Claims (14)

[Claims] 1. An antioxidant using kefir, which comprises an antioxidant in a water-soluble fraction of kefir. 2. The antioxidant according to claim 1, wherein the antioxidant has a molecular weight of 100 to 700, and the antioxidant using kefir is used. 3. The antioxidant according to claim 2, wherein the antioxidant has a hydroxyl group, and an antioxidant using kefir is used. 4. The antioxidant according to claim 2, wherein the antioxidant has an aromatic group, and the antioxidant uses kefir. 5. The antioxidant according to any one of claims 2 to 4, wherein the antioxidant substance is acidic or neutral and stable, and is stable even under high pressure steam sterilization. An antioxidant using kefir. 6. A therapeutic agent for diabetes which comprises the antioxidant according to any one of claims 1 to 5. 7. A tissue repair agent having the antioxidant according to claim 1 and having a DNA repair action. 8. A health food comprising the antioxidant according to claim 1. 9. The first step of centrifuging kefir to precipitate milk fat to obtain a supernatant, and the supernatant having a molecular weight cut off of 1000.
Second step of dialysis with a dialysis membrane of 1 to obtain an external solution having a molecular weight of 1000 or less, and a third step of neutralizing a concentrated solution obtained by concentrating the external solution with an alkali to remove precipitates and obtaining a separated solution Then, the separated liquid was dialyzed with a dialysis membrane having a molecular weight cut off of 100 to obtain a molecular weight of 1
Antioxidation using kefir, characterized in that it has a fourth step of obtaining an internal solution of 00 or more and a fifth step of identifying and fractionating an antioxidant substance from the internal solution by using an online HPLC method. Method of manufacturing agent. 10. The method for producing an antioxidant using kefir according to claim 9, wherein the online HPLC method is a DPPH-HPLC method, and the antioxidant substance of kefir collected in the fourth step is gel filtration. A method for producing an antioxidant using kefir, which is characterized by being purified. 11. A therapeutic agent for diabetes mainly comprising an antioxidant using kefir produced by the method for producing an antioxidant using kefir according to any one of claims 9 and 10. 12. A tissue repair having a DNA repairing action mainly composed of an antioxidant using kefir produced by the method for producing an antioxidant using kefir according to any one of claims 9 and 10. Agent. 13. A health food mainly composed of an antioxidant using kefir, which is produced by the method for producing an antioxidant using kefir according to any one of claims 9 and 10. 14. A first step of centrifuging kefir to precipitate milk fat to obtain a supernatant, and the supernatant having a molecular weight cut off of 1,000.
Second step of dialysis with a dialysis membrane of 1 to obtain an external solution having a molecular weight of 1000 or less, and a third step of neutralizing a concentrated solution obtained by concentrating the external solution with an alkali to remove precipitates and obtaining a separated solution Then, the separated liquid was dialyzed with a dialysis membrane having a molecular weight cut off of 100 to obtain a molecular weight of 1
A fourth step of obtaining an internal solution of 00 or more, a fifth step of identifying and fractionating an antioxidant substance from the inner solution by using an online HPLC method, and a sixth step of identifying the fractionated antioxidant substance A method for identifying an antioxidant using kefir, which comprises steps.