JP2009102397A - Antioxidant using kefir, diabetes treating agent using this, tissue repairing agent having dna repairing action, and health food - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an antioxidant from kefir: fermented milk, a method of identifying the same, and a diabetes treating agent, a tissue repairing agent having DNA repairing action and a health food each using kefir. <P>SOLUTION: The antioxidant production method by using kefir comprises a first process for obtaining a supernatant by precipitating milk fat component by centrifuging the kefir, a second process for obtaining an outer liquid of molecular weight 1,000 or less by dialyzing the supernatant with a dialyzing film of molecular weight cut-off 1,000, a third process for obtaining a separated liquid by removing precipitate generated by neutralizing with an alkali a concentrated liquid generated by concentrating the outer liquid, a fourth process for obtaining an inner liquid of a molecular weight 100 or more by dialyzing the separated liquid with a dialyzing film of molecular weight cut-off 100, and a fifth process for identifying and fractionally collecting an antioxidant substance by using an on-line HPLC (high-performance liquid chromatography) method; the method of identifying the antioxidant; the antioxidant; the diabetes treating agent; and the tissue repairing agent having DNA repairing action; are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to an antioxidant action of kefir, which is a health food, and a therapeutic agent for diabetes using the effect, a tissue repair agent having a DNA repair action, and a health food.

Kefir is fermented milk that is loved by people in the Caucasus region of Georgia. By accident, lactic acid bacteria and yeast are mixed with lactic acid and alcohol in cows, goats, and sheep milk stored in goat leather bags. It is produced by fermentation. The Caucasus fermented milk kefir is made from stable kefir grains obtained through years of research by the Central Research Institute for Microbiology of the Russian Academy of Sciences, an independent national community, as a bacterial species. It enhances digestive function and is used in medical institutions as a diet for kidney and cardiovascular diseases.
Kefir grains are sponge cakes of about 1 to 3 cm and have about 20 types of lactic acid bacteria, yeast and acetic acid bacteria. In Japan, they are generally called yoghurt mushrooms. Kefir grains are mixed with milk and sealed at room temperature for a predetermined time to obtain kefir mixed with kefir grains (called kefir yogurt), which can be obtained simply by filtering and removing kefir grains. It is also used regularly as a health food by ordinary households.

In recent years, active oxygen generated from one's own metabolic system has been attracting widespread attention as a human aging factor when humans perform life activities. This oxidative stress due to active oxygen increases in vivo due to inappropriate eating habits, stress, ultraviolet rays, etc., and this active oxygen is regarded not only as a cause of aging but also as a cause of or aggravation of various diseases such as cancer. Reducing active oxygen has become a major research subject for maintaining human health.
In Japan, in recent years, non-insulin-dependent type II diabetes has continued to increase from Western diets, afflicting many Japanese. In this type II diabetes, it is impossible to maintain cellular homeostasis due to the inability to take up glucose in cells, which is the core physiological function of maintaining health, resulting in various complications and serious symptoms. Is not uncommon. It is considered that active oxygen is largely involved in the decrease in glucose uptake of cells, which is a factor of this type II diabetes, and the reduction of active oxygen activates cell glucose uptake and is considered to be useful for treatment of type II diabetes. Furthermore, development of foods and drugs having an action of reducing active oxygen has been demanded. It is speculated that fermented milk kefir, which has been drunk in the Russian Caucasus region, which is considered to be longevity for a long time, and has been attracting attention as a health food in Japan in recent years, may also have an action to reduce this active oxygen. Isolating and identifying 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 diabetes treatment, particularly type II diabetes treatment. Conceivable.

The present invention has been made in view of such circumstances. The 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. The second object is to provide a method for isolating and identifying an antioxidant substance, and further to provide a therapeutic agent for diabetes, a tissue repair agent having a DNA repair action, and a health food using the antioxidant substance as a main component. This is the third purpose.

The antioxidant using kefir according to the first invention is composed of an antioxidant substance in the kefir water-soluble fraction.
The antioxidant using the kefir according to the second invention is the antioxidant according to the first invention, wherein the antioxidant substance has a molecular weight of 100 to 700.
An antioxidant using kefir according to a third invention is the antioxidant according to the second invention, wherein the antioxidant substance has a hydroxyl group.
The antioxidant which concerns on 4th invention is the antioxidant which concerns on 2nd, 3rd invention, The said antioxidant substance has an aromatic group.
An antioxidant according to a fifth aspect of the present invention is the antioxidant according to the second to fourth aspects of the present invention, wherein the antioxidant substance is acidic or neutral and stable, and is stable to high-pressure steam sterilization.
The diabetes therapeutic agent which concerns on 6th invention has the antioxidant which concerns on 1st-5th invention.
The tissue repairing agent having a DNA repairing action according to the seventh invention has the antioxidant according to the first to fifth inventions.
The health food according to the eighth invention has the antioxidant according to the first to fifth inventions.

The method for producing an antioxidant according to the ninth invention comprises a first step of centrifuging kefir to precipitate milk fat and obtaining a supernatant, and dialyzing the supernatant with a dialysis membrane having a molecular weight cut off of 1000 to obtain a molecular weight. A second step of obtaining an external solution of 1000 or less, a third step of removing a precipitate formed by neutralizing the concentrated solution obtained by concentrating the external solution with an alkali, and fractionating the separated solution Dialysis with a dialysis membrane having a molecular weight of 100 to obtain an internal solution having a molecular weight of 100 or more, and a fifth step of identifying and separating an antioxidant from the internal solution using an on-line HPLC method ing.
A method for producing an antioxidant according to a tenth invention is the method for producing an antioxidant according to the ninth invention, wherein the online HPLC method is a DPPH-HPLC method, and the kefir fractionated in the fourth step is used. Antioxidants are purified by gel filtration.

Here, the on-line HPLC method is a method capable of simultaneously separating and identifying an antioxidant substance in a mixture and evaluating the activity, and in particular, the on-line HPLC method using DPPH is a DPPH (diphenyl 1, 2-picrylhydrazyl), the components in the mixture are separated by normal HPLC (High Performance Liquid Chromatography) analysis, and then the DPPH solution is mixed in the line and reacted, and the reactants are Through a photodiode array detector capable of detecting wavelengths, the peaks of antioxidant ability are identified by simultaneously measuring the spectrum of each component and the decrease in absorbance due to the reaction of DPPH with antioxidants. . FIG. 1 shows the configuration. As shown in FIG. 1, a DPPH solution containing methanol as a solvent is supplied to the eluent after reverse phase HPLC, and the antioxidant in the mixture is monitored by monitoring the decrease in absorbance at 517 nm due to the reaction between the antioxidant and DPPH. The component having activity is identified, and analysis is possible even with a low pH buffer solution (buffer), and the DPPH absorbance baseline is more stable than that using luminol instead of DPPH. In addition, it is possible to separate and sort the antioxidant substance without losing the antioxidant activity. DPPH has a maximum absorption at 517 nm in methanol solvent and is visually recognized as purple. FIG. 2 shows a conceptual diagram of antioxidant detection by DPPH. As shown in FIG. 2, when DPPH undergoes a reaction that accepts hydrogen from an antioxidant and is reduced, it becomes reduced DPPH and has a maximum at 517 nm. Absorption is significantly reduced and the purple color is decolorized. Antioxidants are detected by measuring the purple decolorization peak by HPLC analysis.

The therapeutic agent for diabetes using kefir according to the eleventh invention is mainly composed of an antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention.
The tissue repair agent having a DNA repair action using the kefir according to the twelfth invention is mainly composed of an antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention.
The health food using the kefir according to the thirteenth invention mainly comprises an antioxidant produced by the method for producing an antioxidant according to the ninth or tenth invention.
According to a fourteenth aspect of the present invention, there is provided a method for identifying an antioxidant using kefir, the first step of centrifuging kefir to precipitate milk fat and obtaining a supernatant, and the supernatant with a dialysis membrane having a molecular weight cut off of 1,000. A second step of obtaining an external liquid having a molecular weight of 1000 or less by dialysis, a third step of obtaining a separated liquid by removing a precipitate formed by neutralizing the concentrated liquid obtained by concentrating the external liquid with an alkali, and the separation A fourth step of dialyzing the liquid with a dialysis membrane having a molecular weight cut off of 100 to obtain an internal solution having a molecular weight of 100 or more, and a fifth step of identifying and fractionating an antioxidant from the internal solution using an on-line HPLC method. And a sixth step of identifying the sorted antioxidant.

Antioxidants using kefir according to claims 1 to 5 are useful not only for aging but also for reducing active oxygen in the living body, which is regarded as a cause of cancer or other diseases. is there.
In particular, the antioxidant using kefir according to claim 5 is easy to use because it is acidic or neutral and stable, and also stable against high-pressure steam sterilization.
The anti-diabetic agent having kefir antioxidant according to claim 6 has an action of enhancing glucose uptake, so that it becomes possible to develop an anti-diabetic agent for diabetes, particularly type II, and to use it as an anti-diabetic agent. It was.
The tissue repair agent having a DNA repairing action comprising a kefir antioxidant according to claim 7 is used to prevent UV damage of skin cells or to prevent skin damage by an error repairing action or DNA repair promoting action of DNA replication of the kefir antioxidant. Can be widely used to prevent photoaging and prevent or treat cancers and genetic diseases caused by DNA mutations.
The health food having the kefir antioxidant according to claim 8 can develop a functional food or a health food that reduces active oxygen in the living body.
Moreover, since the manufacturing method of the antioxidant using the above kefir isolates and manufactures the substance which has an active oxygen reduction action (antioxidant action) from kefir, health food and type II anti-diabetic functionality It was possible to contribute to the development of food and tissue repairing agents having DNA repairing action.

It is a block diagram of an online HPLC-DPPH system. It is a conceptual diagram of the antioxidant substance detection by DPPH. It is a preparation process figure of the fraction sample of molecular weight 1000 or less. It is explanatory drawing of the antioxidant activity of ascorbic acid. It is explanatory drawing of the search result of the antioxidant substance of kefir water-soluble fraction (molecular weight 1000 or less). It is an ultraviolet-visible spectrum figure of a kefir antioxidant substance. 1 is an ultraviolet-visible spectrum diagram of kefir antioxidants and representative flavonoids. FIG. It is an absorbance diagram at the time of purification by gel filtration of an active substance. It is explanatory drawing of the mass spectrum (FAB-MS) of a kefir antioxidant substance. It is an infrared absorption spectrum figure of a kefir antioxidant substance. It is explanatory drawing of the result of the antioxidant activity measurement in a test tube. It is explanatory drawing of the action mechanism of DCFH-DA. FIG. 4 is a process diagram of an intracellular reactive oxygen elimination test using a human leukemia-derived cell line K562. It is a measurement figure of intracellular reactive oxygen scavenging activity by FCM (flow cytometry). It is a measurement figure of the glucose uptake | capture promotion effect to a fat cell of a kefir antioxidant. It is explanatory drawing of the repair enzyme hMLHI gene expression enhancement effect of a kefir antioxidant. It is explanatory drawing of the irregular DNA synthesis (UDS) enhancement effect of kefir antioxidant, and its thermal stability.

Hereinafter, embodiments and examples embodying a method for identifying and producing a kefir antioxidant according to the present invention will be described.
FIG. 3 is a process chart for preparing a fraction sample having a molecular weight of 1000 or less, FIG. 4 is an explanatory diagram of the antioxidant activity of ascorbic acid, FIG. 5 is an analysis diagram of a kefir water-soluble fraction (molecular weight 1000 or less), and FIG. FIG. 7 is an ultraviolet-visible spectrum diagram of kefir antioxidants and representative flavonoids, FIG. 8 is an absorbance diagram during purification by gel filtration of the active substance, and FIG. 9 is a kefir antioxidant substance. Fig. 10 is an infrared absorption spectrum diagram of kefir antioxidant, Fig. 11 is an explanatory diagram of the result of measuring the antioxidant activity in a test tube, and Fig. 12 is a diagram showing the action mechanism of DCFH-DA. FIG. 13 is a process diagram of an intracellular reactive oxygen scavenging test using a human leukemia cell line K562, FIG. 14 is a measurement diagram of intracellular reactive oxygen scavenging activity by FCM, and FIG. FIG. 16 is an explanatory diagram of the effect of enhancing the expression of hMLHI gene, which is a DNA repair enzyme of kefir antioxidant, and FIG. 17 is the non-effect of kefir antioxidant. It is explanatory drawing of a periodical DNA synthesis (UDS) enhancement effect and its thermal stability.

(Example 1)
In this example, first, the water-soluble fraction of kefir was fractionated into several molecular weight ranges using a dialysis membrane, and the fraction having the main antioxidant activity was identified. Furthermore, the components in the fraction were separated by an anion exchange column, and components having antioxidant activity were identified by employing an on-line HPLC method using DPPH. This was further separated and purified. The purified product was measured for molecular weight and infrared absorption spectrum by fast atom bombardment mass spectrometry. The antioxidant activity of the purified product in a test tube was measured using DPPH reducing ability as an index.
First, materials and devices used in the present embodiment will be outlined.
(1) Kefir as a starting material is a kefir fermented liquid provided by Nippon Kefir Co., Ltd. (milk solid content 17%, inner milk fat content 2%, lactic acid 50% solution, pectin 0.6%, pH adjustment 3. 90) is stored refrigerated (4 ° C.).
(2) In order to examine the molecular weight of dialysis membrane and osmotic pressure measurement active ingredient, spectrum molecular weight 2,000 (Spectra / Por Membrane MWCO: 2,000), molecular weight cutoff 1,000 (Spectra) / Por Membrane MWCO: 1,000), molecular weight cut-off 500 (Spectra / Por Membrane MWCO: 500), molecular weight cut-off 100 (Spectra / Por Membrane MWCO: 100). For measuring the osmotic pressure of the water-soluble fraction in each molecular weight range, an osmotic pressure measuring device OM802-D manufactured by VOGEL was used.
(3) In vitro antioxidation ability test DPPH using DPPH was obtained from Wako Pure Chemical Industries, Ltd. DPPH was prepared on the day of the experiment using 80% methanol as a solvent and stored refrigerated in the dark. In addition, in the antioxidant activity test, the absorbance of DPPH was measured at a 492 nm absorbance on a 96-well plate (perforated plate) using a Tecan Spectra Reader to measure the absorbance of multiple samples under the same conditions. 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), an autosampler 13 (Waters 717 plus Autosampler), a photodiode array detector 14 ( Control of the system and data analysis were performed using a Waters 996 Photodiode Array Detector with Waters Chromatography Manager MILLENIUM32. A DPPH pump 15 (Waters 501 HPLC pump) was used as a DPPH solution supply pump. A TSKgel DEAE-5PW (φ7.5 mm × 75 mm) manufactured by Tosoh Corporation was used as the anion exchange column 16 as a separation column. In addition, a fraction collector 17 (SF-2120 SUPER FRACTION COLLECTOR) manufactured by ADVANTEC was used for automating the collection of the target substance.

(5) Gel filtration A column using BioGold / BIO-Gel P-2 resin (separated molecular weight range 100-1800) of BioRad (BIO-RAD) was prepared for gel filtration purification (φ10 mm × 450 mm). The column was used connected to an online HPLC-DPPH system by an FPLC-HPLC conversion adapter.
Furthermore, the molecular weight of the purified product was estimated from the gel filtration elution pattern. As standard products, vitamin B12 (MW1355.4), bromophenol blue (MW669.97), and phenol red (MW354.38) were obtained from Wako Pure Chemical Industries, Ltd. and used for experiments.

(6) Reversed phase HPLC analysis For analysis of the purified product after gel filtration, data analysis was performed by controlling a separation tube (Waters 2695 Separation Module) with a chromatography manager 18 of Waters. As a separation column, TSKgel ODS-80Ts (φ4.6 mm × 150 mm), which is a reverse phase column manufactured by Tosoh Corporation, was used.

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

Experimental Method and Results (1) Preparation and Kefir Water-Soluble Fraction and Molecular Weight Fraction 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 are shown in FIG.
As shown in FIG. 3, 275 g of kefir fermentation liquid (milk solid content 17%, inner milk fat content 2%, lactic acid 50% solution, pectin 0.6%, pH adjustment 3.90) was centrifuged (11000 g, 30 min) Then, milk fat content was precipitated to obtain 115 g of kefir supernatant (kefir water-soluble fraction sample A) (first step).
The obtained supernatant was transferred to a dialysis membrane having a fractional molecular weight of 1000, 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 liquid was concentrated by an evaporator. The concentrated solution was neutralized with a 4N aqueous sodium hydroxide solution, and the resulting mainly protein precipitate was removed by centrifugation (550 g, 20 min) (third step). The supernatant (separate) obtained here was transferred to a dialysis membrane with a molecular weight cut off of 100 for the purpose of desalting, and dialyzed with 1000 ml of ultrapure water for 24 hours to obtain an internal solution with a molecular weight of 100 or more (No. 1). 4 steps). A sample (B) having a molecular weight of 1000 or less was prepared by concentrating the obtained internal solution with an evaporator and adjusting the osmotic pressure to 0.300 osmol / kg.

In addition, dialysis was repeated twice while keeping the amount of the internal solution 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 internal solution containing the substance having a molecular weight of about 1000 or more thus prepared was transferred to a dialysis membrane having a molecular weight cut off of 2000, and dialyzed against 1000 ml of ultrapure water for 24 hours. A sample (C) having a molecular weight of 1000 to 2000 was prepared by concentrating the obtained external liquid with an evaporator and adjusting the osmotic pressure to 0.300 osmol / kg.
A sample (D) having a molecular weight of 2000 or more was prepared by concentrating the obtained internal solution with an evaporator and adjusting the osmotic pressure to 0.300 osmol / kg.

(2) Qualitative antioxidant capacity 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), the sample with molecular weight of 1000 or less (B), the sample with 1000 to 2000 (C), the sample with molecular weight of 2000 or more (D) were separated into three kefir water-soluble fractions. A qualitative antioxidant capacity test using DPPH was performed on four 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 aliquots into 1.5 ml Eppendorf tubes. Thereto, 100 ml of methanol was dispensed 50 ml at a time and mixed to precipitate the protein component. This is because the remaining protein in the kefir water-soluble fraction is precipitated with methanol.
The eppendorf tube with a total volume of 100 ml was centrifuged to completely precipitate the protein component (4500 g, 10 min). Next, 50 μl of each supernatant was taken from each sample and dispensed into a new Eppendorf tube. As a negative control, a sample in which 50 μl of 50% methanol was dispensed was prepared. 100 μl of the above-mentioned 0.1 mM DPPH solution was quickly dispensed to all 6 samples prepared in this way, and left at 37 ° C. in the dark. After leaving for 30 minutes, 50 μl of each sample was dispensed into a 96-well plate and the absorbance at 492 nm was measured. The results are shown in Table 1. Here, the strongest decrease in absorbance was observed in a sample having a molecular weight of 1000 or less, and the strongest antioxidant ability was confirmed.

(3) Identification of Antioxidant by Online HPLC-DPPH System As the eluent 11 of the online HPLC-DPPH system 10, a 0.1M ammonium acetate aqueous solution was used. As shown in FIG. 1, an eluent 11 having a single composition was supplied to the online HPLC-DPPH system 10 at a flow rate of 0.7 ml / min.
DPPH was dissolved in 80% methanol solvent on the day of the experiment and stocked at a concentration of 0.1 mM. DPPH solution 19 diluted 10-fold with 80% methanol solvent to 0.01 mM is mixed with the line of online HPLC-DPPH system 10 after separation from anion exchange column 16 by DPPH pump 15 at a flow rate of 0.7 ml / min. I let you.
From the day before the experiment, the anion exchange column 16 of the online HPLC-DPPH system 10 was equilibrated at a flow rate of 0.7 ml / min. On the day of the experiment, a DPPH solution 19 was prepared and the DPPH pump 15 was operated. Simultaneously with the pump operation, 200 ml of 80% methanol solvent was injected and the system was operated until the baseline of DPPH absorbance at 517 nm was stabilized. After confirming the stability of the baseline, 200 ml of 0.1 mM ascorbic acid aqueous solution prepared on the day was injected from the autosampler 13 in order to confirm that this system can detect water-soluble antioxidants. The results are shown in FIG. When ascorbic acid was monitored at a wavelength of 280 nm, a peak was detected from around 10 minutes, and a decrease in absorbance of DPPH at 517 nm was confirmed corresponding to the peak.
Thus, 200 ml of a kefir water-soluble fraction sample having a molecular weight of 1000 or less was injected to search for antioxidant substances. The results are shown in FIG. When the component was monitored at a wavelength of 280 nm, a plurality of peaks were observed. Among them, only the peak (Q) confirmed from around 38 minutes showed a corresponding decrease in DPPH absorbance, and was found to have antioxidant activity. Moreover, the result of having acquired the ultraviolet-visible spectrum of the target substance in the time of a peak (Q) with the photodiode array detector 14 which is an apparatus which can detect the light absorbency in various wavelengths simultaneously is shown in FIG. The target substance was found to be a substance having maximum absorption at 236.0 nm and 291.4 nm (λmax). Moreover, as shown in FIG. 7, among natural antioxidants, compounds having a spectrum pattern similar to those of flavonoids were found.
In this example, an on-line HPLC method using an on-line HPLC-DPPH system was adopted as an on-line 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, and an antioxidant substance in a state where the activity was maintained was separated (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 a large amount of target peaks was collected using a program of 60 minutes per cycle. In the case of large-scale fractionation, the fraction collector 17 was used, and the range from 35 minutes to 50 minutes was divided into five fractions for 3 minutes each and automatically separated. After 20 hours of continuous operation at each time, 200 ml of 80% methanol solvent and 200 ml of 0.2 M aqueous sodium hydroxide solution were injected in this order to wash the anion exchange column 16. Further, the anion exchange column was washed with 1M 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 an evaporator. Moreover, the concentration by an evaporator and the reconcentration operation by adding ultrapure water again were repeated, and ammonium acetate having volatility was removed as much as possible. Finally, the sample (B) was concentrated to about 5 ml, stored frozen, and lyophilized.

(4) Purification by gel filtration and molecular weight estimation The self-made gel filtration column was connected to the online HPLC-DPPH system 10 and equilibrated by supplying a 10% aqueous acetic acid solution at a flow rate of 0.5 ml / min from the day before the experiment.
The freeze-dried sample (B) was dissolved in as little 10% aqueous acetic acid solution as possible, and 100 ml was injected by an autosampler. From the results of the ultraviolet-visible spectrum in FIG. 6, the elution of the target substance was monitored at 290 nm in gel filtration. The results of purification and desalting by gel filtration are shown in FIG.
As shown in FIG. 8, the target peak eluting from around 110 minutes was collected and concentrated by an evaporator and re-concentrated by adding ultrapure water again, and acetic acid, which is a volatile acid, was completely removed. Finally, the sample was concentrated to about 5 ml, stored frozen, and lyophilized.
At the same time, various substances having a known molecular weight were subjected to gel filtration under the same conditions, and the size of the target substance on the molecular structure was estimated from the elution time. First, a purified product after gel filtration was prepared to 1 mg / ml, and 50 ml was supplied. In addition, vitamin B12 (MW1355.4), bromophenol blue (MW669.97), and phenol red (MW354.38) were tested at the same concentration and supply amount. When vitamin B12 was 32.2 minutes, phenol red was 157.9. The minute was eluted as a peak. Bromophenol blue could not be detected under these conditions. The results are shown in Table 2. On the other hand, the molecular weight of kefir antioxidant estimated from the data of vitamin B12 and phenol red was 650. Since the target substance can be concentrated in the external liquid obtained using the dialysis membrane having a fractional molecular weight of 500 and the result here, it was estimated that the molecular weight of the target substance was in the range of about 500 or less. However, since the concentration by the dialysis membrane can change depending on the shape and charge of the substance, the numerical 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 using a calibration curve created using a molecular weight marker as a molecular weight position label.

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

(6) Molecular weight measurement by fast atom bombardment mass spectrometry (FAB-MS / mass spectrum) We requested fast atom bombardment mass spectrometry (FAB-MS) to the Department of Medicinal Resource Control, Department of Pharmaceutical Sciences, Kyushu University Graduate School of Pharmaceutical Sciences The results of molecular weight measurement are shown in FIG. As a result, a maximum peak with a molecular weight of 607.8 m / z was detected. In addition, a strong peak was observed at 341.4 m / z. From this, it was estimated that the molecular weight of this substance was 607.8 and that the metastable degradation product would have a molecular weight of 341.4.

(7) Infrared absorption spectrum measurement FIG. 10 shows the result of infrared (IR) absorption spectrum (microscopic IR) of the antioxidant in kefir requested to Shimadzu General Analysis Center. To summarize the report, there is no spectrum similar to the requested kefir antioxidant in the Sadler library that stores infrared (IR) absorption spectra (microscopic IR) analysis, and the closest structure is ethylene-like. It may be a compound having a secondary bond of From the partial structure analysis, it was reported that there was a benzene ring with a substituent in the ortho position. The absorption at 1125,1029,991,783,744Cm ^-1 indicates out-of-plane deformation vibration of a benzene ring having adjacent trisubstituted group, absorption at 3300～2800Cm ^-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 substance and ascorbic acid which is a water-soluble antioxidant substance was examined. DPPH was dissolved in 80% methanol solvent on the day of the experiment and prepared at a concentration of 0.5 mM. The aqueous ascorbic acid solution was prepared at a concentration of 10 mM on the day of the experiment.
First, 10 mM ascorbic acid aqueous solution was diluted to prepare samples of 0, 50, 100, 150, 200, 250, 300, 350, 400, 600, 800, and 1000 μM. Also, 1 mg / ml kefir antioxidant was diluted to prepare samples of 0, 0.2, 0.4, 0.6, 0.8, 1.0 mg / ml. All samples were then dispensed in 50 ml aliquots into 1.5 ml Eppendorf tubes. 50 μl of 100% methanol was dispensed and mixed there. 100 μl of 0.5 mM DPPH solution was quickly dispensed to all samples in a total volume of 100 μl, and left at 37 ° C. for 30 minutes in the dark. Three 50 ml portions of each sample were dispensed into a 96-well plate, and the absorbance at 492 nm was measured. The results are shown in Table 3, Table 4, and FIG.

As shown in Tables 3 and 4 and FIG. 11, the 50% DPPH reduction concentration of ascorbic acid was 300 μM, and that of kefir antioxidant was 0.4 mg / ml. Assuming that the molecular weight of kefir is 608, 0.4 mg / ml corresponds to 660 μM, and kefir antioxidants have an antioxidant activity about half that of ascorbic acid at a weight concentration based on 50% DPPH reduction. I understood it.

(9) Discussion When the active substance was searched from the water-soluble fraction for the antioxidant action of fermented milk kefir, it was found that it was mainly present in the molecular weight range of 1000 or less. For the confirmation, the active ingredient was searched using the on-line HPLC-DPPH method for samples having molecular weights of 1000 and 2000, and no new ingredient was found. In addition, the same search was conducted for the antioxidant effect with respect to the molecular weight of 100 or less previously classified, but no new component having an antioxidant effect was found. It can be said that the active substance whose activity was confirmed by the DPPH-HPLC method of on-line HPLC method is negatively charged because it can be separated by an anion exchange column. The analysis result by reverse phase HPLC strongly suggested that this kefir antioxidant is a very hydrophilic substance (high polarity). Furthermore, even if a kefir water-soluble sample fractionated to a molecular weight of 1000 or less, which is a starting material for purification, is maintained under high-pressure steam sterilization, it is considered to be a heat-stable substance.
Further, by separating the on-line DPPH supply system in the above-described embodiment from the system and keeping the activity in an isolated state, kefir antioxidants can be produced. Furthermore, it became possible to produce a more purified antioxidant substance by a method such as gel filtration.
In this example, the acidic substance after the on-line HPLC-DPPH method was purified by gel filtration. However, other purification methods are also included in the method of the present invention.

Next, in order to know the molecular characteristics of kefir antioxidants, we first examined the molecular weight. The molecular weight was estimated to be 500 or less from the molecular weight fraction sample at the initial stage of the experiment and the experimental results by gel filtration. However, this numerical value of 500 or less is not accurate, and it is difficult to estimate the molecular weight accurately if the molecular weight estimation by gel filtration changes depending on the shape of the molecule. Next, when the molecular weight measurement by high-speed atom bombardment mass spectrometry (mass spectrum) was requested for the kefir antioxidant purified by gel filtration, a peak having a maximum molecular weight of 607.8 was detected. The molecular weight estimation by this mass spectrum was considered to be the most accurate molecular weight estimation, and it was estimated that the molecular weight of this substance was 607.8 and 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 substance, we requested the infrared absorption spectrum measurement. Results There is no spectrum similar to the requested kefir antioxidant in the Sadler library, and it is highly likely that the compound has novelty. The assignment corresponding to the spectrum suggested the existence of a benzene ring having multiple substituents and an ethylene-like double bond as the molecular skeleton. The existence of hydroxyl groups, which are characteristic of natural antioxidants, was also suggested. This result is consistent with the fact that DPPH is reduced mainly by accepting hydrogen from hydroxyl groups.
Therefore, at the present 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, is a substance that is stable in acidity and neutrality, and stable in high-temperature steam sterilization. You can say that.

(Example 2)
(10) Physiological activity of kefir antioxidant in cultured animal cells In general, even compounds that exhibit antioxidant activity in vitro do not necessarily have antioxidant activity in an appropriate amount of action in the cell. Therefore, the water-soluble kefir antioxidant produced by the above-described embodiment of the method of the present invention, that is, purified from the kefir water-soluble fraction and having a molecular weight of 100 to 700 m / z is actually an appropriate amount of action in the cell. Whether or not it has an antioxidant activity was examined below by examining the physiological activity in animal cultured cells.
Here, the intracellular oxidation state was measured using a fluorescent dye DCFH-DA (2,7-dichlorofluorescein diacetate).

FIG. 12 is an explanatory diagram of the mechanism of action of DCFH-DA, and FIG. 13 is a process diagram of an intracellular reactive oxygen scavenging test using the human leukemia-derived cell line K562.
As shown in FIG. 12, DCFH-DA, which is a membrane-permeable probe, moves from outside the cell to inside the cell, and is hydrolyzed by esterase in the cytoplasm to become DCFH. Since this DCFH loses membrane permeability, it stays in the cell, where it reacts with intracellular reactive oxygen species such as hydrogen peroxide and is oxidized to become DCF. Since this DCF emits fluorescence (λex = 502 nm, λem = 526 nm), the oxidation state in the cell can be measured by measuring the DCF fluorescence intensity of the cell administered with DCFH-DA.
In the case of adherent cells, DCFH-DA is treated while the cells are adhered, and the DCF fluorescence intensity is measured with a confocal laser microscope. However, it is difficult to determine the observation position and to quantify the experimental results. Therefore, in this study, human leukemia cell line K562, a non-adherent cell, was used to measure the size, DNA content, and distribution of membrane antigen expression by irradiating cells stained with fluorescent dyes with laser light. In this method, the intracellular oxidation state was measured by analyzing the fluorescence intensity distribution.

(11) Experimental material cell culture
RPMI 1640 medium (basic nutrient medium) was obtained from Nissui Pharmaceutical Co., Ltd.
As shown in FIG. 13, human leukemia-derived cells K562 were cultured in a 90 mm dish in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and the number of cells was diluted to 1/10 every time the saturation density was reached. Teenager.
Reagent fluorescent dye DCFH-DA Hanks solution was obtained from Nissui Pharmaceutical Co., Ltd.

(12) Equipment used (FCM)
The DCF fluorescence intensity was measured using EPICS XL / XL-MLL System II (also known as FACS) manufactured by Beckman Coulter.
(13) Results and Discussion DCF fluorescence intensity analysis by FCM (flow cytometry) FIG. 13 shows the procedure of the intracellular reactive oxygen elimination test using the human leukemia-derived cell line K562.
As shown in FIG. 13, human leukemia-derived cells K562 were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and subcultured by diluting the number of cells to 1/10 each time saturation was achieved. . A cell having a cell number of 1 × 10 ⁶ was taken into a test tube, treated with 5 μM DCFH-DA fluorescent dye for 7 minutes, and water-soluble at a molecular weight of 100 to 700 produced by the above-described example of the method of the present invention. Of kefir antioxidant was added and treated for 10 minutes. A control without kefir antioxidant treatment was used as a control. After 10 minutes, fluorescence detection by FCM was performed on the human leukemia cell line K562 treated with control and kefir antioxidant.
The result is shown in FIG. As can be seen from FIG. 14, the fluorescence intensity of the cells shifted to the left as compared with the control by the kefir antioxidant treatment. This indicates that the fluorescence intensity per cell was reduced overall, and it was speculated that the hydrogen peroxide in the cell was erased by the kefir antioxidant.
Assuming that the fluorescence intensity of the control is 100%, 50-60% of intracellular active oxygen was eliminated at concentrations of 1 and 10 μg / ml. Therefore, kefir antioxidants have strong intracellular reactive oxygen scavenging ability. I can say that.
These results indicate that oxidative stress causes various functional declines in animal cells and causes many diseases, so that kefir antioxidants are expected to be effective in the prevention and treatment of various diseases. It was.

(Example 3)
(14) Activity to promote sugar uptake of kefir-derived antioxidants into adipocytes Whether or not kefir antioxidants purified by the above-mentioned online HPLC-DPPH method promote sugar uptake into adipocytes is tested. The following experiment was conducted to confirm the effect on diabetes, particularly type II diabetes, caused by failure of uptake.

(Experimental example)
3T3 / L1 preadipocytes were cultured in 10% FBS-DMEM medium, and after reaching half saturation, 0.5 mM isobutyl metylxanthine, 0.25 μM dexamethasone, and 10 μg / ml insulin were added. The cells were added, cultured for 72 hours, cultured for 48 hours in a medium containing only 10 μg / ml insulin, and then differentiated by returning to a medium containing only 10% FBS-DMEM. Cells differentiated by culturing in a normal medium for 72 hours after completion of the differentiation treatment were used for a glucose uptake assay.
Differentiated cells were cultured in a medium containing kefir-derived antioxidants for 4 hours, followed by insulin stimulation for 20 minutes, then rinsed twice with 300 μl of HBS solution without glucose, and 500 μl of 1 μCi / ml 2-Deoxy per well. -D- [1-3H] glucose-containing HBS was added and treated for 10 minutes. After 10 minutes, the radioactive solution was aspirated off and rinsed quickly 3 times with 300 μl of ice-cold 0.9% NaCl solution. 500 μl of 0.5N caustic soda was added to lyse the cells and then neutralized with 100 μl of 2N acetic acid. 500 μl of each sample was placed in a 2 ml eppen, 800 μl of aquasol liquid scintillation cocktail (fluorescent dye-containing liquid for radiation measurement) was added, and 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 measurement diagram of intracellular active oxygen scavenging activity by FCM.
As shown in FIG. 15, the kefir antioxidant substance has a promoting effect of about 2.0 times at a concentration of 0.1 μg / ml and about 2.5 times at a concentration of 10 μg / ml, and promotes sugar uptake in a concentration-dependent manner. It became clear to do. From these results, it was expected that the development of a new antidiabetic therapeutic agent and a functional food containing the kefir-derived antioxidant as a main component was possible.
In addition, health foods using kefir antioxidants can be added with other nutrients and substances in order to increase the nutritional value and to expect further effects in order to make them tasty.

Next, the effect of kefir as a tissue repair agent having a DNA repairing action on the antioxidant was also searched.
Example 4
Effect on Gene Expression of Mismatch Repair Enzyme hMLH1 The mismatch repair gene maintains the stability of the genome by repairing errors that occur during DNA replication, and suppresses carcinogenesis by suppressing the accumulation of error bases. Furthermore, since a defect in which a mismatch repair gene does not function normally is observed to have a defect in the excision repair function, the mismatch repair gene is considered to be involved in the excision repair mechanism. However, the details are not clear. Since the kefir antioxidant substance was highly purified by the HPLC-DPPH method, the effect of enhancing the gene expression of the mismatch repair gene hMLH1 of this substance was examined. In addition, the thermal stability by heating at 100 ° C. for 10 minutes was also examined.

Methods Antioxidants were added to HMV-1 cells (melatoma cells / skin model cells) and cultured for 15 hours, and the mismatch repair gene hMLH1 was expressed by RT-PCR (messenger RNA was extracted from the cells, reverse transcriptase The phase-corrected DNA (cDNA) was synthesized by the method described above, and then the specific cDNA was amplified using the cDNA partial sequence fragment (primer) of the specific gene and analyzed using a method for examining the degree of gene expression.
The results and discussion results are shown in FIG. The numerical data is 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, it was speculated that the gene expression of the mismatch repair gene hMLH1 was subjected to redox control (a biological reaction in which gene expression is controlled by oxidative stress). The addition of this kefir antioxidant, which has about 1/3 the antioxidant property of vitamin C, has made it possible to develop new anticancer agents that repair DNA replication errors.

(Example 5)
Next, the promotion effect of kefir antioxidant and heat-treated kefir antioxidant on unscheduled DNA synthesis was examined.
When the target DNA is damaged, DNA synthesis for DNA repair independent of DNA replication is performed. This is called unscheduled DNA synthesis (UDS). It can be inferred that when the irregular DNA synthesis is promoted, the DNA repair activity of the cells is promoted.
When irradiated with ultraviolet rays (UVA), DNA bases are damaged and damaged bases are repaired by a base excision repair mechanism.
Method After irradiating 3400 J / m ^{2 of} UVA to HMV-1 cells spread in a 96-well plate, kefir antioxidant was added at a concentration of 0.4 μg / ml and cultured for 1 hour. Unscheduled DNA synthesis (UDS) was measured by bromodeoxyuridine (BrdU) incorporation. In order to minimize DNA replication caused by cell division, the cell cycle was stopped by treatment with 50 mM hydroxylurea (Wako), which has an effect of stopping the cell cycle causing cell division.
Results As shown in FIG. 17, UDS was clearly increased in the cells to which the kefir antioxidant was added. Moreover, even if it heated at 100 degreeC for 10 minute (s), since the UDS promotion effect of kefir antioxidant did not fall, it was thought that this kefir antioxidant was heat-stable.
Discussion From the above results, it was considered that the DNA repair was promoted by the activation of the cell mismatch repair mechanism and the like by the kefir antioxidant. Here, the kefir antioxidant used was presumed to be highly purified because it showed a single peak even when the solvent was changed in HPLC purification. It is considered that this material is heat-stable and advantageous in terms of application to functional foods.
Moreover, from these results, it became possible to develop new tissue repair agents and functional foods that promote the DNA repair activity of cells and have a DNA repair action. Kefir antioxidants can be widely used for the development of UV damage prevention methods for skin cells or skin photoaging prevention methods, and the prevention and treatment of cancer and genetic diseases caused by DNA mutations.

As mentioned above, although this invention was demonstrated by the Example, this invention is not limited to these Examples.
In the above examples, the kefir used kefir (kefir fermentation liquid) provided by Nippon Kefir Co., Ltd., but kefir using kefir bacteria manufactured and sold in Japan, those derived from other countries other than Russia, or You may use what was cultivated by yourself in search of kefir grains.
Moreover, the apparatus and chemical | medical agent which were used in the said Example are not limited to this, As long as it does not affect a result, it can change.

10: Online HPLC-DPPH system, 11: Eluent, 12: System controller, 13: Autosampler, 14: Photodiode array detector, 15: DPPH pump, 16: Anion exchange column, 17: Fraction collector, 18: Chromatography manager, 19: DPPH solution

Claims (8)

An antioxidant using kefir, comprising an antioxidant substance in a kefir water-soluble fraction. The antioxidant according to claim 1, wherein the antioxidant substance has a molecular weight of 100 to 700. The antioxidant according to claim 2, wherein the antioxidant substance has a hydroxyl group. The antioxidant according to any one of claims 2 and 3, wherein the antioxidant substance has an aromatic group. The antioxidant according to any one of claims 2 to 4, wherein the antioxidant substance is acidic or neutral and stable, and is stable to high-pressure steam sterilization. Was an antioxidant. The diabetes therapeutic agent which has an antioxidant of any one of Claims 1-5. A tissue repair agent comprising the antioxidant according to any one of claims 1 to 5 and having a DNA repair action. A health food comprising the antioxidant according to any one of claims 1 to 5.

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