JPH10212297A - Active oxygen free radical eliminator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an active oxygen free radical eliminator useful for oils and fats and a food for utilizing oils and fats as an antioxidant, comprising a specific fraction in a soybean whey as an active ingredient. SOLUTION: This medicine comprises a water-soluble component as an active ingredient obtained by removing oils and fats, acid precipitable protein, a water- insoluble component (usually called bean-curd refuse), etc., from soybean. The medicine comprises especially a fraction having 94KDa molecular weight and/or a fraction having 30KDa molecular weight in a soybean whey and preferably synergistically increases active oxygen free radical eliminating action by further using gallic acid. The fraction having 94KDa molecular weight and the fraction having 30KDa molecular weight in a soybean whey are obtained by a known fraction means of a soybean whey such as gel filtration fraction or membrane fraction. The soybean whey component is usable as an antioxidation promoter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an active oxygen radical scavenger containing a specific fraction in soybean whey as an active ingredient.

[0002]

2. Description of the Related Art In a series of studies to date, the present inventors have studied the weak light emission phenomenon in the presence of non-enzymatic X (active oxygen species), Y (catalytic species) and Z (accepting species). Kinetic analysis using natural radical scavengers such as flavonoids revealed that this XYZ system is an active oxygen radical scavenging system ((1) Yoshiki Y, Okub
oK, Onuma S and Igarashi Y (1995): Chemiluminescenc
e of benzoic acid andcinnamic acid, and flavonoids
in the presence of aldehyde and hydrogenperoxide
or hydroxyl radical by Fenton reaction.Phytoche
mistry, 39, 225-229, (2) Yoshiki Y and Okubo K (199
5): Oxygen radical scavenging activity of DDMP
(2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-on
e) conjugated saponin in soybean seed.Biosci.Bio
tech.Biochem., 59, 1556-1557, (3) Yoshiki Y, Okub
o K and Igarashi K (1995): Chemiluminescence of an
thocyanins in the presence of aldehyde and tert-bu
tyl hydroperoxide. J. Biolumin. Chemilumin., 10,
335-338, (4) Yoshiki Y, Kinumi M, Kahara T and Okub
o K (1996): Chemiluminescence of soybean saponins i
n the presence of active oxygen species.Plant Sci
ence, 116, 125-129, (5) Yoshiki Y, KaharaT, Okubo
K, Igarashi K, Yotsuhashi K and Fenwick GR (1956):
Chemiluminescence mechanism of catechins in the
presence of active oxygen.J. Biolumin. Chemilumi
n., in press).

[0003]

DISCLOSURE OF THE INVENTION The present invention is based on the premise that XYZ-based Z is a component which synergistically promotes antioxidant properties, a typical component of which is DDMP saponin, and proteins and the like which fall within the category. As a result, preliminary results were obtained in which unidentified Z components were present in soybean foods such as miso, soy sauce, and bean sprouts. The aim was to get.

[0004]

Means for Solving the Problems The present inventors have searched the Y and Z components from soybean protein and the like in order to achieve the above-mentioned object. As a result, the activity was found in the whey category, the weak light emission characteristics, and the ESR spin trapping. The radical scavenging ability and the antioxidant activity by the method were clarified, and the present invention was completed.

[0005] That is, the present invention relates to a soybean whey having a molecular weight of 94
It is an active oxygen radical scavenger containing a kDa fraction and / or a 30 kDa molecular weight fraction as an active ingredient. It is preferred to include gallic acid.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The soybean whey of the present invention is a water-soluble component obtained by removing oils, fats, acid-precipitable proteins, water-insoluble components (usually called okara) and the like from soybean.

For example, soymilk (WSP: Water Soluble) obtained by extracting soybeans or defatted soybeans with water and removing the okara component
Protein) is adjusted to around the isoelectric point (about pH 4.5) to obtain the remaining soluble fraction from which the protein component has been removed (APP: Acid Precipitated Protein). If necessary, low molecular components such as salts can be removed by membrane separation such as dialysis. Alternatively, the soymilk is brought to a pH slightly higher than the isoelectric point (pH
The soluble fraction excluding the fraction that precipitates by adjusting to 5.8) is dialyzed, and then the pH is further adjusted to around the isoelectric point to further precipitate the fraction (mainly the 7S fraction). It can be obtained as a soluble fraction removed. Whey obtained by the former method is preferable because of its excellent effect of eliminating active oxygen radicals.

[0008] The fraction of 94 kDa molecular weight and 30 kDa molecular weight in soybean whey can be obtained by known fractionation of soybean whey by gel filtration fractionation, membrane fractionation (ultrafilter fractionation, reverse osmosis membrane fractionation, etc.). Can be obtained by using.

[0009] In the soy whey, both the 94 kDa fraction and the 30 kDa fraction have a reactive oxygen radical scavenging effect.
Heating reduces the effect of scavenging active oxygen radicals, so unheated ones are preferred.

[0010] While the fraction of 94 kDa in molecular weight has characteristics of Y and Z, the fraction of 30 kDa has only Z characteristics in the fraction. Both can commonly promote the properties of Z, that is, antioxidant properties. Conventionally, the Z system alone acting on oxidation promotion can be changed to an antioxidant system by combining it with the Y system or the like. Therefore, Z
These fractions having the following characteristics promote antioxidant properties by being combined with a Y type or the like.

The fraction of 94 kDa in molecular weight and / or the fraction of 30 kDa in soybean whey have an active oxygen radical scavenging effect by themselves, but when gallic acid is used in combination, the active oxygen radical scavenging effect increases synergistically. For example, in the case of soy whey to which gallic acid has been added (including a 94 kDa fraction and a 30 kDa fraction), autoxidation of linoleic acid can be significantly suppressed.

The active oxygen radical scavenger of the present invention can be used as an antioxidant in fats and oils, foods utilizing fats and oils, and the like.

[0013]

The embodiments of the present invention will be described below by way of examples. Example 1 (Preparation of experimental method sample) A 10-fold hydrolyzed water extract was obtained from low-temperature defatted soybeans, and water extraction (water extracted p
rotein, WEP), whey (whey), acid precipitated protein (acid pr
ecipitated protein, APP), crude 11S (11S), crude 7S (7
Each fraction of S) and whey (whey) was prepared. (Gel filtration) about 5 dispersed in 50 mM phosphate buffer (pH 7.0)
ml of the sample solution was injected onto a Sepharose 6B or 2B column (6.5 × 2.7 cm) which had been equilibrated with the same buffer, and eluted with the same buffer. The eluate was collected every 5 ml, and the absorbance at 280 nm was measured. (Electrophoretic analysis) Studier et al. (Studier FW (1973): Anal
ysis of bacteriophage T7 early RNAsand proteins on
slab / gels. J. Mol. Biol., 79, 237-248)
After electrophoresis on S-PAGA, 0.1% Coomassie Brilliant Blue R
Gel electrophoresis analysis was performed by staining at -250. (Measurement of antioxidant properties) Rodin iron method (Nakatani N and Kikuz
aki H (1987): A new antioxidative glucoside isolate
d from Organo (Origanum vulgare L.). Agric. Biol.
Chem., 51, 2727-2731), and the antioxidant property was determined as follows. That is, add 2 ml to 1 ml of sample solution (50 mM phosphate buffer).
Add 1.026 ml of 51% linoleic acid ethanol and add 5% with distilled water.
ml. After each of the linoleic acid-containing sample solutions was allowed to stand at 37 ° C., 0.1 ml thereof was collected, and 4.7 ml of 75% ethanol was added, followed by 0.1 ml of 30% ammonium thiocyanate. 0.02M ferrous prepared with 3.5% hydrochloric acid
The color was developed by adding chloride, and the absorbance at 500 nm was measured after 3 minutes. (Measurement of weak light emission) The measurement of weak light emission was carried out in the following manner according to the report 1). That is, the liquid sending pump (Waters M
odel 510) and an injector (Waters U6K) attached photon counting type CL detector (Tohoku Electronics CLD-11
0, 300-650 nm) using 50 mM phosphate buffer (pH 7.0).
The solution was sent at 1.0 ml / min, and active oxygen species (X) such as H2O2, catalytic species (Y) such as gallic acid, and CH3CHO
Photons generated by injecting the acceptor species (Z) were measured. (Measurement of SOD activity) Kono et al. (Hiramatsu M and Kohno M (1
987): Determination of superoxide dismutase activi
ty by electron spin resonance spectrometry using t
The SOD activity was measured as follows by the ESR spin trapping method (JEOL JES-RE1X) of he spin trap method. JEOL NEWS, 23, 6-9). That is, 9.2M DMPO (5,5-dimethyl-1-pyrroline-N-
oxide, LABOTEC CO., LTD) 20μl, sample aqueous solution 50μ
l, 2mM hypoxanthine solution (SIGMA CHEMICAL) 50
μl, 0.4 U / ml xanthine oxidase solution (Behringer
After mixing and stirring 50 μl (manufactured by Mannheim), the mixed solution was aspirated into an aqueous flat cell, incubated for 45 seconds, and then measured. (Results and Discussion) Comparison in XYZ Weak Light Emission System Comparison of each protein fraction in the XYZ weak light emission system was performed as follows. Prepare each protein solution of 40 mg / ml as Y, use 2% CH3CHO as Z, and use H as X to affect the weak luminescence intensity.
The effect of 2O2 concentration was investigated. As a result (FIG. 2 (a)), almost no light was observed for APP, but remarkable weak light emission depending on the H2O2 concentration was observed in the order of whey>whey〓> WEP. Z
Each fraction solution of 40 mg / ml was prepared, and 50 mM gallic acid was used as Y, and the effect of H2O2 concentration on the weak luminescence intensity was examined. As a result (FIG. 2. (a)), it was low in WEP, APP, 11S and 7S, but remarkable weak emission depending on the H2O2 concentration was observed in the order of whey> whey〓. However, the emission intensity was about half the value of Y. The effect of H2O2 concentration on the weak luminescence of the same fraction heat-treated at 40 mg / ml whey in the presence of 2% CH3CHO or 50 mM gallic acid was examined. As a result (FIG. 3), the weak luminescence of whey as Y in the presence of CH3CHO and Z in the presence of gallic acid increased linearly in proportion to the H2O2 concentration.
The weak light emission of ey showed only a slight linear increase as Y. From these results, wh in the presence of H2O2
It is presumed that the weak light-emitting component of ey is an enzyme system and exhibits a more linear H2O2 concentration dependency, so that it has Z characteristics. In addition, the presence of an enzymatic Y characteristic component is also presumed in whey. Each fraction solution having SOD activities of 5, 10, and 20 mg / ml was prepared, and the SOD activity was examined by the ESR spin trapping method. As a result (Fig. 4), almost SO was found in APP and 11S.
Although no D activity was observed, the SOD activity was 0.1 U / W in WEP and 7S.
The activity was as high as 0.3 U / mg for mg and whey〓, especially as high as 1.7 U / mg for whey. Although not shown in the figure, the addition of gallic acid enhanced these activities as well as the weak luminescence behavior. Effect of Y on the antioxidant properties of Whey The effects of whey, gallic acid as Y and CH3CHO as Z on the autoxidation of linoleic acid were investigated by the rhodane iron method. The result (Fig. 3), 1 mg / m
lwhey and its addition of CH3CHO promoted autoxidation of linoleic acid, whereas gallic acid and gallic acid added
In whey, autoxidation of linoleic acid was significantly suppressed. Therefore, it became clear that whey has the characteristic of Z in the XYZ system. Fractionation of whey by gel filtration with Sepharose 6B fractionated by Whey, and gel electrophoresis analysis by SDS-PAGE (Fig. 4). 2 fractions. As a result of examining the weak emission in the XYZ system for the obtained peaks 1 and 2 (FIG. 5), peak 1 showed the characteristics of Y and Z, whereas peak 2 showed the characteristics of only Z. Was. Therefore, attention was paid to peak 1, and as a result of re-gel filtration using Sephadex 4B and 2B having a large fractionation range (not 6), a single peak was detected in each gel filtration. Weak luminescence properties of the isolated preparation and comparison with the lipoxygenase preparation Gel filtration from Whey 10, 20, and 40 mg / ml solutions of the isolated preparation were prepared, and the weak The effect of H2O2 concentration on luminescence was investigated. As a result (FIG. 7), in each case, a concentration-dependent weak emission was observed, and the weak emission as Y in the presence of CH3CHO was about twice as much as the weak emission as Z in the presence of gallic acid. Was. However, although the effect of H2O2 concentration was not observed for Y, the dependence was clearly observed for Z. 94 kDa
Were detected and compared with a lipoxygenase sample from Merck.

As a result (FIG. 8), the main band of the Merck sample also corresponded to 94 kDa, and it was inferred that the main component of the isolated sample was lipoxygenase. Lipoxygenase is a typical oxidase that adds oxygen to a substrate, and is known to be an enzyme causing unpleasant odor in soybean food processing. However, this study has SOD activity,
It is strongly speculated that its oxidizing property is an enzyme that converts to antioxidant property in the presence of Y, and interest in further detailed research on the XYZ system is of interest.

[0015]

According to the present invention, a soybean whey component having an average molecular weight of 94 kDa and / or 30 kDa can be used as an antioxidant accelerator.

[Brief description of drawings]

FIG. 1 is a drawing showing the preparation of each fraction from low temperature defatted soybeans. "

FIG. 2 is a drawing showing the effect of H2O2 concentration on weak light emission intensity. "

FIG. 3 is a drawing showing the effect of H2O2 concentration on weak light emission of heat treatment in the presence of WEP. "

FIG. 4 is a drawing showing the results of examining SOD activity by the ESR spin trapping method. "

FIG. 5 is a drawing showing the results of examining weak light emission in the XYZ system for peaks 1 and 2. "

FIG. 6 is a drawing showing the results of regel filtration on Sephadex 4B and 2B having a large fractionation range. "

FIG. 7 is a drawing showing the results of examining the effect of H2O2 concentration on weak light emission in the presence of CH3CHO or gallic acid. "

FIG. 8 is a drawing showing the results of comparison with a lipoxygenase standard product of Merck.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Kazuyoshi Okubo 1-1, Atsumiyamachi, Tsutsudori Amiya-ku, Aoba-ku, Sendai City, Miyagi Prefecture Inside Tohoku University Faculty of Agriculture

Claims (2)

[Claims] 1. An active oxygen radical scavenger comprising, as an active ingredient, a 94 kDa molecular weight fraction and / or a 30 kDa molecular weight fraction in soy whey. 2. The active oxygen radical scavenger according to claim 1, comprising gallic acid.