JPH0899888A - Glutathione peroxidase activator - Google Patents

Abstract

PURPOSE: To obtain an activator for a glutathione peroxidase having effects on effective scavenging of hydrogen peroxide in the living body. CONSTITUTION: This glutathione peroxidase activator is obtained by using a culture supernatant of Bifidobacterium longum or Lactobacillus acidophilus as an active ingredient thereof. Since the glutathione peroxidase activator has effects on removal of hydrogen peroxide in the living body, preventing effects on the production and accumulation of peroxylipids considered as a causative substance for senescence, tumor, circulatory diseases, various hepatopathies, etc., can be expected. Furthermore, synergistic effects can be expected due to the activation of catalase and superoxide dismutase having similar effects, on the removal of the hydrogen peroxide.

Description

Detailed Description of the Invention

[0001]

The present invention relates to Lactobacillus acidophilus or Bifidobacterium longum .
The present invention relates to a glutathione peroxidase activator containing the culture supernatant of 1. as an active ingredient. Since the glutathione peroxidase activator of the present invention effectively eliminates hydrogen peroxide in vivo, it prevents aging due to the production and accumulation of lipid peroxide in vivo, tumors, cardiovascular diseases, various liver diseases, etc. Expected to be effective.

[0002]

2. Description of the Related Art In recent years, the production and accumulation of lipid peroxides in the living body is caused by human aging such as wrinkles, senile pigments, presbyopia, senile cataracts, gray hair, amnesia, dementia and aging, and arteriosclerosis. , Cardiomyopathy, cerebral thrombosis and other cardiovascular diseases have been reported to affect the onset of various liver diseases and tumors, for the purpose of treatment and prevention of these,
Methods for suppressing the production of lipid peroxide in vivo have been investigated.

Conventionally, as lipid peroxidation inhibitors in vivo, radical scavengers such as vitamin E, vitamin C, vitamin A and uric acid, and enzyme agents such as catalase, peroxidase, glutathione peroxidase and superoxide dismutase have been known. However, vitamin E, which is a radical scavenger, is only used as a substance that directly protects lipid peroxidation in vivo. This vitamin E has an excellent effect of suppressing lipid peroxide in the living body, but has a problem that it is fat-soluble and poorly soluble in water and its application range is limited.

On the other hand, an antioxidant intended to improve the preservability of cosmetics, pharmaceuticals, foods, feeds, etc. by utilizing the oxidation inhibiting effect of microbial cells, cell cultures, cell extracts, etc. Proposals have been made [JP-A-58-198584, JP-A-2-117349, JP-A-2-210490, JP-A-5-276912]. In vivo lipid peroxide inhibitors include Streptococcus lactis lister (S
treptococcus lactis lister) containing a lactic acid bacterium preparation utilizing the antioxidative effect of a digestion product of bacteria (JP-A-3-4768) and Lactobacillus rhamnosus (Lac
tobacillus rhamnosus), Lactobacillus casei subsp pseudoboehmite plantarum (Lactobacillus
casei subsp. pseudoplantarum) , Lactobacillus
Delbrucky Subspecies Bulgaricus (Lac
tobacillus delbrueckii subsp. bulgaricus), Lactobacillus Buhineri (Lactobacillus buchneri), Lactobacillus fermentum (Lactobacillus fermen
such as those bacteria in tum) and / or the cell hydrophilic solvent extract as an active ingredient [Japanese Patent 4-264034 discloses] have been proposed. However, when producing an in vivo lipid peroxide inhibitor using these lactic acid bacteria, it is necessary to separate and purify the active ingredient through many steps, which is a problem in terms of cost.

By the way, hydrogen peroxide is known as a substance involved in the peroxidation of lipids in the living body. This hydrogen peroxide does not directly oxidize lipids, but it serves as a substrate for peroxidase to oxidize organic substances, or it undergoes one-electron reduction to form hydroxyl radicals, and exerts a strong oxidizing action. Glutathione peroxidase and catalase are known as enzymes that decompose this hydrogen peroxide. It is considered that as the activity of glutathione peroxidase or catalase is increased in vivo, lipid peroxide is less likely to be generated in vivo. In particular, glutathione peroxidase has an extremely low Km value for hydrogen peroxide as compared to catalase, and thus can be said to be an enzyme capable of effectively eliminating hydrogen peroxide in vivo.

As a disease caused by a decrease in glutathione peroxidase activity, a disease in which glutathione reductase is innately deficient, glucose-6-phosphate dehydrogenase is innately deficient in red blood cells. Disease, γ
-It is known that glutathione synthetase and γ-glutamylcysteine synthetase in the glutamyl cycle are congenitally deficient, and hydrogen peroxide generated in erythrocytes cannot be sufficiently treated, resulting in hemolytic anemia. May present with symptoms.

[0007]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have conducted extensive studies on substances that activate glutathione peroxidase, which is an enzyme capable of effectively eliminating hydrogen peroxide in the living body. Lactobacillus Bifidobacterium longum ( Lifobabacterium longum ) or Lactobacillus acidophilus (Lactobacillus ac
The strong activating effect of glutathione peroxidase was found in the culture supernatant of ( idophilus) , and the present invention was completed. Accordingly, the present invention provides lactic acid bacteria of Bifidobacterium longum (Bifidobacteriumlongum) or Lactobacillus acidophilus (Lactobacillus acidophilu
It is an object to provide a glutathione peroxidase activator containing the culture supernatant of s) as an active ingredient.

[0008]

In the present invention, Bifidobacterium longum or Lactobacillus acidophilus is used as a lactic acid bacterium for obtaining a culture supernatant. As a strain producing a culture supernatant that activates glutathione peroxidase of the present invention, Bifidobacterium longum SBT2928 (FERM) obtained by separating from the feces of an infant according to a conventional method. P-10
657) and SBT 2933R (FERM P-8743) or Lactobacillus acidophilus SBT 2062 (FER
M P-10730) can be exemplified.

[0009] Bifidob
acterium longum ) SBT 2928 (FERMP-10657) and SBT 2
The mycological properties of 933R (FERM P-8743) are as follows.

A Morphological properties (1) Cell shape: Bacillus (2) Motility: None (3) Presence of spores: None (4) Gram stainability: Positive

Growth condition on B medium (1) Vigorous growth in Brix / River broth culture (37)
(2) Cultivated vigorously in GAM agar culture to form white, flat colonies about 2 mm in diameter (16 to 37 ° C under anaerobic conditions)
24 hours culture)

C Physiological Properties (1) Nitrate Reduction: Negative (2) Methyl Red Test: Negative (3) Indole Production: Negative (4) Hydrogen Sulfide Production: Negative (5) Starch Hydrolysis: Negative ( 6) Pigment formation: Negative (7) Urease: Negative (8) Oxidase: Negative (9) Catalase: Negative (10) Attitude toward oxygen: Obligatory anaerobic (11) Yeast extract-added litmus milk produces acid when cultured And curd. (12) Growth range: It does not grow at 15 ° C, but grows at 22 ° C to 45 ° C, and the optimum growth temperature is 35 ° C to 38 ° C. Also, pH 6.0
Grow at ~ 7.0. (13) Degradability of various carbohydrates 1. Arabinose + 2. Xylose + 3. Glucose + 4. Fructose + 5. Mannose + 6. Galactose + 7. Sucrose + 8. Maltose + 9. Lactose + 10. Trehalose-11. Sorbit-12. Mannit-13. Inosit-14. Starch-

In addition, it decomposes ribose, melibiose, raffinose and melezitose, but does not decompose cellobiose, glycogen and inulin.

From the above-mentioned mycological properties, Bergey's manual
of the systematic bacteriology, vol.2 (1986), SBT 2928 (FERM P-10657) and SBT 2933R (FER
M P-8743) is a bifidobacterium longum (Bifid
obacterium longum ).

Further, Lactobacillus acidophilus (L
actobacillus acidophilus) SBT 2062 (FERM P-10730)
The mycological properties of the are as follows.

A Morphological properties (1) Cell shape: Bacillus (2) Motility: None (3) Presence or absence of spores: None (4) Gram stainability: Positive

Growth condition on B medium (1) Vigorous growth in Brix / River broth culture (37)
(2) Cultivated vigorously in MRS agar culture, forming white or grayish white colonies with a diameter of around 2 mm (cultivated at 37 ° C for 24 to 48 hours under aerobic or anaerobic conditions).

C Physiological Properties (1) Nitrate Reduction: Negative (2) Methyl Red Test: Negative (3) Indole Formation: Negative (4) Hydrogen Sulfide Formation: Negative (5) Starch Hydrolysis: Positive ( 6) Dye formation: Negative (7) Urease: Negative (8) Oxidase: Positive (9) Catalase: Negative (10) Attitude toward oxygen: Positive (11) Optical rotation of lactic acid: DL (12) Growth range: 15 ° C (13) Decomposition of various carbohydrates 1. Arabinose-2. Xylose-3. Rhamnose-4. Ribose-5. Glucose + 6. Mannose + 7. Fructose + 8. Galactose + 9. Sucrose + 10. Maltose + 11. Cellobiose + 12. Lactose + 13. Trehalose + 14. Meribiose-15. Raffinose-16. Melettitose-17. Mannitol-18. Sorbitol-19. Esculin + 20. Salicin + 21. Amygdalin +

From the above-mentioned mycological properties, Bergey's manual
When identified according to of systematic bacteriology, vol.2 (1986), SBT 2062 (FERM P-10730) was in agreement with the mycological properties of Lactobacillus acidophilus .

The above Bifidobacterium longum (B
Ifidobacterium longum ) or Lactobacillus acidophilus (Lactobacillus acidophilus) , using a milk medium or a medium containing milk components as a culture medium, after culturing according to a method for culturing a normal lactic acid bacterium, an operation such as centrifugation is performed. Collect the culture supernatant. And in this invention, this culture supernatant is used as an active ingredient of a glutathione peroxidase activator. Further, this culture supernatant may be subjected to a treatment such as freeze-drying, spray-drying, and heat-drying, if necessary, to be used in the form of powder.

When the glutathione peroxidase activator of the present invention is administered, the active ingredient, Bifidobacterium longum, or Lactobacillus acidophilus (Lactobacillus ) is administered.
Acidophilus culture supernatant can be used as it is, but it can also be used by formulating powders, tablets, pills, capsules, granules and the like according to a conventional method. Furthermore, this glutathione peroxidase activator is various nutritional supplements and soft drinks, fruit juice drinks, fermented drinks, jellies,
It can also be used by blending with foods and drinks such as ice cream and confectionery such as gums and candies.

The dose of the glutathione peroxidase activator of the present invention is 50 g to 500 g per day for an adult in the case of a liquid culture supernatant, and 5 g to 50 g per day for an adult in the case of a dry powder culture supernatant. . It is added to food and drink so that this required amount can be secured once to several times a day,
It may be taken as a medicine.

Next, the present invention will be described in detail with reference to examples.

[Reference Example 1] Non-fat milk solid content containing 0.5% by weight of yeast extract
Bifidobacterium longum (Bifidobacterium longum ) pre-cultured in 4,000 ml of 12% by weight skim milk medium in the same medium
longum ) SBT 2928 (FERM P-10657) and Bifidobacterium longum SBT 2933
2.5% by weight of R (FERM P-8743) each and inoculate at 37 ℃
After culturing for a period of time, centrifugation was performed at 5,000 rpm for 20 minutes to collect the culture supernatant, which was freeze-dried to obtain 200 g of the culture supernatant powder.

[0024]

[Reference Example 2] Non-fat milk solid content containing 0.5% by weight of yeast extract
To 12% by weight of skim milk medium 4,000 ml, Lactobacillus acidophilus which was previously cultured in the same medium (Lactobacillus ac
idophilus) SBT 2062 (FERM P-10730) was inoculated at 5% by weight, cultured at 37 ° C for 16 hours, centrifuged at 5,000 rpm for 20 minutes to collect the culture supernatant, freeze-dried and then cultured. 200 g of fine powder was obtained.

[0025]

[Test Example 1] Animal experiments were carried out using the culture supernatant powders obtained in Reference Examples 1 and 2. Breeding of experimental animals Six 6-week-old SD male rats, each group consisting of 6 rats, were administered with the culture supernatant powder of Bifidobacterium longum ( Group A), Lactobacillus acidophilus <br / > The test feed shown in Table 1 was ingested for 5 weeks by dividing into 3 groups of Lactobacillus acidophilus (group B) and control group (group C). In addition, AI and vitamin mixture
N-76 composition (Journal of Nutrition, vol.107, pp.1340-1
348, 1977]. Also, during the 5-week breeding period, no significant difference was observed in the amount of increase in body weight and the intake of feed in each group.

[0026]

[Table 1]

Rats bred for 5 weeks were sacrificed under ether anesthesia, and blood was collected from the posterior aorta and subjected to each test. Further, the liver was removed and its weight was measured.

Preparation of red blood cells After heparinization of blood collected from rats, at 4 ° C.
Centrifugation was performed at 3,000 rpm for 10 minutes to separate plasma and blood. After collecting the plasma, the buffy coat on the blood clot was removed with a Pasteur pipette, and the rest was used as the red blood cell fraction. To this erythrocyte fraction was added 4 times the volume of physiological saline, and the mixture was sufficiently stirred so as not to destroy the erythrocyte membrane.
The upper layer was removed by centrifugation at 000 rpm for 10 minutes. This operation was repeated 3 times to wash the red blood cells, and then 4-fold volume of distilled water was added to the red blood cells to hemolyze the red blood cells to obtain hemolyzed blood.

Preparation of liver extract Exactly 1 g of the liver extracted from the rat was collected, 10 ml of 50 mM phosphate buffer (pH 7.0) was added for homogenization, and the extract was filtered and recovered. Further, 3 ml of the same buffer solution was added to the extraction residue for homogenization, and the extract solution was separated by filtration and collected. This operation was repeated once to recover the extract, and the same buffer was added to make the total volume 20 ml. The extract was diluted 10 times with the same buffer to obtain a liver extract.

Preparation of plasma lipoprotein fraction Blood collected from a rat was separated and collected, and then plasma was added with ethylenediaminetetraacetic acid disodium to a final concentration of 1 mg / ml and dissolved. Specific gravity of 500 μl of this plasma 1.
Overlay with 500 μl of 063 g / ml potassium bromide solution at 10 ° C.
Ultracentrifugation was performed at 105,000 × g for 18 hours, and the upper layer was collected. Then, add 0.9% sodium chloride and 0.1% to the recovered solution.
10 mM containing μM ethylenediaminetetraacetic acid / sodium
It was dialyzed against a phosphate buffer (pH 7.0) to obtain a plasma lipoprotein fraction. In addition, in this plasma lipoprotein fraction,
Includes very low density lipoproteins and low density lipoproteins.

Measurement of hemoglobin concentration of hemolyzed blood To 20 μl of the above hemolyzed blood, 5 ml of 6.7 mM phosphate buffer (pH 7.2) containing 35 mM sodium lauryl sulfate was added, mixed and allowed to stand at room temperature for 5 minutes. It was measured. The hemoglobin concentration (mg / 100 μl) of the hemolyzed blood was calculated from this absorbance and the absorbance of the equine erythrocyte standard solution (hemoglobin concentration 150 g / l) used as the standard.

Glutathione peroxidase activity of erythrocytes
The hemolysate 100μl of sexual measuring the, 50 mM phosphate buffer (pH 7.0),
1 mM ethylenediaminetetraacetic acid, 1 mM sodium azide,
Add 800 μl of reaction solution containing 0.2 mM reduced nicotinamide adenine dinucleotide phosphate, 1 mM reduced glutathione and 1 EU / ml oxidized glutathione reductase, mix and leave at room temperature for 5 minutes, then 1.5 mM cumin hydroperoxide solution 100 μl Was added and the change in absorbance at 340 nm over 5 minutes was recorded. Then, the glutathione peroxidase activity of erythrocytes was calculated by the following formula 1.

[0033]

[Formula 1]

Table 2 shows the average value of glutathione peroxidase activity of erythrocytes of each group.

[0035]

[Table 2]

Measurement of catalase activity of erythrocytes 1/15 M phosphate buffer (pH 7.0) containing 12.5 mM hydrogen peroxide 3
ml to a quartz cell for measuring absorbance, and 40 μl of the hemolyzed blood described above was added and mixed.
The time to decrease from 50 to 0.400 was measured. Then, the catalase activity of erythrocytes was calculated by the following equation 2.

[0037]

[Formula 2]

Table 3 shows the average value of the catalase activity of erythrocytes of each group.

[0039]

[Table 3]

Liver catalase activity measurement 1/15 M phosphate buffer (pH 7.0) containing 12.5 mM hydrogen peroxide 3
ml to a quartz cell for measuring absorbance, and 0.01 ml of the above liver extract was added and mixed.
The time to decrease from 0.450 to 0.400 was measured. And
Liver catalase activity was calculated by the following equation 3.

[0041]

(Equation 3)

Table 4 shows the average values of liver catalase activity in each group.

[0043]

[Table 4]

Erythrocyte superoxide dismuter
Xe activity measurement 1 ml of cooled ethanol and 0.6 ml of chloroform were added to 2 ml of the above-mentioned hemolyzed solution, and the mixture was shake-extracted for 15 minutes. The supernatant was collected. Distilled water was added to this supernatant to bring the total volume to 2 ml, and the supernatant was used for measurement of superoxide dismutase activity.

850 μl of 0.01 M phosphate buffer (pH 7.5) containing 0.013 mM riboflavin was added to a quartz cell for measuring absorbance, and 50 μl of the above supernatant and 2 mM dianisidine solution were added.
After adding 100 μl and mixing and irradiating with a 20 W fluorescent lamp of a spectrophotometer for 5 minutes, the absorbance at 460 nm was measured. Then, the superoxide dismutase activity of erythrocytes was calculated by the following formula 4 from this absorbance and the absorbance of the hemolyzed bovine erythrocyte (superoxide dismutase activity 3,000 U / ml) used as a standard.

[0046]

[Formula 4]

Table 5 shows the superoxide of red blood cells in each group.
The average value of dismutase activity is shown.

[0048]

[Table 5]

Measurement of lipid peroxide content of plasma lipoprotein fraction
Plasma lipoprotein fractions 100μl constant the addition of 12mM solution of ascorbic acid 10μl and 1.2mM ferrous heptahydrate solution 10μl sulfate, after mixing, was shaken 12 h at 37 ° C.. Furthermore, 280 μM butylhydroxytoluene solution 10
μl and 10 μl of 28 mM ethylenediaminetetraacetic acid / sodium solution were added and mixed, and then 100 μl of this reaction solution was added.
0.67% thiobarbituric acid 1 ml and 20% trichloroacetic acid
0.5 ml was added, and the mixture was kept at 100 ° C. for 20 minutes, cooled, centrifuged at 4,000 rpm for 5 minutes to collect the supernatant, and the absorbance at 532 nm was measured. This absorbance and 1,1,3,3-tetraeoxypropane (0-
The amount of lipid peroxide in the plasma lipoprotein fraction was calculated as malondialdehyde from the absorbance (16 μM). Table 6 shows the average values of lipid peroxides in the plasma lipoprotein fractions of each group.

[0050]

[Table 6]

From these test results, the culture supernatant of Bifidobacterium longum and Lactobacillus acillus (Lactobacillus ac
idophilus) culture supernatant was found to activate both glutathione peroxidase activity of erythrocytes, catalase activity of erythrocytes, catalase activity of liver and superoxide dismutase activity of erythrocytes. In addition, the amount of lipid peroxide in the plasma lipoprotein fraction was significantly reduced, indicating that Bifidobacterium longum (Bifidobacterium longum )
It was found that the culture supernatant of longum ) or the culture supernatant of Lactobacillus acidophilus has an inhibitory effect on lipid peroxide in vivo.

[0052]

Example 1 Granules containing the glutathione peroxidase activator of the present invention were produced by a conventional method.

Culture supernatant powder obtained in Reference Example 1 or 2
After adding lactose 883g and corn starch 221g to 380g,
The powder was mixed with a V-type mixer to obtain a mixed powder. To this mixed powder and 16 g of hydroxypropyl cellulose, 150 ml of sterilized purified water
After adding and stirring, the dissolved solution is kneaded together with a twin-screw kneader, and after granulation with an extrusion granulator, 60 ° C. with an aeration dryer,
The particles were sized by drying for 50 minutes to produce 1,480 g of a granular glutathione peroxidase activator.

[0054]

Example 2 A soft drink containing the glutathione peroxidase activator of the present invention was produced according to a conventional method.

5 kg of the culture supernatant powder obtained in Reference Example 1 or 2, 20 kg of apple frozen concentrated juice, 92 kg of granulated sugar, 2.5 kg of malic acid, 0.5 kg of citric acid, 3 kg of apple aroma,
After adding 900 kg of water to 1 kg of essence, mixing and dissolving,
1,000 kg soft drink containing glutathione peroxidase activator sterilized by plate sterilizer at 95 ℃ for 5 seconds
Was manufactured.

[0056]

EFFECTS OF THE INVENTION Bifidobacterium longum or Lactobacillus of the present invention
A glutathione peroxidase activator containing the culture supernatant of Lactobacillus acidophilus as an active ingredient activates glutathione peroxidase, which is an enzyme that can effectively eliminate hydrogen peroxide in vivo, and induces peroxidation in vivo. Since it has a lipid-suppressing action, it is expected to be effective in preventing aging, tumor, cardiovascular disease, various liver diseases, etc. due to the production and accumulation of lipid peroxide.

Further, the glutathione peroxidase activator of the present invention activates catalase and superoxide dismutase, which are considered to have a hydrogen peroxide suppressing effect. Therefore, the lipid peroxide due to the synergistic effect of these enzymes and glutathione peroxidase. Can also be expected to suppress the effect of

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Claims (1)

[Claims] 1. A glutathione peroxidase activator comprising a culture supernatant of a lactic acid bacterium Bifidobacterium longum or Lactobacillus acidophilus as an active ingredient.