JP2008050296A - Physiologically active composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a physiologically active composition having physiological activity such as superior antiallergic activity, antihypertensive activity, and immunopotentiation activity. <P>SOLUTION: The physiologically active composition having antiallergic activity, antihypertensive activity, and immunopotentiation activity has, as active ingredients, a γ-aminobutyric acid-containing powder which is obtained by adding water to asparagus, crushing the asparagus, adding a yeast extract and sodium glutamate to the obtained extracted solution to form a medium, and adding lactic acid bacteria to the resulting medium to culture the lactic acid bacteria, and freeze-drying the obtained culture medium and crushing the freeze-dried culture medium and a powder of Sparassis obtained by freeze-drying a commercially available fruit body of Sparassis and thereafter crushing the freeze-dried fruit body. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composition having physiological activities such as antiallergic activity, antihypertensive activity, and immunostimulatory activity comprising γ-aminobutyric acid and a mushroom-derived component.

  γ-Aminobutyric acid (hereinafter abbreviated as GABA) is an inhibitory neurotransmitter present in the brain and spinal cord of mammals, and it has been confirmed in many mammals that blood pressure is lowered by ingestion. . This blood pressure lowering effect is effective only for those who have developed hypertension, and does not lower blood pressure for healthy individuals and does not cause hypotension. GABA is also contained in plants such as germinated brown rice and tea leaves, fermented foods such as kimchi and takuan, in addition to the brain and spinal cord of mammals. However, there are no reports of side effects due to GABA intake.

  In addition, mushrooms have been used for food since ancient times, but recently the physiological activity of the components has been clarified. Mushroom fruit bodies are made of sugar, protein, dietary fiber, vitamins, minerals, etc., and it has been found that this mushroom component has a high effect on the prevention and treatment of diseases. Health food was born. It has been elucidated that some types of mushrooms are also effective in the prevention and treatment of lifestyle-related diseases that are closely related to diets such as hyperlipidemia, hypertension, and diabetes (for example, non-patent literature) 1).

On the other hand, asparagus is a vegetable that is rich in fiber and low in calories, and about 28,000 tons are harvested annually throughout the country, and imports from overseas are also popular. Prefectures with high yields in Japan include Nagano Prefecture, Hokkaido, Saga Prefecture, Fukushima Prefecture, Kagawa Prefecture, Nagasaki Prefecture, and Akita Prefecture. Until now, it has been known that asparagus contains many useful components such as aspartic acid, vitamin U (methylmethionine), rutin, folic acid, saponins, and glutathione in addition to dietary fiber. (For example, refer to Patent Document 1) and the like have been reported. It has also been reported that GABA is contained in a relatively large amount.
Japanese Patent Laid-Open No. 5-271405 “Mushroom physiological activity and function”, p. 16-25 (2005) CMC Publishing

  Foods that are said to have excellent antiallergic activity, antihypertensive activity, or immunostimulatory activity such as mushrooms have been used, but sufficient effects are not necessarily exhibited.

  The object of the present invention is to provide a physiologically active composition having physiological activities such as superior antiallergic activity, antihypertensive activity, and immunostimulatory activity.

  As a result of intensive studies to develop a composition having the above-mentioned desirable properties, the present inventors have found that GABA having various physiological activities including blood pressure lowering action, hyperlipidemia, hypertension, and diabetes. As a result, it was found that by taking in combination with mushrooms effective for the prevention and treatment of lifestyle-related diseases such as anti-allergic activity, antihypertensive activity or immunostimulatory activity, The invention has been completed.

  That is, the gist of the present invention is a physiologically active composition characterized by comprising γ-aminobutyric acid and a mushroom-derived component as active ingredients, and preferably further includes lactic acid bacteria. Also preferably, γ-aminobutyric acid is a culture containing γ-aminobutyric acid obtained by adding lactic acid bacteria to a medium containing an asparagus extract and then culturing, and preferably physiologically active Is the aforementioned bioactive composition having antiallergic activity, antihypertensive activity or immunostimulatory activity.

  According to the present invention, antiallergic activity, antihypertensive activity, and immunostimulatory activity can be obtained safely and effectively.

  Hereinafter, the present invention will be described in detail.

  GABA used in the present invention is a kind of amino acid contained in trace amounts in tea, vegetables, cereals and the like as ingredients of food. In the present invention, a pure GABA product or a composite containing GABA may be used. The GABA production method used in the present invention is not particularly limited, and includes a chemical synthesis method, a method of culturing a microorganism having GABA production ability, a fermentation method using glutamate decarboxylase and animals and plants containing the enzyme, and the like. Any method may be used, but since GABA can be obtained in large quantities and at low cost, a method using a culture obtained by adding and cultivating lactic acid bacteria capable of producing GABA to a medium containing food raw materials Is preferred.

  Examples of microorganisms having GABA-producing ability include lactic acid bacteria, and lactic acid bacteria are not particularly limited as long as they have glutamic acid decarboxylase-producing ability and can be added to foods without any problem. , Lactobacillus hilgardi, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus bilis, Lactobacillus bilis, Lactobacillus bilis, Lactobacillus bilis, Lactobacillus Bulgaricus (Lactobacillus bulgaricus), Streptococcus -Lactis (Streptococcus lactis), Lactobacillus acidophilus (Lactobacillus acidophilus), etc.

  The medium for culturing the above-mentioned microorganisms is not particularly limited, and is a GYP medium (D-glucose 1%, peptone 0.5%, yeast extract 1%, sodium acetate trihydrate 0.2), which is commonly used for lactic acid bacteria culture. %, Twin 80 0.05%, Magnesium sulfate heptahydrate 0.02%, Manganese sulfate tetrahydrate 10 ppm, Iron sulfate heptahydrate 10 ppm, Sodium chloride 10 ppm) FYP medium (GYP medium sugar is D- In addition to fructose), commercially available GAM medium (Nissui Pharmaceutical), MRS medium (Difco), etc. can also be used, yeast extract, meat extract, malt extract, fish extract, soybean degradation product, peptone In addition, it is also possible to use a culture medium in which polypeptone, potato leachate and the like are used alone or in a combination of two or more thereof in water.

  Moreover, a food raw material may be added to the above-described medium, or lactic acid bacteria may be cultured using the food raw material as a medium instead of the above-described medium. Such a food material may be any material as long as lactic acid bacteria can grow, and examples include vegetables, fruits, seeds, cereals, potatoes, beans, algae, mushrooms, seafood, meats, Examples include milk and eggs. These various food raw materials may be used as they are, or may be treated by combining known techniques such as washing, crushing, shredding, extraction, pressing, concentration, solid-liquid separation, heat sterilization, filter sterilization alone or in combination of two or more. You may use after. Moreover, you may perform operations, such as adjustment of a water content, pH adjustment, sugar addition, and addition of the component used for an above-described culture medium as needed. Among these, a method of inoculating a lactic acid bacterium having GABA producing ability into a vegetable extract is preferable, and a method of inoculating a lactic acid bacterium having GABA producing ability into an asparagus extract is more preferable.

  For example, a method of enriching GABA by adding glutamic acid and / or glutamic acid sodium salt to an extract obtained from asparagus and adding lactic acid bacteria capable of producing GABA can be mentioned. Any asparagus may be used for the enrichment treatment as long as it does not impair the effects of the present invention. Asparagus includes green asparagus grown in sunlight, white asparagus cultivated while shading with soil, etc., mini asparagus that harvests in a short period of time, and purple asparagus that is different from green asparagus. Among them, green asparagus rich in nutritional components such as amino acids and rutin is more preferable. The production area is not particularly limited, and it may be domestically produced or imported from overseas. The part to be used is not particularly limited, and young stems, above-ground stems, and storage roots can be used. Among these, young stems are preferable. The young stem may be the root portion or the tip portion, but the root portion cut when aligning the length of the asparagus as the product is most preferable because it can be obtained at low cost. Asparagus may be used as it is, or may be used after processing such as crushing, cutting, freeze-drying and dehydration.

  The culture obtained by adding GABA-producing ability lactic acid bacteria to the medium containing the asparagus extract obtained as described above, and culturing by a normal method in culturing lactic acid bacteria includes GABA, other It contains monosaccharides such as amino acids, polysaccharides, oligosaccharides, D-glucose, D-fructose, D-galactose, proteins, peptides, saponins, polyphenols, and the like. As amino acids, aspartic acid, glutamic acid, leucine, isoleucine, lysine, alanine, serine, proline and the like are relatively contained.

  The mushroom-derived component used in the present invention is a component obtained from a mushroom, and the type of mushroom is not particularly limited as long as it is edible. Specifically, mushrooms can be used as raw, dried, dried powder or solvent extract, and the form of use is not limited as long as it contains mushroom-derived components. The mushrooms may be natural or obtained by artificial cultivation.

  The types of mushrooms used in the present invention include, for example, nameko, mushroom, yamabushitake, mushroom, kishimeji, shiitake, enokitake, maitake, agaricus, oyster mushroom, octopus bamboo, jellyfish, agarisk, eringi, mushroom, mushroom, mushroom, At least one type of mushroom selected from the group consisting of Bunaharitake, Beechhimeji, Kuritatake, Kawaratake, Suehirotake, Mannentake, Honjimeji, Truffle, and Cordyceps can be used. Among these, Hanabiratake, Hatakeshimeji, Honshimeji, Shakashimeji are preferred.

  Examples of the solvent used to obtain the solvent extract from the above mushrooms include water, methanol, ethanol, isopropyl alcohol, butanol, glycerin, ethylene glycol, 1,3-butylene glycol, acetone, chloroform, ethyl acetate, hexane, ether, and the like. Can be used. In addition, after further diluting or concentrating the extract to further increase the effectiveness and safety, ultrafiltration or reverse osmosis membrane treatment or those treated with activated carbon or various resins, or those treatment solutions are diluted or A concentrated product can also be used.

  The amount of each component contained in the physiologically active composition of the present invention is not particularly limited and may be appropriately selected. When the mixing ratio is dry weight and the lactic acid bacteria culture is 1, the mushroom-derived component is It is 0.1-1000, More preferably, it is 0.5-800, More preferably, it is 1-400.

  In the physiologically active composition of the present invention, it is preferable to contain lactic acid bacteria in addition to the above-mentioned GABA and mushroom-derived components. As the lactic acid bacterium used here, the lactic acid bacterium described above as a microorganism capable of producing GABA is preferably used. The lactic acid bacteria are not limited to live bacteria, and dead bacteria may be used.

  The amount of lactic acid bacteria to be included in the physiologically active composition of the present invention is not particularly limited and may be appropriately selected, but is 0.005 to 20%, more preferably 0, based on the solid content weight of the physiologically active composition. 0.02 to 10%, more preferably 0.01 to 5%.

  The bioactive composition of the present invention can be processed into any food in combination with general food processing raw materials. For example, it can be processed into any food such as tablets, capsules or beverages, confectionery, bread, and does not impair the function of the bioactive composition according to the present invention. When combined with a general food processing raw material, the blending amount of the physiologically active composition of the present invention is not particularly limited, but is preferably blended so as to be 20 to 2000 mg per day. If it is less than this range, the effect may not be expected, and if it is more than this range, the increase in the effect can no longer be expected.

  The physiologically active composition of the present invention exhibits antiallergic activity, antihypertensive activity or immunostimulatory activity when ingested. The mechanism of action of the antiallergic activity, antihypertensive activity and immunostimulatory activity in the present invention is unknown, but antihypertensive activity, brain function improving effect by GABA, a sympathetic nerve inhibitory substance, antiallergic effect by mushroom-derived components, It seems that a synergistic effect is exhibited by the complementary action by the hypertensive action and the immunostimulatory action. Furthermore, when lactic acid bacteria are included, it is considered that allergic ameliorating action, antihypertensive action, and immunostimulatory action by lactic acid bacteria are added by complementary actions.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

In the examples, the contents of GABA and amino acids are values obtained by the following method. That is, it measured on the following conditions by the high performance liquid chromatography method (HPLC method), and detected using the fluorescence detector.
HPLC: Shimadzu Corporation LC-9A
Column: Shim-pack ISC-07 / S1504
Mobile phase: 0.2 N sodium citrate buffer (pH 2.2)
Flow rate: 0.3 ml / min Temperature: 55 ° C
Reaction solution: ortho-phthalaldehyde Detection wavelength: excitation wavelength 348 nm, fluorescence wavelength 450 nm

Production Example 1 [GABA powder containing lactic acid bacteria]
To 5 L of GYP medium containing 1.0% by mass of sodium glutamate, 100 ml of a pre-cultured lactic acid bacterium (FERM P-20710) cell suspension was added, and the culture was started at 30 ° C., 20 rpm, without aeration. . One day after the start of the culture, 2.5 L of 40 mass% sodium glutamate aqueous solution was added, and 2.5 L of 2 times concentration of GYP medium was added at the same time to make the total volume 10 L. The culture was continued under the same conditions while maintaining 8 ± 0.5. The culture broth after 40 hours had passed since the culture broth volume was 10 L was freeze-dried and then crushed to obtain 960 g of a light brown powder containing 55% GABA.

Production Example 2 [Asparagus-derived GABA powder]
10 L of water was added to 10 kg of green asparagus young stalks, crushed with a mixer, and squeezed using a nonwoven fabric to obtain an asparagus pressing solution. This was further subjected to suction filtration using filter paper (ADVANTEC Toyo No. 5C) using diatomaceous earth as a filter aid to obtain a light brown extract. 1% by mass of yeast extract (manufactured by Asahi Food and Healthcare) was added to the extract and sterilized at 121 ° C. for 15 minutes in an autoclave. After allowing to cool, 2% by mass of sodium glutamate was added and dissolved by stirring well. A total amount of lactic acid bacteria (FERM AP-20710) pre-cultured for 1 day in 150 ml of MRS medium (manufactured by Difco) was added thereto, and static culture was performed at 30 ° C. for 24 hours. Bacterial cells were collected from the obtained culture solution with a centrifuge, and the supernatant was filtered through a 0.45 μm membrane filter to remove the bacterial cells. The supernatant thus obtained was freeze-dried and then crushed to obtain 515 g of a brown powder containing 39% GABA.

Production Example 3 [GABA powder containing asparagus-derived lactic acid bacteria]
Culturing was carried out in exactly the same manner as in Production Example 2, and the obtained culture broth was lyophilized as it was and then crushed to obtain 550 g of a brown powder containing 36% GABA.

Production Example 4 [Hanabiratake powder]
2 kg of a commercially available fruit body (manufactured by Unitika) was freeze-dried and then crushed to obtain 213 g of a powder of Hanabira bamboo.

Example 1
Hanabira bamboo powder obtained in Production Example 4 and a commercially available GABA product (manufactured by Kyowa Hakko, trade name: GABA Kyowa) were mixed at a mass ratio of 1: 1 to prepare a composition.

Example 2
Hanabira bamboo powder obtained in Production Example 4 and GABA powder containing lactic acid bacteria obtained in Production Example 1 were mixed at a mass ratio of 1: 1 to prepare a composition.

Example 3
Hanabira bamboo powder obtained in Production Example 4 and asparagus-derived GABA powder obtained in Production Example 2 were mixed at a mass ratio of 1: 1 to produce a composition.

Example 4
The composition was prepared by mixing the Hanabira bamboo powder obtained in Production Example 4 and the GABA powder containing asparagus-derived lactic acid bacteria obtained in Production Example 3 at a mass ratio of 1: 1.

Example 5
Hanabira bamboo powder obtained in Production Example 4 and a commercially available GABA product (manufactured by Kyowa Hakko, trade name: GABA Kyowa) were mixed at a mass ratio of 399: 1 to prepare a composition.

Example 6
The composition was prepared by mixing Hanabira bamboo powder obtained in Production Example 4 and lactic acid bacteria-containing GABA powder obtained in Production Example 1 in a mass ratio of 399: 1.

Example 7
The composition was prepared by mixing the Hanabira bamboo powder obtained in Production Example 4 and the asparagus-derived GABA powder obtained in Production Example 2 in a mass ratio of 399: 1.

Example 8
Hanabira bamboo powder obtained in Production Example 4 and asparagus-derived lactic acid bacteria-containing GABA powder obtained in Production Example 3 were mixed at a mass ratio of 399: 1 to prepare a composition.

Comparative Examples 1-5
The Hanabira bamboo powder obtained in Production Example 4 was used as Comparative Example 1, the commercially available GABA product (manufactured by Kyowa Hakko, trade name: GABA Kyowa) was used as Comparative Example 2, and the GABA powder containing lactic acid bacteria obtained in Production Example 1 was compared. In Example 3, the asparagus-derived GABA powder obtained in Production Example 2 was used as Comparative Example 4, and the asparagus-derived lactic acid bacteria-containing GABA powder obtained in Production Example 3 was used as Comparative Example 5.

Test Example 1 [Antiallergic Activity Test]
As an index of antiallergic activity, blood IgE amount, interleukin 4 (IL-4) production amount and interferon γ (IFN-γ) production amount were used. 72 mice (BALB / c, 6 weeks old, female) were divided into 9 groups of 8 mice, and ovalbumin (OVA) was sensitized by intraperitoneal administration to day 0, and additional sensitization was performed to Day 5 by subcutaneous administration on the back. Local sensitization was performed every day for 7 days from day 18. From the day 25, foods containing the samples of Examples 1 to 4 and Comparative Examples 1 to 5 at an addition amount of 100 mg / ml were fed. Blood was collected on day 38, and blood OVA-specific IgE concentration was measured according to a conventional method using enzyme immunoassay (ELISA). As a control, only a standard feed to which no sample was added was given. After the measurement, the spleen was isolated and cultured for 7 days in a medium containing 100 μg / ml of OVA. The culture supernatant was collected, and IFN-γ and IL-4 were collected according to a conventional method using enzyme immunoassay (ELISA). Was measured.

  The obtained measurement results are shown in FIGS. It has been clarified that anti-allergic activity is increased by simultaneously taking Hanabiratake and GABA having antiallergic action. Moreover, it turned out that it is more effective in GABA derived from asparagus containing lactic acid bacteria.

Test Example 2 [Antihypertensive Activity Test]
Obtain 63 5-week-old SHR rats (male), divide them into 9 groups of 7 each, and after 2 weeks of acclimatization, measure blood pressure at the point of breeding until 16 weeks of age and develop hypertension I confirmed that Three times after 24, 48, and 72 hours (once a day), the samples of Examples 5 to 8 and Comparative Example 1 were added in an amount of 200 mg / ml, and the samples of Comparative Examples 2 to 5 were set to 0. Each feed was fed at an addition amount of 5 mg / ml. As a control, only a standard feed to which no sample was added was given. Blood pressure was measured after 0, 4, and 8 hours from the time of food intake at 24, 48, and 72 hours, respectively. The measurement was performed by the tail-cuff method (non-invasive automatic blood pressure measurement method).

  The measurement results are shown in FIGS. The blood pressure in each group is an average value. As is clear from these figures, the antihypertensive effect is low only with Hanabiratake, but the immediate effect increases by ingesting simultaneously with GABA and normal blood pressure also becomes normal by continuous administration. Approaching the. Further, as can be seen from the results of Example 8, it has been proved that such effects can be remarkably enhanced by simultaneously ingesting Hanabiratake and lactic acid bacteria-containing asparagus-derived GABA.

Test Example 3 [immunostimulatory activity test]
As an index of immunostimulatory activity, activation of natural killer cells (NK cells) was used as an index. 72 mice (BALB / c, female, 12 weeks old) divided into 9 groups of 8 mice, 1 week (once a day), samples of Examples 1-4 and Comparative Examples 1-5 were 200 mg / ml The food was added in an added amount. Only the standard feed with no sample added was given to the control. Thereafter, the spleen was removed and immediately transferred to an EMEM-10% FBS culture solution to disrupt a large cell mass. Thereafter, centrifugation was performed, and the precipitate (cells) was washed several times with an EMEM-10% FBS culture solution, and then adjusted to a concentration of 2 × 10 ⁷ cells / ml or less. Further, a lymphocyte separation liquid was added to this solution, and centrifugation was performed to collect lymphocytes. The obtained lymphocytes were washed several times with RPMI 1640-10% FBS culture solution, adjusted to a concentration of 5 × 10 ⁵ cells / ml, and cultured overnight in a 5% CO ₂ incubator.

YAC-1 cells were used as target cells, and BCECF-AM reagent was added to YAC-1 cells prepared at a concentration of 1 × 10 ⁹ cells / ml and labeled for 30 minutes. Thereafter, centrifugation was performed to collect the precipitate. The obtained precipitate was washed several times with 1640-10% FBS culture solution and adjusted to a concentration of 5 × 10 ⁴ cells / ml.

250 μl of the spleen lymphocyte cell solution (5 × 10 ⁵ cells / ml concentration) and 25 μl of BCECF-AM-labeled YAC-1 cell solution (5 × 10 ⁴ cells / ml) were mixed and allowed to stand at 37 ° C. for 2 hours. Centrifugation was then performed and the supernatant was collected (solution A). In addition, 275 μl of RPMI1640-10% FBS culture solution containing 0.25% Triton X-100 was added to the precipitate, and the supernatant was collected by centrifugation (Solution B). The solutions A and B were subjected to fluorescence measurement with an excitation wavelength of 500 nm and a fluorescence wavelength of 540 nm. Moreover, the fluorescence measurement was performed about the thing (blank A and B) which processed only the BCECF labeling YAC-1 cell similarly as a blank. Cytotoxic activity was calculated from the following equation.
Cytotoxic activity (%) = [{solution A ÷ (solution A + solution B)} − {blank A ÷ (blank A + blank B)}] × 100
Solution A: Sum of fluorescence due to spontaneous release and free fluorescence due to cell damage Blank A: Fluorescence due to spontaneous release

  The measurement results are shown in Table 1. As is clear from Table 1, it was revealed that Hanabiratake showed a very strong NK cell activity. It was also found that this effect is improved by simultaneous intake with GABA. Furthermore, the effect was found that the NK cell activity was dramatically improved by ingestion with lactic acid bacteria-containing asparagus-derived GABA.

It is a figure which shows the IgE density | concentration in the blood of the OVA sensitized mouse | mouth in the test example 1. FIG. It is a figure which shows the production amount of IL-4 in Test Example 1. It is a figure which shows the production amount of IFN-γ in Test Example 1. It is a figure which shows the time-dependent change of the blood pressure value of the hypertensive mouse | mouth in Experiment 2 (comparative example). It is a figure which shows the time-dependent change of the blood pressure value of the hypertensive mouse | mouth in Experiment 2 (Example).

Claims (6)

  A physiologically active composition comprising γ-aminobutyric acid and a mushroom-derived component as active ingredients.   The bioactive composition according to claim 1, further comprising lactic acid bacteria.   The bioactive composition according to claim 1 or 2, wherein the γ-aminobutyric acid is a culture containing γ-aminobutyric acid obtained by adding lactic acid bacteria to a medium containing an asparagus extract and then culturing.   The physiologically active composition according to any one of claims 1 to 3, wherein the physiological activity is antiallergic activity.   The physiologically active composition according to any one of claims 1 to 3, wherein the physiological activity is antihypertensive activity. The physiologically active composition according to any one of claims 1 to 3, wherein the physiological activity is immunostimulatory activity.