JP4085141B2 - Microbial culture obtained on medium containing green tea, black tea and mugwort extract - Google Patents

Description

As a raw material for cosmetics, beverages, foods, feeds, etc., or by adding to these products or raw materials, it suppresses melanin production and prevents quality deterioration due to their oxidation, and occurs in the living body of humans or animals using them. a microorganism culture having antioxidant properties from erasure or inhibit natural product generation of active oxygen and free radicals, obtained by culturing green tea, black tea and mugwort extract in a medium to Ingredients Relates to microbial cultures.

  Skin darkening due to sunburn or the like, spots, freckles, etc. are thought to be due to the production of melanin, an amorphous pigment of blackish brown color. Therefore, melanin production-suppressing substances are often added to cosmetics.

  For such purposes, melanin-inhibiting substances containing extracts from animals and plants such as kojic acid and its derivatives, ascorbic acid and its derivatives, hydroquinone and its glycosides are known.

  Recently, malignant neoplasms (such as cancer), lifestyle-related diseases such as cerebrovascular diseases and diabetes occupy the top causes of death. There are active oxygen and free radicals as major causes of lifestyle-related diseases. Antioxidants can be added to cosmetics, beverages, foods, feeds, etc. and used for the purpose of preventing quality deterioration and maintaining quality, and eliminate the generation of active oxygen and free radicals generated in the body. Some are used for the purpose of suppression.

  Oxidation of fats and oils contained in cosmetics, beverages, foods, feeds, etc. not only deteriorates the color and flavor of the product, but also causes browning, fading and a decrease in nutritional value. Therefore, antioxidants are often added to cosmetics, beverages, foods or feeds to maintain quality.

  For such purposes, as antioxidants used in the field of beverages, foods or feeds, for example, L-ascorbic acid (vitamin C, VC), erythorbic acid (isoascorbic acid), Natural antioxidants such as tocopherol (vitamin E, VE) and phenolic synthetic antioxidants such as dibutylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) are known.

  Conventionally, cosmetics (skin lotions) having a skin-whitening effect are composed of extracts from animals and plants such as kojic acid and derivatives thereof, ascorbic acid and derivatives thereof, hydroquinone and glycosides thereof, and the like. There were concerns about adverse effects on the human body and rough skin. Further, a sufficient whitening effect was not obtained.

On the other hand, skin darkening due to sunburn, stains, freckles, etc. are thought to be due to the production of melanin, a black-brown amorphous pigment.
This melanin is considered to be produced by oxidative polymerization in melanosomes in pigment cells called melanocytes existing in the basal layer of the epidermis by various melanin intermediate metabolites from tyrosine by tyrosinase.
And it is thought that the active oxygen produced | generated by the ultraviolet-ray etc. activates this reaction system, melanin is produced | generated excessively, and the pigmentation to the skin is accelerated | stimulated.

  The antioxidant substance L-ascorbic acid is often used because it is easily soluble in water and has an acidic and strong reducing action, but it is difficult to use for oils and fats, so acidity changes the taste of food. Limited to processed fruit products and pickles. In addition, tocopherol is also often used because it prevents unnecessary oxidation of fat and oil components, but it has to be used in combination with other antioxidants because its antioxidant ability is not so high.

  On the other hand, natural antioxidants have the disadvantage that their antioxidant ability is inferior to synthetic antioxidants. However, synthetic antioxidants such as dibutylhydroxytoluene (BHT) and butylhydroxyanisole, which have strong antioxidant ability, are regarded as suspicious food additives that induce carcinogenicity due to genetic damage, mutagenicity, chromosomal abnormalities, etc. Has come to be questioned.

As cosmetics, natural antioxidants and synthetic antioxidants are used to prevent the oxidation of fats and oils, but it is considered to be a cause of skin effects and rough skin, and safety is questioned. I came.
For these reasons, it has been desired to develop an antioxidant having strong antioxidant ability, high versatility, and high safety derived from natural products.

  As natural antioxidants, browning substances, amino acids, sugar alcohols, fructose, pericarp, wasabi and catechins have been reported. Moreover, it has been shown that an extract of lactic acid bacteria has an antioxidant effect as an antioxidant substance of a microorganism culture.

  These antioxidants have been added to cosmetics, beverages, foods, feeds, etc., and have been used for the purpose of preventing quality degradation and maintaining quality, but they eliminate active oxygen and free radicals generated in the living body. Or it became clear that it suppresses, and the search of the natural extract with strong antioxidative ability in a living body has come to be performed.

  There are four types of active oxygen generated by in-vivo oxidation: singlet oxygen, superoxide, hydroxyl radical, and hydrogen peroxide, and more than 10 types of free radicals such as alkyl hydroperoxide and hydroperoxyl radical. ing. Antioxidant ability can generally be determined by measuring radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH).

  It is thought that aging, lifestyle-related diseases and cancer are caused by in vivo oxidation as an effect on the living body. Aging is not limited to illness such as decreased motor function and visceral function, forgetfulness, eye cataracts, skin wrinkles, etc., and blood vessels tend to suffer from arteriosclerosis. As lifestyle-related diseases, arteriosclerosis and diabetes are considered to be particularly closely related, and other examples include liver dysfunction and rheumatoid arthritis.

  In addition, cancer is considered to be one of the causes of mutation when a substance constituting DNA is altered and damaged by oxidation and another cell is regenerated. In order to prevent such diseases, antioxidant substances that prevent or suppress in-vivo oxidation have attracted attention and have been researched and developed as functional foods, foods for specified health use and supplements.

  Antioxidants for such applications include carotenoids (beta-carotene, lycopene, fucoxanthin), phenolic compounds (flavonoids, anthocyanins, catechins), plant-derived sulfides and beta-diketones, and microbial cultures. Examples include DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid).

The object of the present invention is such a natural extract, which is highly safe, has a strong anti-oxidant ability, has an excellent melanin production inhibitory effect, has a high skin whitening effect, and provides various cosmetics, various beverages, foods, feeds, etc. , highly versatile, original taste and flavor products can maintain, also obtained by culturing green tea that is effective in vivo antioxidant properties, black tea and mugwort extract in a medium to Ingredients microorganisms To provide a culture.

Traditional lactic acid bacteria and yeast combined fermented milk kefir (Kefir), considered to be the birthplace of the Caucasus region, is produced by culturing kefir grains, the starter, in milk.
The present inventor has found that when this kefir grain, which is a natural product, is used in cosmetics, beverages, foods, feeds, etc., it has been found that it is safe for humans and animals. It was. Then, the kefir grains are cultured with green tea extract, and the culture solution obtained by removing the cells from the microorganism culture is found to have an excellent skin whitening effect, and the kefir grains are found to have an antioxidant ability. The culture solution was used as a cosmetic (see Patent Document 1).
JP 2004-149442 A

An application has already been filed for a microorganism isolated from kefir grains, a microorganism culture obtained by culturing this microorganism or a group of microorganisms containing the microorganism, and a product using them. (Japanese Patent Application No. 2004-175969, filing date: June 14, 2004).
In addition, we have already filed applications for microorganism cultures obtained by culturing microorganisms isolated from kefir grains and microorganisms containing these microorganisms or microorganisms containing them in a medium mainly composed of mugwort extract and products using them. Have finished. (Japanese Patent Application No. 2004-311983, filing date: October 27, 2004).

As a result of further extensive research, the microorganisms isolated from kefir grains, green tea, black tea, Yeo simulated extract is 6.67: 1.665: culturing in a medium prepared at a ratio of 1.665 By adding the microbial culture obtained by the above as raw materials for beverages, foods, cosmetics, feeds, etc., or by adding them to these products or raw materials, the melanin production inhibitory effect and the quality deterioration prevention due to their oxidation, or use them It has been found that the generation of active oxygen and free radicals generated in human or animal organisms is eliminated or suppressed. Then, a microorganism culture having antioxidant properties derived from the natural products, green tea, black tea, Yeo simulated extract is 6.67: 1.665: culturing in a medium prepared at a ratio of 1.665 It has been found that the above-mentioned object can be achieved by using the microorganism culture obtained by the above.

The present invention is an invention having the following configuration. That is,
(1) A new species of microorganism belonging to Lactobacillaceae having antioxidative activity isolated from kefir grains (Lactobacillus sp.) SIID1719-6b (original accession number FERM AP-20062, new accession) the number FERM ABP-10299)], green tea, black tea, Yeo simulated extract is 6.67: 1.665: in medium prepared at a ratio of 1.665 to a microorganism culture obtained by culturing The present invention relates to a microorganism culture having an inhibitory effect on melanin production.
(2) A new species of microorganism belonging to Lactobacillaceae isolated from Kefir grains (Lactobacillus sp.) SIID1719-6b (original accession number FERM AP-20062, new accession number FERM ABP-10299 the)], green tea, black tea, Yeo simulated extract is 6.67: 1.665: a microorganism culture obtained by culturing in a medium prepared at a ratio of 1.665, the antioxidant capacity The present invention relates to a microorganism culture.

  Here, the “melanin production inhibitory effect” refers to an effect of inhibiting the production of melanin pigment, which is a causative substance of spots and freckles generated in the skin by ultraviolet rays and aging.

Here, the “antioxidant ability” means an antioxidant ability that maintains the quality of the product, and eliminates active oxygen and free radicals generated in the living body when a beverage, food or feed is administered to humans or animals. It refers to the ability to inhibit in vivo oxidation. Moreover, the "microbial culture", the new species of microorganism separated from Kefir grains, green tea, black tea, Yeo simulated extract is 6.67: 1.665: medium prepared at a ratio of 1.665 Refers to a culture obtained by culturing in

According to the first aspect of the present invention, a novel microorganism belonging to Lactobacillaceae having antioxidative ability isolated from Kefir grains [Lactobacillus sp. SIID1719-6b (original accession number) FERM AP-20062, a new accession number FERM ABP-10299)], green tea, black tea, Yeo simulated extract is 6.67: 1.665: obtained by culturing in a medium prepared at a ratio of 1.665 This is an effective microbial culture that can provide a microbial culture having an inhibitory effect on melanin production. The culture is safe without toxicity and side effects and can be used as a cosmetic.

According to the invention described in claim 2, a new species of microorganism belonging to Lactobacillaceae isolated from Kefir grains (Lactobacillus sp.) SIID1719-6b (original accession number FERM AP-20062, new accession number FERM ABP-10299)], green tea, black tea, Yeo simulated extract is 6.67: 1.665: a microorganism culture obtained by culturing in a medium prepared at a ratio of 1.665 Thus, there is an effect of providing a microorganism culture having an antioxidant ability. The culture is safe without toxicity and side effects, and can be used in various fields as a raw material or additive for cosmetics, beverages, foods or feeds.

A preferred embodiment of the present invention will be described in detail.
Microorganisms isolated from kefir grains include strain Acetobacter cerevisiae SIID 1719-2b, strain Gluconobacter oxydans SIID 1719-3b, and Lactobacillaceae, a new species of the family Lactobacillaceae. The genus (Lactobaccilus sp.) SIID1719-6b, the strain Pichiamembrani faciens SIID1719-1y, the strain Saccharomyces cerevisiae SIID1719-4y, and the strain Pichia anomala SID1719-1 are present.

(. Lactobacillus sp) Lactobacillus from kefir grains are strains isolated new Lactobacillus Basi piracy (Lactobacillaceae) family of SIID1719-6b, green tea, black tea, Yeo simulated extract is 6.67: 1.665: 1 Whether the microbial culture obtained by culturing alone in a medium prepared at a ratio of .665 has a melanin production inhibitory effect and an antioxidant ability was evaluated as follows. As a result, Lactobacillus is a new species Lactobacillus Basi piracy (Lactobacillaceae) family SIID1719-6b green tea, black tea, Yeo simulated extract is 6.67 (Lactobacillus sp.): 1.665 : Preparation in a ratio of 1.665 The microorganism culture obtained by culturing in the prepared medium had a melanin production inhibitory effect and an antioxidant ability .
In addition, Lactobacillus sp. SIID1719-6b (original accession number FERM AP-20062, new accession number FERM ABP-10299) which is a new species of the Lactobacillaceae family has already been incorporated into the National Institute of Advanced Industrial Science and Technology. Have been deposited at the Patent Biological Depositary.

  As a representative example, cultivation of Lactobacillus sp. SIID1719-6b, a new species of the strain Lactobacillaceae, in a medium mainly composed of green tea, black tea, and mugwort extract, and melanin production inhibitory effect And the antioxidant evaluation test method was implemented as follows.

(Melanin production inhibitory effect test)
First, B16mouse melanoma cell was added to a cell culture well so as to be 2.0 × 10 ⁵ cells / well and cultured in RPMI1640 medium. After 24 hours, a culture (B3 solution) obtained by cultivating SIID1719-6b with green tea, black tea, mugwort extract at a ratio of 6.67: 1.665: 1.665 for 48 hours was used as B16mouse melanoma cell. The final concentration confirmed to have good growth was added to 1/50 of the total culture solution. As a positive control, kojic acid, which is a typical melanin inhibitor, was added to a final concentration of 100 μg / ml, and distilled water was added to a 10-fold dilution as a negative control, and cultured for 3 days. Next, melanin was quantified by dissolving melanin from the cells using 10% DMSO-1N NaOH and measuring the absorbance at 470 nm. The melanin production inhibitory effect of each sample was determined by comparing the amount of melanin (S) of cells cultured with 1 / 50B3 solution and kojic acid and the amount of melanin of cells obtained with distilled water (C) (next) (See Equation (1)). The result is shown as a melanin production inhibitory effect in the graph of FIG.
It was confirmed that the 1/50 B3 solution medium in which the final concentration of the whole culture solution of the B3 solution was 1/50 was more effective than the kojic acid known for the melanin production suppressing effect.

(Antioxidant ability test)
Antioxidant ability test was carried out as follows.
Lactobacillus sp. SIID1719-6b, a new strain of the family Lactobacillaceae, is prepared with a green tea, black tea, mugwort extract at a ratio of 6.67: 1.665: 1.665. After culturing for 48 hours, this culture (B3 solution) and only a medium without addition of SIID1719-6b (B3 medium) as a control were added to a final concentration of 0.2%, and the DPPH radical scavenging rate was confirmed. . The antioxidant capacity of the B3 solution was compared using distilled water as a blank.

First, 199.6 μl of 0.1 mM DPPH (dissolved in 50% ethanol) is added to each well of a 96-well microplate. Next, 0.4 μl of each of B3 solution, B3 medium, and blank was added to each well (final concentration 0.2%) to initiate radical scavenging reaction. The absorbance at λ = 517 nm immediately after addition (0 minutes) and after 30 minutes was measured with a microplate reader, and the radical scavenging rate was determined from the absorbance of the B3 solution, B3 medium and blank. The results are shown as antioxidant ability in the graph of FIG.
Since the antioxidant capacity of the B3 solution was approximately 3.2 times higher than that of the B3 medium, it was confirmed that a high antioxidant capacity was obtained by adding the strain.

It is a graph which shows the comparison of the melanin production inhibitory effect. It is a graph which shows the comparison of antioxidant ability.

Claims (2)

A new kind of microorganism belonging to Lactobacillaceae having antioxidative activity isolated from kefir grains (Lactobacillus sp.) SIID1719-6b (original accession number FERM AP-20062, new accession number FERM ABP the -10299)], green tea, black tea, Yeo simulated extract is 6.67: 1.665: a microorganism culture obtained by culturing in a medium prepared at a ratio of 1.665, melanogenesis A microorganism culture having an inhibitory effect. A new species of microorganism belonging to Lactobacillaceae isolated from Kefir grains (Lactobacillus sp.) SIID1719-6b (original accession number FERM AP-20062, new accession number FERM ABP-10299)] , green tea, black tea, Yeo simulated extract is 6.67: 1.665: 1.665 a microorganism culture obtained by culturing in a medium prepared at a ratio of, microbial culture having antioxidant properties object.