JP6793692B2 - Uses of fermentation products in combination with thermath thermophilus and yeast - Google Patents

Description

The present invention belongs to the field of fermented raw materials used for skin care, and particularly relates to fermentation methods and applications of combinations of thermath thermophilus and yeast.

The application of biofermented raw materials in the field of skin care agents including cosmetics is increasing day by day, and enzyme cosmetics have become very popular with consumers in recent years. Fermentation is a pure biological process with no added chemical constituents. Fermentation makes it easier to elute bioactive substances, and extracting all active ingredients by fermentation produces new nutrients, improves the balance of nutrients, lowers the molecular weight of nutrients, and makes the activity stronger. Can be rapidly absorbed by the skin. Fermenting microorganisms themselves contain various enzymes and trace elements, which have replenishment and synergistic effects. As such, it meets consumer expectations for "natural, healthy, highly efficient" skin care products.

Yeast is currently the most applied strain in the field of fermented cosmetic raw materials, and yeast extract has a natural, safe and strong moisturizing effect. Yeast cells contain abundant natural moisturizing factors (NMFs), such as amino acids, peptides, and inorganic components such as sodium, potassium, and magnesium. Here, amino acids make up 40% of the natural moisturizing factor (NMF), and inorganic components such as sodium, potassium and magnesium make up 12% of the natural moisturizing factor (NMF). The major components of the yeast cell wall include yeast β- (1,3) / (1,6) -glucan and mannan. Yeast β- (1,3) / (1,6) -glucan contains a large amount of hydrophilic groups and can absorb water or retain water in the stratum corneum of the skin. The unique molecular structure of yeast mannan is capable of retaining water molecules and acts to moisturize for a long time. For example, the patent documents of Chinese patent application publication numbers CN105434319A, CN107184411A, and CN10655182A disclose the production of cosmetics using yeast fermentation products as raw materials for cosmetics.

Thermus Thermophilus is a thermophilic bacterium capable of producing thermostable superoxide dismutase (SOD) and B vitamins. Superoxide dismutase is, O ₂ ^- is oxidized to H ₂ O _2, the living body can be prevented from being damaged by oxidation, by prevention member that the infringement of free radicals on the skin, the efficacy of preventing aging Fulfill. The B vitamins can reduce the inflammatory response of the skin, prevent damage from sunlight, and promote cell regeneration. The B vitamins are also part of the molecular structure of various enzymes and coenzymes, which can promote amino acid metabolism to maintain skin health. The patent document of Chinese patent application No. 2017107329367 also discloses that the fermentation product of thermath thermophilus is adopted as a skin care ingredient, but the skin care ingredient is a recombinant human other than the fermentation product of thermath thermophilus. It also contains more collagen. However, the patent document only mentions the fermentation product of Thermath thermophilus and does not disclose how it ferments. Further, for example, a Chinese patent application, Japanese Patent Application No. 20070031387 also discloses a sunscreen composition using a bioenzyme, and the composition contains a bioenzyme of Thermath thermophilus, and a proportion thereof is provided. .. However, all common thermophilas are produced by fermentation and do not disclose the activity of the enzyme or the amount of the enzyme produced, and also the stability when such an enzyme is combined with other components. It is one of the important factors. Since sunscreen products contain enzymes, it is uncertain whether the enzymes are affected by UV light and inactivated, and whether they have enzyme activity.

In the prior art, each single bacterial species is fermented and then two types of fermented liquids are mixed. The actual effect is often unfavorable because the activity of a large amount of flora and enzymes is lost due to the dissolution of different flora and enzymes together to cause a neutralization reaction. Therefore, it is expected that conventional fermentation methods and products must be improved to obtain more bioactive substances and reduce the interaction and neutralizing effects of mixing single fermentation products.

In order to solve the above problems, the present invention improves the physiological activity of the thermophilus fermentation product by providing a fermentation step of a combination of thermath thermophilus and yeast, and the effect of applying the thermophilus fermentation product. Achieving the purpose of enriching, especially the mild anti-inflammatory, moisturizing and skin barrier repair of thermath thermophilus fermentation products, as well as improving the effect in yellowing elimination, pursuit of "natural, healthy, high efficiency" of cosmetics Meet.

If the inventors of the present invention ferment a plurality of types of bacteria in a controllable manner by symbiotic fermentation of a plurality of bacteria, not only can a larger number of beneficial enzymes be fermented, but also the amount of each type of enzyme produced can be reduced. It was found that the amount of fermentation of a general single bacterial species can be multiplied by several times. The more enzymes there are, the stronger the catalytic health management function for the human body. Yeast extracts and thermath thermophilus fermentation products are commercially available on the market, but both are added to cosmetics as a single product, and fermentation by a combination of yeast and thermophilus is pioneered and used in cosmetics. That is a new challenge.

In addition, the inventors of the present invention can co-culture yeast and thermophilic bacteria, and according to a special culture step, can exert an activation-inducing action on the culture of yeast against thermophilic bacteria. It was found that the activity of the product obtained by culturing thermophilic bacteria alone was significantly improved, while the activity of the culture product of the yeast itself was not significantly affected. Therefore, the release of the physiologically active substance of the thermophilic bacterium is enhanced, or the activity of the physiologically active substance is enhanced.

Although some beneficial bioactive substances can be produced by culturing thermophilic bacteria alone, the amount of such bioactive substances is very small and the activity is not high. When co-culturing yeast and thermophilic bacteria, we have found a product that can significantly improve the bioactive substances of thermophilic bacteria according to a special culture method, and in particular, the activity of some bioactive substances is remarkable. Found to be strengthened.

The present invention also provides a method for culturing a fermentation product of a thermophilic bacterium.
Step (1) of fermenting Saccharomyces cerevisiae once or multiple times under medium temperature and acidic conditions to grow yeast, and 0 days or 0 days after the temperature is raised after the yeast has reached the late logarithmic growth phase. One day later, the step (2) of inoculating Saccharomyces cerevisiae and performing a second fermentation under alkaline conditions to obtain a fermentation product is included.

In some preferred forms, the yeast is brought into a logarithmic growth phase by performing at least two growth cultures, or at least three, or at least four, five, six growth cultures.

In some preferred forms, thermus thermophilus is also subjected to one or more growth cultures prior to mixed fermentation.

In some preferred embodiments, the method performs sterilization, decolorization, deodorization, and filtration to remove impurities from the fermentation broth of step (2) to obtain a fermentation product from a combination of thermath thermophilus and yeast. (3) is further included.

In some preferred forms, the strain of Saccharomyces cerevisiae is CICC1596, purchased from the China Industrial Microbial Species Conservation and Management Center, and the preservation number is CICC1596.

In some preferred embodiments, the Thermus Thermophilus HB27 used in the present invention is purchased from the American Type Culture Collection (ATCC) and has a storage number of BAA-163.

In some forms, yeast is cold cultured and the temperature is between 20 ° C and 30 ° C.

In some forms, yeast is cultured in an acidic region and has a pH value of 4.0-5.0.

In some forms, yeast begins to heat up 0 or 1 day after entering the late logarithmic growth phase. Preferably, the temperature is raised to 55 ° C. to 75 ° C., or 60 ° C. to 65 ° C. Alternatively, the temperature is raised to 60 ° C., 65 ° C., 70 ° C., 58 ° C., and 61 ° C. The time for raising the temperature from low temperature to high temperature generally takes several minutes, and the temperature is raised by, for example, an electric heating method.

In some forms, thermophilic bacteria are started inoculated 0 or 1 day after the temperature is raised, and at the same time or 1 day after inoculation, the pH value of the medium is adjusted to alkaline, and the pH value is 7. It is 0 to 8.5.

In some embodiments, the condition of the secondary fermentation, aeration is maintained ^{5m 3 / h / 50L~10m 3 /} h / 50L, 100rpm~200rpm, fermentation days is 1-3 days.

In some forms, the method for expanding the culture of yeast is to take one loop (the amount of bacterial solution that can be collected at one time using the inoculation loop) of Saccharomyces cerevisiae CICC1596 on a test tube slope on a clean bench and take 50 ml. Place in a 250 ml Erlenmeyer flask containing a culture solution (pH 4.8 ± 0.2), incubate at 200 rpm (200 rotations per minute) at 30 ° C. for about 10 hours, and bring the cells into a logarithmic growth phase (1). ) And the cells in the logarithmic growth phase are inoculated into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and the inoculation amount is 10% (volume percentage, 200 ml step (1)). In the step (2) of culturing for about 10 hours at 200 rpm and 30 ° C., and 30 L of the fermented solution (pH 4.8 ± 0) of the 3 L bacterial species cultivated in the step (2). Inoculate into a 50L tank of a first-class species containing .2), maintain the aeration rate at 5 m ³ / h / 50 L, and incubate for about 10 hours at 200 rpm and 30 ° C. Step (3) and 300 L of 30 L bacterial solution This includes the step (4) of transferring to a 500 L tank containing the secondary seed, maintaining the aeration rate at 5 m ³ / h / 50 L, and culturing at 200 rpm and 30 ° C. for about 10 hours.

Further, the present invention provides a method including the following steps.
(Step 1)
Propagation culture is performed on Saccharomyces cerevisiae and Saccharomyces cerevisiae, respectively.
Here, the method of breeding Saccharomyces cerevisiae is as follows.
(1) Take one loop of Saccharomyces cerevisiae CICC1596 on the slope of a test tube on a clean bench, place it in a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and place it at 200 rpm (200 per minute). Rotation), incubate at 30 ° C. for about 10 hours to bring the cells into the logarithmic growth phase;
(2) Inoculate the cells in the logarithmic growth phase into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and inoculate the inoculation amount at 10% (volume percentage, 200 ml step (1)). (Yeast culture solution in the logarithmic growth phase) and inoculate at 200 rpm and 30 ° C. for about 10 hours;
(3) 3 L of the bacterial species cultured in step (2) is inoculated into a 50 L tank of a first-class species containing 30 L of fermented liquid (pH 4.8 ± 0.2), and the aeration volume is 5 m ³ / h / 50 L. Incubate at 200 rpm, 30 ° C. for about 10 hours;
(4) Transfer 30 L of the bacterial solution to a 500 L tank of a secondary species containing 300 L, maintain the aeration rate at 5 m ³ / h / 50 L, and incubate at 200 rpm and 30 ° C. for about 10 hours.
The method for growing thermath thermophilus is as follows.
Take 2 ml of a frozen solution of Thermus Thermophilus HB27 on a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and maintain the aeration volume at 5 m ³ / h / 50 L. Then, the cells are cultured at 150 rpm and 65 ° C. for about 12 hours, and after the cells have reached the logarithmic growth phase, 30 L of the bacterial solution is inoculated into a 500 L tank of the secondary species containing 300 L of the culture solution.
(Step 2)
The primary fermentation method for yeast is as follows.
The yeast culture solution (after step (4)) in the 300 L secondary seed tank was transferred to a 5000 L fermentation tank containing 2400 L culture solution (pH 4.8 ± 0.2), and the aeration volume was 5 m ^3. Maintain at / h / 50 L and incubate at 200 rpm and 30 ° C. for about 14 hours.
(Step 3)
As the secondary fermentation, the temperature of the culture solution in the primary fermentation (step 2) was raised from 30 ° C. to 65 ° C., and after the temperature was raised to 65 ° C. (0 days), 300 L of thermath thermophilus was immediately grown. Transfer the liquid to this fermentation tank, maintain the aeration volume at 5 m ³ / h / 50 L, stir at 150 rpm, adjust the pH value to 7.8 ± 0.2 with alkaline liquid, and adjust the pH value to 7.8 ± 0.2 at 150 rpm and 65 ° C for about 18 h. Incubate.

In some preferred embodiments, the components of the culture are 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, with 20 L of the culture. For 1 g of anhydrous iron chloride and 5 g of sodium chloride, the pH value is adjusted and controlled with an acid or alkali, and water is replenished to 20 L. Different amounts of culture medium are increased or decreased according to the formulation example.

Further, the method for symbiotic fermentation of thermophilic bacteria and yeast is used in the production of a reagent for extinguishing inflammation of the skin or moisturizing the skin or eliminating yellowing of the skin of fermentation products by symbiotic fermentation culture of thermophilic bacteria and yeast. First, at low temperature and acidity, yeast is subjected to one or more liquid growth fermentations to bring it into a logarithmic growth phase, and then 0 days after changing the temperature of the fermentation broth from low temperature to high temperature. Alternatively, one day later, the thermophilus is added to perform secondary fermentation, and the fermentation conditions are set to alkaline conditions.

Preferably, the fermentation method is any of the fermentation methods described above.

In some preferred forms, the fermentation method with a combination of Thermus thermophilus and yeast is a step (1) of fermenting Saccharomyces cerevisiae once or multiple times under low temperature, acidic conditions to grow yeast and yeast. Includes the step (2) of raising the temperature, inoculating Saccharomyces cerevisiae, and performing a second fermentation under alkaline conditions to obtain a fermentation product after the late logarithmic growth phase.

In some preferred forms, the yeast is brought into a logarithmic growth phase by performing growth culture on the yeast at least twice in step (1), and in step (2), the thermophilus is added to the yeast culture medium. Prior to inoculation, the thermath thermophilus is subjected to one or more growth cultures.

In some preferred embodiments, the Saccharomyces cerevisiae strain is CICC1596, purchased from the China Industrial Microbial Species Conservation and Management Center, the preservation number is CICC1596, and the Thermophilus HB27 used is American Type Culture. -Purchased from the Collection (ATCC), the storage number is BAA-163.

In some preferred forms, yeast is subjected to low temperature culture, the temperature is 20 ° C. to 30 ° C., yeast is subjected to acidic culture, the pH value is 4.0 to 5.0, and yeast. Is started to raise the temperature 0 to 1 day after the late logarithmic growth phase, and the temperature is raised to 55 ° C. to 70 ° C.

In some preferred embodiments, the thermophilic bacterium is started inoculated 0 days after the temperature is raised, and the pH value of the culture is adjusted to alkaline at the same time or 1 day after the inoculation, and the pH value is 7.00 to 7.0. It is 8.5.

In some preferred forms, the anti-inflammatory has the effect of reducing an inflammatory factor, which comprises IL-1α, IL-6, IL-8 or TNF-α.

In some preferred forms, the reagent is a skin care agent, which can be present in any form, solution, aqueous solution, suspension, pack, emulsion, cream, cream, gel, dry powder, wet powder. , It may be one of the spray agents.

Furthermore, the method for co-fermenting thermophilic bacteria and yeast, which is used in the production of anti-inflammatory or skin moisturizing reagents for fermentation products by co-fermentation culture of thermophilic bacteria and yeast, includes the following.
(Step 1)
Propagation culture is performed on Saccharomyces cerevisiae and Saccharomyces cerevisiae, respectively.
Here, the method of breeding Saccharomyces cerevisiae is as follows.
(1) Take one loop of Saccharomyces cerevisiae CICC1596 on the slope of a test tube on a clean bench, inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and inoculate it at 200 rpm (every minute). 200 rpm), incubate at 30 ° C. for about 10 hours to bring the cells into the logarithmic growth phase;
(2) Inoculate the cells in the logarithmic growth phase into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and inoculate the inoculation amount at 10% (volume percentage, 200 ml step (1)). (Yeast culture solution in the logarithmic growth phase) and inoculate at 200 rpm and 30 ° C. for about 10 hours;
(3) 3 L of the bacterial species cultured in step (2) is inoculated into a 50 L tank of a first-class species containing 30 L of fermented liquid (pH 4.8 ± 0.2), and the aeration volume is 5 m ³ / h / 50 L. Incubate at 200 rpm, 30 ° C. for about 10 hours;
(4) Transfer 30 L of the bacterial solution to a 500 L tank of a secondary species containing 300 L, maintain the aeration rate at 5 m ³ / h / 50 L, and incubate at 200 rpm and 30 ° C. for about 10 hours.
The method for growing thermath thermophilus is as follows. Take 2 ml of a frozen solution of Thermus Thermophilus HB27 on a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and maintain the aeration volume at 5 m ³ / h / 50 L. Then, the cells are cultured at 150 rpm and 65 ° C. for about 12 hours, and after the cells have reached the logarithmic growth phase, 30 L of the bacterial solution is inoculated into a 500 L tank of the secondary species containing 300 L of the culture solution.
(Step 2)
The primary fermentation method for yeast is as follows.
The yeast culture solution (after step (4)) in the 300 L secondary species tank was transferred to a 5000 L fermentation tank containing 2400 L culture solution (pH 4.8 ± 0.2), and the aeration volume was 5 m. Maintain at ³ / h / 50L and incubate at 200 rpm and 30 ° C. for about 14 hours.
(Step 3)
As the secondary fermentation, within 0 to 1 day after raising the temperature of the primary fermentation culture solution from 30 ° C. to 65 ° C., 300 L of the Thermophilus growth solution was transferred to this fermentation tank, and the aeration amount was 5 m. Maintain at ³ / h / 50L, stir at 150 rpm, adjust the pH value to 7.8 ± 0.2 with an alkaline solution, and incubate at 150 rpm at 65 ° C. for about 18 hours.
Here, the components of the culture solution are 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, and 1 g of anhydrous chloride. For iron, 5 g of sodium chloride, the pH value is adjusted and controlled with acid or alkali, the rest is water.

Preferably, the strain of Saccharomyces cerevisiae is CICC1596, purchased from the China Industrial Microbial Species Conservation and Management Center, the preservation number is CICC1596, and the Thermus Thermophilus HB27 used is the American Type Culture Collection (ATCC). ), And the storage number is BAA-163.

In some preferred embodiments, the reagent is a cosmetic reagent and is used, for example, in a skin care reagent.

In addition, a reagent according to the present invention, which can reduce the outflow of transdermal water in the skin, includes a mixture of fermentation products obtained by the method according to the present invention.

In some preferred forms, in said applications or reagents, the thermophilic bacterium is Thermath Thermophilus HB27, purchased from the American Type Culture Collection (ATCC), and storage number BAA-163.

In some preferred forms, the yeast is CICC1596, purchased from the China Industrial Microbial Species Conservation and Management Center, and the preservation number is CICC1596.

In some other preferred embodiments, the reagent may further comprise other components in addition to the fermentation product, eg, components of other cosmetic reagents such as moisturizers, emulsifiers, light stabilizers, augmentations. It may contain one or a mixture of one or more of a mucilage, a solubilizer, an antiseptic, an antioxidant, a sunscreen, a pH adjuster, a penetration enhancer, a liposome, a skin nutrient, a fragrance, and a pigment. By some in vitro and human studies on a mixture of fermentation products of the invention, the fermentation products of the invention are against a single yeast fermentation product, a thermophilic bacterial fermentation product and a reagent after fermentation mixing. It has a remarkable improving effect, which explains that the co-culture of the two types of bacteria has a promoting effect on each other, and the activity content or activity of the physiologically active substance is significantly improved. It seems.

In some preferred forms, the cosmetic reagent may be in the following combination form, and here are some prominent examples, such as a pack, a lotion, a milky lotion, a facial cream, and the like. You may.

In some forms, the ingredients of the pack are as follows.

(1) When calculated by weight, it depends on a combination of at least 0.05 to 1 part by weight of sodium hyaluronate, 1 to 5 parts by weight of white jellyfish polysaccharide, 0.01 to 3 parts by weight of albutin, thermath thermophilus and yeast. It contains 0.1 to 20 parts by weight of fermentation product and 70 to 90 parts by weight of deionized water.

(2) When calculating by weight, at least 0.1 to 3.0 parts by weight of albutin, 0.05 to 1 part by weight of sodium polyglutamic acid, 5 to 20 parts by weight of aloe gel, coenzyme Q10 0.1 to 20 parts by weight. It contains 1 to 10 parts by weight of sodium gluconate, 1 to 10 parts by weight of a fermentation product of a combination of thermath thermophilus and yeast, and 30 to 50 parts by weight of deionized water.

In some forms, it may be a compounding component of a lotion, as in any of the following forms.

(Example 1) When calculating by weight, at least the following components, 1 to 10 parts by weight of a water-soluble moisturizer, 2 to 10 parts by weight of cactus extract, 0.1 to 1 part by weight of disodium ethylenediaminetetraacetate, thermath Includes 1-10 parts by weight of fermentation product from a combination of thermophilus and yeast, and 50-90 parts by weight of deionized water.

(Example 2) When calculated by weight, at least the following components, 0.1 to 5 parts by weight of a water-soluble moisturizer, 0.1 to 5 parts by weight of a fermentation product of a combination of thermophilus and yeast, pH It contains 1 to 10 parts by weight of a regulator and 50 to 80 parts by weight of deionized water.

In some embodiments, it may be in the form of a milky lotion, for example, as follows.

(Example 1) When calculating by weight, at least the following components, 1 to 5 parts by weight of white oil, 0.1 to 10 parts by weight of petrolatum, 1 to 5 parts by weight of stearate, 0.1 to 5 parts by weight of polyethylene glycol monooleyl ether It contains 5 parts by weight, 0.1 to 0.8 parts by weight of methyl paraoxybenzoate, 3 to 10 parts by weight of the fermentation product of the combination of Thermus thermophilus and yeast, and 3 to 80 parts by weight of deionized water.

In some forms, it may be in the form of a facial cream, for example, the following ingredients.

(Example 1) When calculating by weight, at least the following components, 0.01 to 1 part by weight of hyaluronic acid, 1 to 10 parts by weight of glycerin monostearate, 0.1 to 10 parts by weight of lanolin, 1 to 20 parts by weight of olive oil. By weight, 1-10 parts by weight of fermentation product from the combination of thermophilus and yeast, 1-3 parts by weight of antioxidant, 0.1-0.5 parts by weight of natural fragrance, and 1-70 parts by weight of deionized water. Including part.

In addition, the present invention does not substantially change the amount of B vitamins produced by the thermophilus when yeast and thermath thermophilus are co-cultured, but the production of some physiologically active substances is promoted. It has been found that such bioactive substances can improve the activity of B vitamins. Therefore, the present invention provides a new use of yeast as a bioactive substance that promotes the production of B vitamins by thermath thermophilus. Therefore, the present invention provides an application in which Saccharomyces cerevisiae, whose strain is CICC1596, is used as a fermentation co-energetic agent in which the HB27 strain Thermus thermophilus produces a physiologically active substance that enhances group B microorganisms.

According to the results of in vitro experiments, the fermentation product of the combination of Thermus thermophilus and Saccharomyces cerevisiae has a stronger ability to promote the growth of skin fibroblasts than the fermentation product of a single Thermus thermophilus. It was revealed that the growth rate of skin fibroblasts can be improved by about 20% at a concentration of%.

Fermentation products from a combination of thermophilus and yeast have a stronger anti-inflammatory effect than fermentation products of a single thermophilus, and 10% of thermophilus when skin keratinocytes are stimulated by inflammatory effects. The addition of fermentation products from the combination of yeast and yeast clearly reduced the secretion of the inflammatory factors IL-1α, IL-6, IL-8 and TNF-α, which corresponds to the anti-inflammatory effect of 100 ppm dexamethasone, but dexamethasone. It is milder and the anti-inflammatory effect is clearly superior to the fermentation product of a single Thermus thermophilus of 10% or a single yeast fermentation.

According to the results of human clinical trials, serum containing fermentation product of 1% thermophilus and yeast combination is remarkable for reducing transdermal water outflow after 7 days of use. It was found that the fermentation product produced by the combination of thermath thermophilus and yeast had an effect of repairing and strengthening the skin barrier function. After using a serum containing a fermentation product of 10% thermophilus and yeast combination for 7 days, the yellowing of the skin can be significantly reduced, depending on the thermophilus and yeast combination. Fermentation products have been shown to have the effect of improving skin color and removing yellowing.

It is a comparative diagram concerning removing the yellowing of the facial skin of the fermentation product according to Example 1 of the present invention, the left side is the effect of the face before use, and the right side is 7 days by the fermentation product according to Example 1. This is the effect of the face after use. On the contrary, there is no substantial change in Examples 2 to 6.

Hereinafter, how the present invention is realized by a specific embodiment will be described, but these explanations are for explaining the technical idea of the present invention and not for limiting the present invention. Absent. The specific scope of protection follows the scope of claims.

(Example 1)
[Fermentation method using a combination of thermophilus and yeast (1)]
The materials are as follows.
<Preparation and components of culture medium>
The composition of 20 L of the culture solution is 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. The value is adjusted and controlled with acid or alkali (hydrochloric acid or sodium hydroxide solution), replenished with water up to 20 L, and different amounts of culture solution are increased or decreased depending on the above formulation. In the present invention, unless otherwise specified, all the examples adopt the medium component of the above-mentioned composition and use it as a culture solution for bacterial culture. Of course, in actual industrial production, they can be blended according to different requirements, but all of these blending ingredients are common blending ingredients for culturing yeast and / or thermath thermophilus.

Saccharomyces cerevisiae is CICC1596, purchased from the China Industrial Microbial Species Conservation and Control Center, and the preservation number is CICC1596 (obtained by commercial purchase).

Thermus Thermophilus HB27 was purchased from the American Type Culture Collection (ATCC) and has a storage number of BAA-163 (obtained by commercial purchase).

1. Bacterial species expansion culture (step 1)
<1.1 Proliferation of Saccharomyces cerevisiae>
(1) Take one loop of Saccharomyces cerevisiae CICC1596 on the slope of a test tube on a clean bench, inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and inoculate it at 200 rpm (every minute). 200 rpm), cultured at 30 ° C. for about 10 hours, and the cells are in the logarithmic growth phase;
(2) Inoculate the cells in the logarithmic growth phase into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and inoculate the inoculation amount at 10% (volume percentage, 200 ml step (1)). (Yeast culture solution in the logarithmic growth phase) and inoculate at 200 rpm and 30 ° C. for about 10 hours;
(3) The 3 L bacterial species cultured in step (2) are inoculated into a 50 L tank of a first-class species containing 30 L of fermented liquid (pH 4.8 ± 0.2), and the aeration volume is 5 m ³ / h / 50 L. Maintain and inoculate at 200 rpm, 30 ° C. for about 10 hours;
(4) Transfer 30 L of the bacterial solution to a 500 L tank of a secondary species containing 300 L, maintain the aeration rate at 5 m ³ / h / 50 L, and incubate at 200 rpm and 30 ° C. for about 10 h.

<1.2 Proliferation of Thermophilus>
Take 2 ml of a frozen solution of Thermus Thermophilus HB27 on a clean bench and inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2) to increase the aeration rate to 5 m ³ / h / 50 L. The cells are maintained at 150 rpm and cultured at 65 ° C. for about 12 hours, and after the cells have reached the logarithmic growth phase, 30 L of the bacterial solution is inoculated into a 500 L tank of the secondary species containing 300 L of the culture solution.

The components of the culture solution here are 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, and 1 g of anhydrous iron chloride. 5 g of sodium sulphate, pH value adjusted and controlled with acid or alkali, replenished to 20 L with water. Different amounts of culture medium are increased or decreased depending on the above formulation.

2. (Step 2)
<Primary fermentation>
The yeast culture solution (after step (4)) in the 300 L secondary seed tank was transferred to a 5000 L fermentation tank containing 2400 L culture solution (pH 4.8 ± 0.2), and the aeration volume was 5 m ^3. Maintain at / h / 50 L and incubate at 200 rpm and 30 ° C. for about 14 hours.

At this time, the components of the culture solution were 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, and 1 g of anhydrous chloride. Iron, 5 g of sodium sulphate, pH value adjusted and controlled with acid or alkali, replenished with water up to 20 L. Different amounts of culture medium are increased or decreased depending on the above formulation.

3. (Step 3)
<Secondary fermentation>
Primary fermentation (step 2) After raising the temperature of the culture broth from 30 ° C to 65 ° C, immediately after the temperature reaches 65 ° C, 300 L of the thermophilus growth solution is transferred to this fermentation tank, and the aeration volume is 5 m. Maintain at ³ / h / 50 L, stir at 150 rpm, adjust the pH value to 7.8 ± 0.2 with an alkaline solution, and incubate at 150 rpm at 65 ° C. for about 18 hours.

At this time, the components of the culture solution were 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, and 1 g of anhydrous chloride. Iron, 5 g of sodium sulphate, pH value adjusted and controlled with acid or alkali, replenished with water up to 20 L. Different amounts of culture medium are increased or decreased depending on the above formulation.

4. Separation and purification <Sterilization by centrifugation>
The culture broth that has undergone the secondary fermentation of step 3 is filtered through a plate frame to block the cells on a filtration membrane (0.22 um) to obtain a fermentation supernatant.

<Purification>
Fermentation supernatant is added to activated carbon to adsorb impurities, remove malodor, and filter cinder using multiple layers of defatted gauze and diatomaceous earth (activated carbon addition ratio is 6%, activated carbon is purchased from Kanyu Coal Industry). ..

<Filtration>
The purified fermentation broth is filtered through a 0.22 um filtration membrane to obtain a fermentation product from a combination of Thermus thermophilus and yeast.

(Example 2 : Reference example )
[Method of yeast fermentation alone]
Specific embodiments of the product obtained by yeast fermentation alone are as follows.

<Preparation of culture solution>
About 20 L of culture broth containing 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. , The pH value is adjusted and controlled with acid or alkali, water is replenished up to 20 L, and different amounts of culture medium are increased or decreased depending on the above formulation.

1. Bacterial species expansion culture (1) Take one loop of Saccharomyces cerevisiae CICC1596 on the slope of a test tube on a clean bench and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2). , 200 rpm (200 rpm), cultivated at 30 ° C. for about 10 hours, and the cells are in the logarithmic growth phase;
(2) Inoculate the cells in the logarithmic growth phase into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and inoculate the inoculation amount at 10% (volume percentage, 200 ml step (1)). (Yeast culture solution in the logarithmic growth phase) and inoculate at 200 rpm and 30 ° C. for about 10 hours;
(3) The 3 L bacterial species cultured in step (2) are inoculated into a 50 L tank of a first-class species containing 30 L of fermented liquid (pH 4.8 ± 0.2), and the aeration volume is 5 m ³ / h / 50 L. Maintain and inoculate at 200 rpm, 30 ° C. for about 10 hours;
(4) Transfer 30 L of the bacterial solution to a 500 L tank of a secondary species containing 300 L, maintain the aeration rate at 5 m ³ / h / 50 L, and incubate at 200 rpm and 30 ° C. for about 10 h.

2. Fermentation Transfer the yeast culture solution in the 300 L secondary seed tank to a 5000 L fermentation tank containing 2700 L culture solution (pH 4.8 ± 0.2), and maintain the aeration rate at 5 m ³ / h / 50 L. Then, incubate at 200 rpm and 30 ° C. for about 14 hours.

<Preparation of culture solution>
About 20 L of culture broth containing 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. The pH value is adjusted and controlled with acid or alkali, 20 L of water is replenished, and different amounts of the culture solution are increased or decreased depending on the above formulation.

3. Separation and purification <Sterilization by centrifugation>
The culture broth that has undergone fermentation in step 2 is filtered through a plate frame to block the cells on a filtration membrane (0.22 um) to obtain a fermentation supernatant.

<Purification>
Fermentation supernatant is added to activated carbon to adsorb impurities, remove malodor, and filter sinda using multiple layers of defatted gauze and diatomaceous earth (activated carbon addition ratio is 6%, activated carbon is purchased from Kanyu Coal Industry). ).

<Filtration>
The purified fermentation broth is filtered through a 0.22 um filtration membrane to give a single yeast fermentation product.

(Example 3 : Reference example )
[Method of fermenting thermophilic bacteria alone]
Specific embodiments of a single thermophilus fermentation product are as follows.

<Preparation of culture solution>
About 20 L of culture broth containing 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. , The pH value is adjusted and controlled with acid or alkali, replenished with water up to 20 L, and different amounts of culture broth are increased or decreased depending on the above formulation.

1. Bacterial species expansion culture Take 2 ml of a frozen solution of Thermophilus HB27 on a clean bench and inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and inoculate the air volume to 5 m. Maintain at ³ / h / 50L, incubate for about 12 hours at 150 rpm and 65 ° C., and inoculate 30 L of bacterial solution into a secondary species 500 L tank containing 300 L of culture solution after the cells have reached the logarithmic growth phase. To do.

2. Fermentation Transfer 300 L of thermath thermophilus growth solution to a fermentation tank containing 2700 L of culture solution, maintain the aeration rate at 5 m ³ / h / 50 L, stir at 150 rpm, and set the pH value to 7.8 ± 0. Adjust to .2 and incubate for about 18 hours at 150 rpm and 65 ° C.

3. Separation and purification <Sterilization by centrifugation>
The culture broth that has undergone fermentation in step 2 is filtered through a plate frame to block the cells on a filtration membrane (0.22 um) to obtain a fermentation supernatant.

<Purification>
Fermentation supernatant is added to activated carbon to adsorb impurities, remove malodor, and filter sinda using multiple layers of defatted gauze and diatomaceous earth (activated carbon addition ratio is 6%, activated carbon is purchased from Kanyu Coal Industry). ).

<Filtration>
The purified fermented liquor is filtered through a 0.22 um filtration membrane to give a single thermophilus fermentation product.

(Example 4 : Reference example )
[Reagent obtained by mixing the fermentation products of Examples 2 and 3]
The fermentation products of Examples 2 and 3 are taken and mixed at a weight ratio of 1: 1 to obtain a composite reagent.

(Example 5 : Reference example )
[Method of co-culturing yeast and thermophilic bacteria (2)]
Specific embodiments of the fermentation product of the combination of thermath thermophilus and yeast are as follows.

<Preparation of culture solution>
About 20 L of culture broth containing 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. , The pH value is adjusted and controlled with acid or alkali, replenished with water up to 20 L, and different amounts of culture broth are increased or decreased depending on the above formulation.

1. Expansion culture of bacterial species <Growth of yeast>
Take one loop of Saccharomyces cerevisiae CICC1596 on the slope of a test tube on a clean bench, inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and inoculate at 200 rpm at 30 ° C. for about 10 hours. , The cells are in the logarithmic growth phase. Inoculate into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2) and inoculate the inoculation amount at 10% (volume ratio, where 200 ml of yeast culture solution in the logarithmic growth phase is inoculated). Then, inoculate at 200 rpm and 30 ° C. for about 10 hours. Inoculate 3 L of bacterial species into a 50 L tank of a first-class species containing 30 L of fermented liquid (pH 4.8 ± 0.2), maintain the aeration rate at 5 m ³ / h / 50 L, and maintain the aeration rate at 200 rpm and 30 ° C. for about 10 h. Incubate. Transfer 30 L of bacterial solution to a 500 L tank of secondary species containing 300 L of culture solution. The aeration rate is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 200 rpm and 30 ° C. for about 10 hours.

<Proliferation of Thermus Thermophilus>
Take 2 ml of a frozen solution of Thermath Thermophilus HB27 on a clean bench and inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2) to increase the aeration rate to 5 m ³ / h / 50 L. The cells are maintained and cultured at 150 rpm and 65 ° C. for about 12 hours. After the cells have reached the logarithmic growth phase, 30 L of the bacterial solution is inoculated into a 500 L tank of a secondary species containing 300 L of the culture solution, cultured, and aerated. The amount is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 150 rpm and 65 ° C. for about 12 hours.

2. Fermentation Transfer the yeast in a 300 L secondary seed tank and the Thermath thermophilus culture in a 300 L secondary seed tank to a 5000 L fermentation tank containing a 2400 L culture solution (pH 4.8 ± 0.2). The aeration volume is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 200 rpm and 30 ° C. for about 14 hours. Further, the temperature of the fermentation broth was raised to 65 ° C., the aeration rate was maintained at 5 m ³ / h / 50 L, the mixture was stirred at 150 rpm, the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution, and 65. Incubate at ° C for about 18 hours.

3. Separation and purification <Sterilization by centrifugation>
The culture broth that has undergone fermentation in step 2 is filtered through a plate frame to block the cells on a filtration membrane (0.22 um) to obtain a fermentation supernatant.

<Purification>
Fermentation supernatant is added to activated carbon to adsorb impurities, remove malodor, and filter sinda using multiple layers of defatted gauze and diatomaceous earth (activated carbon addition ratio is 6%, activated carbon is from Kanyu Coal Industry). Purchase).

<Filtration>
The purified fermentation broth is filtered through a 0.22 um filtration membrane to obtain a fermentation product of a combination of Thermus thermophilus and yeast.

(Example 6 : Reference example )
[Method of co-culturing yeast and thermophilic bacteria (3)]
Specific embodiments of the fermentation product of the combination of thermath thermophilus and yeast are as follows.

<Preparation of culture solution>
About 20 L of culture broth containing 60 g of peptone, 4 g of malt juice, 7 g of anhydrous magnesium sulfate, 2.4 g of potassium dihydrogen phosphate, 40 g of ammonium sulfate, 1 g of anhydrous iron chloride, and 5 g of sodium chloride. , The pH value is adjusted and controlled with acid or alkali, replenished with water up to 20 L, and different amounts of culture broth are increased or decreased depending on the above formulation.

1. Expansion culture of bacterial species <Growth of yeast>
Take one loop of Saccharomyces cerevisiae CICC1596 on a test tube slope on a clean bench, inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and inoculate at 200 rpm at 30 ° C. for about 10 hours. , The cells are in the logarithmic growth phase and are inoculated into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and the inoculation amount is 10% (volume ratio) at 200 rpm and 30 ° C. Incubate for about 10 hours, inoculate 3 L of bacterial species into a 50 L tank of a first-class species containing 30 L of fermented solution (pH 4.8 ± 0.2), maintain the aeration rate at 5 m ³ / h / 50 L, and 200 rpm. Incubate at 30 ° C. for about 10 hours. Transfer 30 L of the bacterial solution to a 500 L tank of a secondary species containing 300 L, maintain the aeration rate at 5 m ³ / h / 50 L, and incubate at 200 rpm and 30 ° C. for about 10 hours.

<Proliferation of Thermus Thermophilus>
Take 2 ml of a frozen solution of Thermus Thermophilus HB27 on a clean bench and inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2) to increase the aeration rate to 5 m ³ / h / 50 L. The cells are maintained and cultured at 150 rpm at 65 ° C. for about 12 hours, and after the cells have reached the logarithmic growth phase, 30 L of the bacterial solution is inoculated into a 500 L tank of the secondary species containing 300 L of the culture solution. Further, the temperature of the fermentation broth was raised to 65 ° C., the aeration rate was maintained at 5 m ³ / h / 50 L, the mixture was stirred at 150 rpm, the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution, and 150 rpm. , Incubate at 65 ° C. for about 18 hours.

2. 2. Fermentation The yeast in the 300 L secondary seed tank and the thermophilus culture in the 300 L secondary seed tank were simultaneously transferred to a 5000 L fermentation tank containing 2400 L of the culture solution (pH 7.8 ± 0.2). The aeration volume is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 150 rpm and 65 ° C. for about 18 hours. The temperature of the fermentation broth was lowered to 30 ° C., the aeration rate was maintained at 5 m ³ / h / 50 L, the mixture was stirred at 200 rpm, the pH value was adjusted to 4.8 ± 0.2 with an acid solution, and the pH value was adjusted to 4.8 ± 0.2 at 200 rpm and 30 ° C. Incubate for about 14 hours.

3. 3. Separation and purification <Sterilization by centrifugation>
The culture broth that has undergone the secondary fermentation in step 2 is filtered through a plate frame to block the cells on a filtration membrane (0.22 um) to obtain a fermentation supernatant.

<Purification>
Fermentation supernatant is added to activated carbon to adsorb impurities, remove malodor, and filter sinda using multiple layers of defatted gauze and diatomaceous earth (activated carbon addition ratio is 6%, activated carbon is from Kanyu Coal Industry). Purchase).

<Filtration>
The purified fermentation broth is filtered through a 0.22 um filtration membrane to obtain a fermentation product of a combination of Thermus thermophilus and yeast.

(Example 7 : Reference example )
[Fermentation method using a combination of thermophilus and yeast (4)]
The difference from Example 1 is that in the secondary fermentation, the temperature of the culture solution in the primary fermentation (step 2) is raised from 30 ° C. to 65 ° C., and then one day after reaching 65 ° C., 300 L The thermophilus growth solution was transferred to this fermentation tank, the aeration rate was maintained at 5 m ³ / h / 50 L, stirred at 150 rpm, the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution, and 150 rpm, 65. It is to be cultured at ° C. for about 18 hours.

(Example 8 : Reference example )
[Fermentation method using a combination of thermophilus and yeast (5)]
The difference from Example 1 is that in the secondary fermentation, the temperature of the culture solution of the primary fermentation (step 2) is raised from 30 ° C. to 65 ° C., and then 2 days after reaching 65 ° C., 300 L The thermophilus growth solution was transferred to this fermentation tank, the aeration rate was maintained at 5 m ³ / h / 50 L, stirred at 150 rpm, the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution, and 150 rpm, 65. It is to be cultured at ° C. for about 18 hours.

(Example 9 : Reference example )
[Fermentation method using a combination of thermophilus and yeast (6)]
The difference from Example 1 is that in the secondary fermentation, the temperature of the culture solution in the primary fermentation (step 2) is raised from 30 ° C. to 75 ° C., and then 3 days after reaching 75 ° C., 300 L The thermophilus growth solution was transferred to this fermentation tank, the aeration rate was maintained at 5 m ³ / h / 50 L, stirred at 150 rpm, the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution, and 150 rpm, 65. It is to be cultured at ° C. for about 18 hours.

(Example 10)
<Cytotoxicity test>
Logistic phase cells (human skin fibroblasts and human skin horny cells) are collected, a single cell suspension is prepared in culture, and the final cell concentration is adjusted to 40,000 cell / mL in 96-well plates. 100 uL of cell suspension was added, hatched at 5% CO ₂ , 37 ° C., and after the cells adhered to the wall, test substances were added (cell culture medium containing each fermentation product of Examples 1 to 9). After culturing and fusing to 80% to 90%, 20 uL of MTT solution is added to each well and the cells continue to hatch for 3 hours, and the culturing is completed. Carefully aspirate the medium in the wells, add 100 uL of DMSO to each well, and vibrate for 10 min to fully thaw the crystals. The absorbance value of each well is measured with a microplate reader, a wavelength of 490 nm is selected, and 630 nm is used as a reference wavelength. Each process is repeated 5 times.

A high-sugar DMEM cell culture solution containing each fermentation product of Examples 1 to 9 was added, and the cell culture solution was a high-sugar DMEM cell culture solution containing 3% fermentation product, and the implementation of 3%. The growth rate of human skin fibroblasts in Example 1 (fermentation product of the combination of Thermophilus and yeast) was 131%, compared to Example 3 (single Thermophilus fermentation product) of 3%. It has been improved by 20%, specifically refer to Table 1 below.

The growth rate of human skin keratinocytes to which 10% of Example 1 (fermentation product of a combination of thermath thermophilus and yeast) was added was 101%, and the growth of human skin keratinocytes to which 100 ppm of dexamethasone was added. The rate is 89%. 10% Example 1 (fermentation product of the combination of Thermus thermophilus and yeast) is even milder than 100 ppm dexamethasone. The results are shown in Table 1 below.

According to the analysis of variance, the difference between Examples 1 and 7 and Examples 2-6 and 8-9 was P <0.01, respectively, reaching a very significant difference, with Examples 2-6 and 8-9. There is no noticeable difference between them. This is because the culture method based on the combination of Example 1 of the present invention can significantly promote the growth rate of human skin fibroblasts and also has a remarkable promoting effect on human skin keratinocytes, and is a mixed culture. The effect of the mixing action of Example 1 or Example 7 is the best, and the effect of the mixing action of Example 7 is the best. At the same time, when performing mixed culture, the timing of mixing is also very important, and the effect is best when mixed immediately after the temperature rises or one day later, but the effect of mixing over two days is fiber. It also explains that the effect on blast cells drops sharply. Similar effects are similar to the effect of cells on the growth rate of human skin keratinocytes.

(Example 11)
<Extinguishing effect test>
Collect log-phase human skin keratinocytes, make a single cell suspension in culture, adjust the final cell concentration to 40,000 cell / mL, and add 100 uL of cell suspension to a 96-well plate. Incubate at 5, 5% CO ₂ , 37 ° C. for 24 hours, suck out the culture solution, wash once with PBS, suck out, add 20 uL of PBS, irradiate with UVB, and control the radiation amount to 60 mJ / cm ^2. , Replaced with the test substance (each fermentation product of Examples 1 to 9 was diluted with a high-sugar DMEM cell culture solution, and each fermentation product having a concentration of 10%), cultured for 24 hours, and then the culture solution. Collect the supernatant. Detect the content of inflammatory factors (IL-1a, IL-6, IL-8, TNF-a) (detected according to the instructions of the finished product reagent kit). Repeat the test 5 times.

According to the results, when a high-sugar DMEM cell culture medium containing a fermentation product (Example 1) of a combination of 10% thermophilus and yeast was added, the inflammatory factor content IL-1α was 110.12 pg. / ML, IL-6 is 16.23 pg / mL, IL-8 is 1300.20 pg / mL and TNF-α is 42.25 pg / mL, and the amount of secretion is 10% single thermus. It is clearly reduced compared to the thermophilus fermentation product and has a clear anti-inflammatory effect (according to analysis, the content of inflammatory factors is significantly reduced), which corresponds to the anti-inflammatory effect of 100 ppm dexamethasone.

This is because the active product obtained by the culture method of the present invention can significantly reduce the production of inflammatory factors, and compared to a single fermentation or a mixture of fermentations, according to analysis of variance, Example 1, Explain that both 7 and the other treatments have reached a high significant difference (specific test data is omitted). This is because the co-culture of yeast and thermath thermophilus uses yeast for primary culture and then secondary mixed culture to promote both of the bioactive substances of the two types of bacteria. Explain that the activity is also significantly improved. On the contrary, such an effect cannot be obtained by culturing alone, mixing after culturing alone, and culturing by other methods. This is thought to be due to the inclusion of B vitamins in the thermophilus, but the main action of the B vitamins is probably skin anti-inflammatory, which is after co-culturing two different bacteria. It can be explained that the activity of the B vitamins in the thermophilus can be improved, or that other unknown substances give the B vitamins greater activity.

In addition, as a result of measuring the B vitamins in the nine treatments at the same time by a usual method, the present invention found that there was no obvious change in the B vitamins of the fermentation product every 100 grams in each treatment. This explains that fermentation by combination improves the activity of some B vitamins by producing a factor having further activity in some B vitamins, and has a further excellent anti-inflammatory effect. The mechanism of production of these specific active factors needs to be further investigated.

(Example 12)
<Skin barrier repair test>
Although the stratum corneum of the skin has an excellent barrier function, it is damaged by the influence of external factors such as temperature, wind, and sunlight, so it is very important to perform barrier repair in a timely manner. In this test, TEWL (Trans epidermal Water Loss, TEWL) was adopted as an index, and the lower the TEWL, the higher the barrier repair ability of the skin. Here, the test equipment is a skin moisture outflow (TEWL) test probe TM300 manufactured by CK of Germany, and the test conditions are that the subject sits in a room at a temperature of 22 ° C. ± 1 ° C. and a humidity of 50% ± 5% for 20 minutes. Is.

<Test method>
Select 20 volunteers aged 22-55 years with no skin disease or severe allergies to past drugs, cosmetics or some chemicals. Serum containing the control group and 1% of the fermentation products of Examples 1 to 9 in half of the face twice in a row in the morning and evening (the main components of the serum are deionized water, Tween-80, propylene glycol, antiseptic). There are 1% fermentation products of Examples 1-9 of the present invention, and the solvent used for the 1% fermentation products is prepared using PBS as a solvent and has a pH of 7.1). After using the product for about 7 days, perform a TEWL test and calculate the rate of change of TEWL. The calculation method of the rate of change of TEWL is [(TEWL value after 7 days-7 TEWL value before 7 days) / TEWL value before 7 days] * 100%. The lower the rate of change in TEWL, the better the barrier effect on the skin. (Repeat 5 times in each example). The results are shown in Table 3.

According to the significant difference analysis, Examples 1 and 7 and Examples 2 to 6 and 8 to 9 reached a significant difference in the repair effect on the barrier (specific test data is omitted), which is the present invention. The co-culture product obtained by culturing by the method of Example 1 in the above can have a remarkable repair function for the skin stratum corneum, and the activity or production amount of a physiologically active substance that repairs the skin stratum corneum cells. Or it may have improved the output or activity of other cofactors.

(Example 13)
<Skin yellowing elimination test>
Using the CIEXYZ system, conversion is performed by a mathematical method to obtain a CIEL * a * b * color space. The space is composed of one luminance (L) and two hue (a, b) axes. Luminance is a measure for displaying grayscale, the value of which is between 0 and 100, where 0 displays black and 100 displays white. a * is the saturation between red and green, the range of change is +60 to -60, and a positive value indicates the change in the intensity of red. b * indicates the saturation between yellow and indigo, the range of change is +60 to -60, and a positive value indicates the change in the intensity of yellow. The skin color is also indicated by L * a * b *, and the lower the b * value, the clearer the effect of eliminating yellowing, and the lower the Δb *, the better the effect of eliminating yellowing.

<Test method>
Twenty volunteers aged 22-55 years were selected and serum containing 10% of the fermentation products of Examples 1-9 in the control group and 10% of the fermentation products of Examples 1 to 9 in half of the face twice in a row in the morning and evening (as the main component of serum). Use deionized water, Tween-80, propylene glycol, preservatives) and use for 7 consecutive days (test repeated 5 times). Before using the product, VISIA imaging is performed after using the product for 7 days, and the average Δb * value of each group is calculated.

Here, the test equipment is the spectrophotometer CM2600d (Minolta, Osaka, Japan) of the LAB test equipment. Before the test, the subject thoroughly washed his face and then wiped it with a tissue, and the temperature was 22 ° C. ± 1 ° C. and the humidity was 50%. Sit for 20 minutes in a ± 5% room.

According to our analysis of variance, the reagents of Example 1 and the reagents of Example 7 were found to be highly different from the other treatments, and the co-culture of the present invention increased the amount of bioactive substances that cancel aging and pigmentation. Explain that it is beneficial to. In particular, it is relatively important that there is an appropriate addition time for co-culture. No significant difference was observed between Examples 2-6 and 8-9 (Fig. 1).

From human skin cell studies, we found that increasing the amount of the raw material (Examples 1 or 7) added increased cell viability (concentration 1.25% -12%), so the raw material proliferated skin cells. It was found to improve the reproduction ability. Conversely, adding the same concentration to the same raw materials of Examples 2-6, 8-9 did not increase the viability of the cells, and some, for example, in Examples 8-9. It reduces the viability of cells. A rational interpretation of this is as follows. The normal regeneration cycle of the skin is 3-4 weeks, and promoting cell proliferation and regeneration promotes metabolism of the epidermal layer of the skin, eliminating aging and pigmented keratin and yellowing the appearance of the skin. It can have the effect of erasing only. Analysis and testing have shown that the raw materials contain large amounts of cell nutrients such as polysaccharides, polyphenols, amino acids, etc., which are responsible for improving the proliferative and regenerative capacity of skin cells. Helps proliferate and regenerate and improves cell viability. This also means that in co-culture fermentation methods, the timing of mixing the two strains is particularly important, and choosing the right timing will help in the production of some beneficial bioactive substances, and vice versa. Hazardous substances may be produced, or may produce harmful substances for certain symptoms.

(Example 14)
[Anti-inflammatory test for skin preparations]
<Extinguishing effect test>
Fermentation products obtained in Examples 1 and 8 were selected and tested by 3 × 160 = 480 patients (80 men and women in each group) with acne (acne level 3 to 4) aged 15 to 35 years. Was tested against.

<How to use>
After washing the face, the fermentation products of Examples 1 and 8 were used twice every morning and evening (a skin preparation of 15% of the fermentation products prepared with sterile water), and the sterile water was used as a control for 10 consecutive days. Use, observe the degree of redness, swelling, and skin damage of the affected area, and based on the expert's score (0-4 points give a serious degree of inflammation, 0-no acne, 1-mild acne Yes, scattered and frequent blackheads, with scattered inflammatory acne; 2-moderate acne, superficial pustules, many areas of inflammatory acne, limited to the face; 3-severe, Deep inflammatory comedones are present; 4-severe, cysts, scars are likely to form).

The specific data is as follows. The fermentation product of Example 1 can significantly reduce the level of acne, where 75 women are varied from level 3 or 4 before use to levels 0-1 after use for 10 days. , 50 females changed to level 0, and 68 male patients changed from pre-use level 4 to post-use level 1. Those who do not have a clear change are acne or ineffective due to other causes. However, when the same method was adopted, there was no obvious change in acne level before and after use in view of the anti-inflammatory effect of the fermentation products of Examples 2 to 6, and the patients who used Examples 3 and 4 were used. Only 6 females changed from level 3 to level 1 and only 4 males changed from level 4 to level 1. From the anti-inflammatory effect of adopting the fermentation product of Example 8, it has a certain anti-inflammatory effect, but the effect is not clear, and only 7 out of 80 female patients changed from level 3 to level 1. Of the 80 male patients, only 6 changed from level 4 to level 1. From the results of human experimentation, Example 1 had a better suppression and antibacterial effect on acne than Comparative Examples 2 to 6 and 8 to 9, and the effect was more clear, and it was compatible with the antibacterial effect in Example 8. It was revealed that there was no obvious change in the sterile water.

The terms and expressions used to describe the methods herein are not unique and constant, and attempts are made to use these terms and expressions to exclude any expression of the same meaning that describes the invention or features. The present invention recognizes various expression methods within the specified range without any intention. Therefore, we believe that the present invention has been expressly developed herein by description of various specific solutions and arbitrary features, but further to change the representation of the design disclosed herein. Experienced professionals must be relied upon and these changes must be consistent with the statement accompanying the invention. The text, patents, and patent applications are combined with the content of all other texts and the useful electronic information referred to and documented herein and should always be referred to as complete content, of which This point must be clearly stated when announcing any part. The Applicant reserves the right to incorporate any or all of these texts, patents, patent applications or other textual information and materials into the application documents and make this specification a part of the disclosure.

Claims (7)

Applications in the production of reagents for anti-inflammatory, skin moisturizing or skin yellowing elimination of fermentation products by symbiotic fermentation culture of thermophilic and yeast.
The symbiotic fermentation process thermophilic bacteria and yeast,
First, the step (1) of fermenting the yeast once or multiple times under acidic conditions at 20 ° C. to 30 ° C. to grow the yeast,
Steps to obtain a fermentation product by inoculating thermophilic bacteria immediately after raising the temperature to 55 ° C to 65 ° C after the yeast enters the late logarithmic growth phase and performing the second fermentation under alkaline conditions. (2) and
Including
The yeast is Saccharomyces cerevisiae,
The thermophile is thermath thermophilus,
It is used in the production of reagents for anti-inflammatory, skin moisturizing or skin yellowing elimination of fermentation products by symbiotic fermentation culture of thermophilic bacteria and yeasts. By performing at least two or more growing culture to yeast, is the yeast in logarithmic growth phase,
Before inoculating Thermus thermophilus in yeast culture, performing one or more times of expansion culture to Thermus thermophilus,
The application according to claim 1, characterized in that. The strain of Saccharomyces cerevisiae is CICC1596, purchased from the China Industrial Microbial Species Conservation and Management Center, and the preservation number is CICC1596.
Thermus thermophilus used are purchased from the American Type Culture Collection (ATCC), storage number is BAA-163, according to any one of claims 1-2, characterized in that application. In step (1), the low temperature cultivation against yeast, the temperature is 20 ° C. to 30 ° C., wherein the acidic condition intends row acidic culture against yeast, a pH value from 4.0 to 5 .0 and
The first aspect of claim 1, wherein in the step (2), the yeast begins to rise in temperature from 0 to 1 day in the late logarithmic growth phase, and the temperature rises to 55 ° C. to 65 ° C. Use. Claim 1 is characterized in that, immediately after the temperature rises, thermophilic bacteria are started to be inoculated, and at the same time, the pH value of the culture solution is adjusted to alkaline, and the pH value is 7.0 to 8.5. Uses described in. The anti-inflammatory has an effect on the reduction of inflammatory factors and includes
The inflammatory factors include IL-1α, IL-6, IL-8 or TNF-α.
The application according to claim 1, characterized in that. The reagent is a skin care formulation, which can be present in any form and is any one of a solution, aqueous solution, suspension, pack, emulsion, cream, cream, gel, dry powder, wet powder, spray. One,
The application according to claim 1, characterized in that.