JP6669822B2 - Fermentation method of the combination of Thermos thermophilus and yeast - Google Patents

Description

  The present invention relates to the field of cosmetic fermentation raw materials, and more particularly to a fermentation method and application of a combination of Thermos thermophilus and yeast.

  The application of biofermented raw materials in the cosmetics field is increasing day by day, and in recent years, enzyme cosmetics have been highly popular with consumers. Fermentation is a purely biological process, with no added chemical components. Fermentation makes it easier to elute physiologically active substances, and extracting all active ingredients by fermentation creates new nutrients, improves the balance of nutrients, lowers the molecular weight of nutrients, and enhances activity And can be quickly absorbed by the skin. The fermenting microorganism itself contains various enzymes and trace elements and has a replenishing and synergistic effect. Therefore, it meets the expectations of consumers for “natural, healthy, and highly efficient” skin care products.

  Yeast is currently the most applied strain in the field of cosmetic fermentation raw materials, and yeast extract has a natural, safe and strong moisturizing effect. Yeast cells contain abundant natural moisturizing factors (NMF), for example, amino acids, peptide systems, and inorganic components such as sodium, potassium, and magnesium. Here, amino acids account for 40% of the natural moisturizing factor (NMF), and inorganic components such as sodium, potassium and magnesium account for 12% of the natural moisturizing factor (NMF). The main components of the yeast cell wall include yeast β- (1,3) / (1,6) -glucan and mannan M60. 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 M60 is capable of retaining water molecules and has a long-term moisturizing action. For example, Chinese Patent Application Publication Nos. CN105543319A, CN107184411A, and CN10655182A disclose the production of cosmetics using a yeast fermentation product as a raw material of cosmetics.

Thermus Thermophilus is a thermophilic bacterium that can produce thermostable superoxide dismutase (SOD) and B vitamins. Superoxide dismutase can oxidize O ₂ ⁻ to H ₂ O ₂ , avoid damage to the living body by oxidation, and prevent aging by preventing free radical invasion of the skin. Fulfill. B vitamins can reduce the inflammatory response of the skin, prevent sun damage and promote cell regeneration. B vitamins are also part of the molecular structure of various enzymes and coenzymes and can promote amino acid metabolism to maintain skin health. Chinese Patent Application No. 2017107329367 also discloses that the fermented product of Thermos thermophilus is used as a skin care component, but the skin care component is a recombinant human fermented product other than the fermented product of Thermos thermophilus. It also contains collagen. However, the patent document only mentions a fermentation product of Thermos thermophilus and does not disclose how to ferment. In addition, for example, the patent document of Chinese patent application No. 20070031387 also discloses a sunscreen composition using a bioenzyme, which composition contains a bioenzyme of Thermos thermophilus, and its ratio is provided. . However, all common Thermos thermophilus are produced by fermentation, and no disclosure is made regarding the activity of the enzyme or the yield of the enzyme, and furthermore, when such an enzyme is combined with other components, the stability is also low. This is one of the important factors. Since sunscreen products contain enzymes, it is also uncertain whether the enzymes will be inactivated by the effects of ultraviolet light or have the activity of the enzymes.

  In the prior art, two types of fermented liquids are mixed after fermenting a single bacterial species. When the different flora and enzymes are dissolved together to cause a neutralization reaction, a large amount of the activities of the flora and enzymes are lost, so that the actual effect is often undesirable. Therefore, it is necessary to improve the conventional fermentation method and product, to obtain a further bioactive substance, and to reduce the interaction and the neutralization effect by mixing a single fermentation product.

  In order to solve the above-mentioned problems, the present invention provides a fermentation process of a combination of a Thermos thermophilus and a yeast, thereby improving the physiological activity of the Thermos thermophilus fermentation product and improving the application effect of the Thermos thermophilus fermentation product. Achieving the enrichment objectives, in particular, improving the effect of the Thermus thermophilus fermentation product on mild anti-inflammatory and fulfilling the pursuit of "natural, healthy, high efficiency" of cosmetics.

  The inventors of the present invention can control not only fermentation of a variety of useful enzymes but also yield of each type of enzyme by fermentation of a plurality of types of bacteria in a controllable manner by co-fermentation of a plurality of bacteria. It has been found that the fermentation amount can be several times the fermentation amount of a general single strain. The more enzymes, the stronger the catalytic health care function for the human body. Yeast extract and Thermos thermophilus fermentation products are marketed on the market, but both are added to cosmetics as a single product, and fermentation by combination of yeast and Thermos thermophilus is used to develop cosmetics That is a new challenge.

  Further, the inventors of the present invention can co-cultivate yeast and thermophilic bacteria, and according to a special culturing step, it is possible to exert an activation-inducing effect on the culture of yeast against thermophilic bacteria, It has been found that the activity of the product obtained by culturing the thermophilic bacterium alone is remarkably improved, while the activity of the culture product of the yeast itself is 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.

  Some useful bioactive substances can be produced by culturing thermophilic bacteria alone, but the amount of such bioactive substances is very small and the activity is not high. When yeast and thermophilic bacteria are co-cultured, according to a special culture method, a product that can significantly improve the physiologically active substance of the thermophilic bacterium is found, and in particular, the activity of some physiologically active substances is remarkably increased. Found that it has been strengthened.

The present invention also provides a method of culturing a fermentation product of a thermophilic bacterium, the method comprising:
Fermenting Saccharomyces cerevisiae one or more times under acidic conditions at medium temperature to grow the yeast (1); and 0 days after the temperature is increased after the yeast has reached the late logarithmic growth phase. One day later, inoculating a Thermus thermophilus and performing a second fermentation under alkaline conditions to obtain a fermentation product (2).

  In some preferred forms, the yeast is subjected to at least two growth cultures, or at least three, or at least four, five, or six growth cultures, to bring the yeast to a logarithmic growth phase.

  In some preferred embodiments, the Thermus thermophilus is subjected to one or more growth cultures prior to mixed fermentation.

  In some preferred embodiments, the method comprises subjecting the fermentation liquor of step (2) to sterilization, decolorization, deodorization, and removal of impurities by filtration to obtain a fermentation product by a combination of Thermos thermophilus and yeast. (3) is further included.

  In some preferred embodiments, the Saccharomyces cerevisiae strain is CICC1596, purchased from the China Center for the Preservation and Control of Microbial Species, and has the storage number of CICC1596.

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

  In some embodiments, the yeast is subjected to low-temperature culturing and the temperature is between 20C and 30C.

  In some embodiments, the yeast is cultured in an acidic region and has a pH value of 4.0 to 5.0.

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

  In some embodiments, 0 or 1 day after the temperature is increased, the inoculation of the thermophilic bacterium is started, and at the same time or 1 day after the inoculation, the pH value of the medium is adjusted to alkaline. 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 embodiments, the method for expanding the yeast comprises removing one loop of Saccharomyces cerevisiae CICC1596 (the amount of bacterial solution that can be collected at one time using the inoculation loop) on a test tube slope on a clean bench, and adding 50 ml of the yeast. A step (1) of placing in a 250 ml Erlenmeyer flask containing a culture solution (pH 4.8 ± 0.2), culturing at 200 rpm (200 rotations per minute) at 30 ° C. for about 10 hours, and allowing the cells to enter 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 inoculum is increased to 10% (volume percentage, 200 ml step (1)). (2) culturing at 200 rpm and 30 ° C. for about 10 hours, and 3 L of the bacterial species cultivated in step (2) are subjected to a 30 L fermentation liquid ( a step (3) of inoculating a 50L tank of a first-class species containing (pH 4.8 ± 0.2), maintaining the aeration at 5 m ³ / h / 50 L, and culturing at 200 rpm for 10 hours at 30 ° C .; (4) transferring the bacterial solution to a second-class 500 L tank containing 300 L, maintaining the aeration at 5 m ³ / h / 50 L, and culturing at 200 rpm, 30 ° C. for about 10 h.

Further, the present invention provides a method including the following steps.
(Step 1)
Growth culture is performed on Saccharomyces cerevisiae and Thermus thermophilus, respectively.
Here, the propagation method of Saccharomyces cerevisiae is as follows.
(1) Take 1 loop of Saccharomyces cerevisiae CICC1596 on the test tube slope in a clean bench, put it in a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2), and put 200 rpm (200 per minute). (Rotation), culturing at 30 ° C. for about 10 h to allow the cells to reach a logarithmic growth phase;
(2) 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 inoculum amount is increased by 10% (volume percentage, 200 ml step (1)). Yeast culture in a logarithmic growth phase), and cultured 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 first-class 50 L tank containing 30 L of fermentation broth (pH 4.8 ± 0.2), and the aeration rate is 5 m ³ / h / 50 L. And culture at 200 rpm, 30 ° C. for about 10 h;
(4) Transfer 30 L of the bacterial solution to a 500 L tank of a second grade containing 300 L, maintain the aeration at 5 m ³ / h / 50 L, and incubate at 200 rpm, 30 ° C. for about 10 h.
The method of multiplying Thermos thermophilus is as follows.
Take 2 ml of a frozen solution of Thermos thermophilus HB27 in 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 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 a secondary species containing 300 L of the culture solution.
(Step 2)
The primary fermentation method for yeast is as follows.
The yeast culture (after step (4)) in the 300 L secondary seed tank was transferred to a 5000 L fermentation tank containing 2400 L culture (pH 4.8 ± 0.2) and the aeration was 5 m ^3. / H / 50L, and culture at 200 rpm and 30 ° C for about 14 hours.
(Step 3)
As the secondary fermentation, the temperature of the culture solution of the primary fermentation (step 2) is raised from 30 ° C. to 65 ° C., and after the temperature is raised to 65 ° C. (day 0), 300 L of Thermos thermophilus is immediately expanded. The solution was transferred to this fermentation tank, the aeration was maintained at 5 m ³ / h / 50 L, the mixture was stirred at 150 rpm, the pH was adjusted to 7.8 ± 0.2 with an alkaline solution, and the solution was stirred at 150 rpm at 65 ° C. for about 18 hours. Incubate.

  In some preferred embodiments, the composition of the culture broth is a 20 L culture broth comprising 60 g peptone, 4 g wort, 7 g anhydrous magnesium sulfate, 2.4 g potassium dihydrogen phosphate, 40 g ammonium sulfate, 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 the water is replenished to 20 L. Different amounts of culture solution will increase or decrease according to the formulation.

  Further, it is an application in the manufacture of a reagent for skin anti-inflammatory or skin moisturization of a fermentation product by a symbiotic fermentation culture of a thermophilic bacterium and a yeast. In acid, the yeast is subjected to one or more liquid growth fermentations to allow for a logarithmic growth phase, and then 0 or 1 day after changing the temperature of the fermentation liquor from low to high, Secondary fermentation is performed by adding Thermos thermophilus, and fermentation conditions are set to alkaline conditions.

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

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

  Also, a reagent according to the present invention, which is capable of reducing the outflow of transdermal water from the skin, said reagent comprising a mixture of fermentation products obtained by the method according to the present invention.

  In some preferred forms, in the use or reagent, the thermophilic bacterium is Thermus thermophilus HB27, purchased from the American Type Culture Collection (ATCC), and has the storage number BAA-163.

  In some preferred embodiments, the yeast is CICC1596, purchased from the Center for the Preservation and Control of Microbial Species of China, and has the storage number of CICC1596.

  In some other preferred embodiments, the reagent may further include other components in addition to the fermentation product, such as components of other cosmetic reagents, such as fruit oil, silicone oil, glyceryl stearate, Other components such as xanthan gum and pure water may be added. Further, for example, a blended serum containing pure water, Tween-80, propanediol, a preservative and the like may be added. According to some in vitro and human studies on the mixture of fermentation products of the present invention, the fermentation products of the present invention can be used for single yeast fermentation products, thermophilic bacterial fermentation products and reagents after fermentation mixing. It has a remarkable ameliorating effect, which explains that the co-cultivation of the two types of bacteria has a stimulating effect on each other, and both the content or activity of the activity of the physiologically active substance is greatly improved. It seems.

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

  According to in vitro experimental results, the fermentation product of the combination of Thermos thermophilus and Saccharomyces cerevisiae has a stronger ability to promote the growth of dermal fibroblasts than the fermentation product of a single Thermos thermophilus. It was found that at a concentration of%, the growth rate of skin fibroblasts could be improved by as much as 20%.

  The fermented product of the combination of Thermos thermophilus and yeast has a stronger anti-inflammatory effect than the fermented product of a single Thermos thermophilus, and skin keratinocytes are stimulated with an inflammatory effect by 10% of Thermos thermophilus. And the addition of a fermentation product from a combination of yeasts, the secretion of the inflammatory factors IL-1α, IL-6, IL-8 and TNF-α is clearly reduced, corresponding to the anti-inflammatory effect of 100 ppm of dexamethasone, It is milder and the anti-inflammatory effect is clearly superior to 10% of a single Thermos thermophilus fermentation product or a single yeast fermentation.

  According to the results of human clinical trials, serum containing 1% of a fermentation product of the combination of Thermus thermophilus and yeast is remarkable for reducing transdermal water outflow after 7 days of use. It has been shown that the fermentation product of the combination of Thermus thermophilus and yeast has an effect of repairing and enhancing the skin barrier function. Serum containing 10% of the fermented product of Thermus thermophilus and yeast combination can significantly reduce the yellowness of the skin after 7 days of use, resulting in a significant decrease in the yellowness of the skin. The fermentation product was found to have an effect of improving skin color and removing yellowness.

Hereinafter, how the present invention is realized by specific embodiments will be described.However, these descriptions are for explaining the technical idea of the present invention and are not for limiting the present invention. Absent. The specific scope of protection is in accordance with the claims. However, Examples 2 to 6 and 8 to 9 are reference examples.

(Example 1)
[Fermentation method using a combination of Thermos thermophilus and yeast (1)]
The materials are as follows.
<Preparation of culture solution and components>
The formulation of the 20 L culture was 60 g peptone, 4 g wort, 7 g anhydrous magnesium sulfate, 2.4 g potassium dihydrogen phosphate, 40 g ammonium sulfate, 1 g anhydrous iron chloride, 5 g sodium chloride, pH The value is adjusted and controlled with an acid or alkali (hydrochloric acid or sodium hydroxide solution), supplemented with water up to 20 L, and different volumes of the culture solution increase or decrease according to the above formulation. In the present invention, unless otherwise specified, all of the Examples employ a medium component having the above composition to prepare a culture solution for bacterial culture. Naturally, in actual industrial production, they can be blended according to different requirements, but all such blending components are common blending components for culturing yeast and / or Thermos thermophilus.

  Saccharomyces cerevisiae is CICC1596, purchased from the China Center for the Preservation and Control of Microbial Species, and the storage number is CICC1596 (obtained by commercial purchase).

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

1. Expansion of bacterial species (Step 1)
<1.1 Propagation of Saccharomyces cerevisiae>
(1) One loop of Saccharomyces cerevisiae CICC1596 on a test tube slope was taken in a clean bench, inoculated into a 250 ml Erlenmeyer flask containing 50 ml of a culture solution (pH 4.8 ± 0.2), and 200 rpm (every minute). Culturing at 30 ° C. for about 10 h, and the cells are in logarithmic growth phase;
(2) 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 inoculum amount is increased by 10% (volume percentage, 200 ml step (1)). Yeast culture in a logarithmic growth phase), and cultured 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 first-class 50 L tank containing 30 L of fermentation broth (pH 4.8 ± 0.2), and the aeration rate is increased to 5 m ³ / h / 50 L. Maintain and incubate at 200 rpm, 30 ° C. for about 10 h;
(4) 30 L of the bacterial solution is transferred to a 500 L tank of a second grade containing 300 L, and 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.

<1.2 Propagation of Thermos thermophilus>
Take 2 ml of a frozen solution of Thermos thermophilus HB27 in a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and adjust the aeration rate to 5 m ³ / h / 50 L. The culture is 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 secondary tank containing 300 L of the culture solution.

  The components of the culture solution were as follows: a mixture of 20 L of the culture solution was 60 g of peptone, 4 g of wort, 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 chloride, pH value is adjusted and controlled with acid or alkali, and replenished to 20 L with water. Different amounts of culture will increase or decrease depending on the formulation.

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

  At this time, the components of the culture solution were as follows: 20 L of the culture solution was 60 g of peptone, 4 g of wort, 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 sodium chloride, pH value adjusted and controlled with acid or alkali, replenish up to 20 L with water. Different amounts of culture will increase or decrease depending on the formulation.

3. (Step 3)
<Secondary fermentation>
Primary fermentation (step 2) After the temperature of the culture solution was raised from 30 ° C. to 65 ° C., and the temperature reached 65 ° C., 300 L of the Thermos thermophilus growth solution was immediately transferred to this fermentation tank, and the aeration rate was 5 m. ^The mixture is maintained at ³ / h / 50 L, stirred at 150 rpm, adjusted to a pH value of 7.8 ± 0.2 with an alkaline solution, and cultured at 150 rpm at 65 ° C. for about 18 hours.

  At this time, the components of the culture solution were as follows: 20 L of the culture solution was 60 g of peptone, 4 g of wort, 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 sodium chloride, pH value adjusted and controlled with acid or alkali, replenish up to 20 L with water. Different amounts of culture will increase or decrease depending on the formulation.

4. Separation and purification <Eradication by centrifugation>
The culture solution that has undergone the secondary fermentation in step 3 is filtered through a plate frame to block the cells on a filter membrane (0.22 μm), thereby obtaining a fermentation supernatant.

<Purification>
The fermentation supernatant is added to activated carbon to absorb impurities, remove odors, and filter synda using multiple layers of defatted gauze and diatomaceous earth (addition ratio of activated carbon is 6%, activated carbon is purchased from Kantan Co., Ltd.) .

<Filtration>
The clarified fermentation liquor is filtered through a 0.22 um filtration membrane to obtain a fermentation product from a combination of Thermos thermophilus and yeast.

(Example 2)
[Method of fermenting yeast alone]
Specific embodiments of the product of yeast fermentation alone are as follows.

<Preparation of culture solution>
About 20 L of culture solution containing 60 g of peptone, 4 g of wort, 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 an acid or alkali, water is replenished up to 20 L, and different amounts of culture are increased or decreased according to the above formulation.

1. Bacterial strain expansion culture (1) One loop of Saccharomyces cerevisiae CICC1596 on a test tube slope was taken on a clean bench and inoculated into a 250 ml Erlenmeyer flask containing 50 ml of culture solution (pH 4.8 ± 0.2). Cultivation at 200 rpm (200 rotations per minute) at 30 ° C. for about 10 h, and the cells are in logarithmic growth phase;
(2) 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 inoculum amount is increased by 10% (volume percentage, 200 ml step (1)). Yeast culture in a logarithmic growth phase), and cultured 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 first-class 50 L tank containing 30 L of fermentation broth (pH 4.8 ± 0.2), and the aeration rate is increased to 5 m ³ / h / 50 L. Maintain and incubate at 200 rpm, 30 ° C. for about 10 h;
(4) 30 L of the bacterial solution is transferred to a 500 L tank of a second grade containing 300 L, and 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.

2. Fermentation The yeast culture in the 300 L secondary seed tank was transferred to a 5000 L fermentation tank containing 2700 L culture (pH 4.8 ± 0.2), and the aeration was maintained at 5 m ³ / h / 50 L. Then, the cells are cultured at 200 rpm and 30 ° C. for about 14 hours.

<Preparation of culture solution>
About 20 L of culture solution containing 60 g of peptone, 4 g of wort, 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 an acid or alkali, supplemented with 20 L of water, and different amounts of the culture solution increase or decrease according to the above composition.

3. Separation and purification <Eradication by centrifugation>
With respect to the culture solution that has undergone the fermentation in Step 2, the bacterial cells are blocked by a filtration membrane (0.22 μm) through plate frame filtration to obtain a fermentation supernatant.

<Purification>
The fermentation supernatant is added to activated carbon to absorb impurities, remove odors, and filter synda using multiple layers of defatted gauze and diatomaceous earth (addition ratio of activated carbon is 6%, activated carbon is purchased from Kantan Co., Ltd.) ).

<Filtration>
The clarified fermentation liquor is filtered through a 0.22 um filtration membrane to obtain a single yeast fermentation product.

(Example 3)
(Method of fermenting thermophilic bacteria alone)
Specific embodiments of a single Thermos thermophilus fermentation product are as follows.

<Preparation of culture solution>
About 20 L of culture solution containing 60 g of peptone, 4 g of wort, 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 , PH value is adjusted and controlled with acid or alkali, water is replenished up to 20L, and different amounts of culture solution increase or decrease according to the above formula.

1. Bacterial species expansion culture Take 2 ml of a frozen solution of Thermos thermophilus HB27 in a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of a culture solution (pH 7.8 ± 0.2), and inhale a volume of 5 m. ³ / h / 50L, culture at 150 rpm, 65 ° C for about 12h, and after the cells reached the logarithmic growth phase, inoculate 30L of bacterial solution into a 500L secondary-grade tank containing 300L of culture solution I do.

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

3. Separation and purification <Eradication by centrifugation>
With respect to the culture solution that has undergone the fermentation in Step 2, the bacterial cells are blocked by a filtration membrane (0.22 μm) through plate frame filtration to obtain a fermentation supernatant.

<Purification>
The fermentation supernatant is added to activated carbon to absorb impurities, remove odors, and filter synda using multiple layers of defatted gauze and diatomaceous earth (addition ratio of activated carbon is 6%, activated carbon is purchased from Kantan Co., Ltd.) ).

<Filtration>
The clarified fermentation liquor is filtered through a 0.22 um filtration membrane to obtain a single Thermos thermophilus fermentation product.

(Example 4)
[Reagents obtained by mixing the fermentation products of Examples 2 and 3]
The fermentation products of Examples 2 and 3 are taken and mixed in a 1: 1 weight ratio to obtain a complex reagent.

(Example 5)
[Method of co-culturing yeast and thermophilic bacteria (2)]
A specific embodiment of a fermentation product with a combination of Thermus thermophilus and yeast is as follows.

<Preparation of culture solution>
About 20 L of culture solution containing 60 g of peptone, 4 g of wort, 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 , PH value is adjusted and controlled with acid or alkali, water is replenished up to 20L, and different amounts of culture solution increase or decrease according to the above formula.

1. Bacterial species expansion culture <Propagation of yeast>
One loop of Saccharomyces cerevisiae CICC1596 on a test tube slope was taken in a clean bench, inoculated into a 250 ml Erlenmeyer flask containing 50 ml of a culture solution (pH 4.8 ± 0.2), and cultured at 200 rpm at 30 ° C. for 10 hours. The cells are in a logarithmic growth phase. Inoculate a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and inoculate 10% (by volume, here, inoculating 200 ml of yeast culture in logarithmic growth phase). And culturing at 200 rpm and 30 ° C. for about 10 hours. 3L of the bacterial species is inoculated into a first-class 50L tank containing a 30L fermentation broth (pH 4.8 ± 0.2), the aeration rate is maintained at 5m ³ / h / 50L, and about 10 hours at 200 rpm and 30 ° C. Incubate. Transfer 30 L of bacterial solution to a second-class 500 L tank 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.

<Propagation of Thermos thermophilus>
Take 2 ml of a frozen solution of Thermos thermophilus HB27 in a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and adjust the aeration rate to 5 m ³ / h / 50 L. The cells were maintained at 150 rpm and cultured at 65 ° C. for about 12 hours. After the cells reached the logarithmic growth phase, 30 L of the bacterial liquid was inoculated into a 500 L secondary-grade tank containing 300 L of culture liquid, and cultured. The volume is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 150 rpm at 65 ° C. for about 12 hours.

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

3. Separation and purification <Eradication by centrifugation>
With respect to the culture solution that has undergone the fermentation in Step 2, the bacterial cells are blocked by a filtration membrane (0.22 μm) through plate frame filtration to obtain a fermentation supernatant.

<Purification>
The fermentation supernatant is added to activated carbon to adsorb impurities, remove odors, and filter synda using multiple layers of defatted gauze and diatomaceous earth. (The activated carbon addition ratio is 6%. Purchase).

<Filtration>
The clarified fermentation liquor is filtered through a 0.22 um filtration membrane to obtain a fermentation product of the combination of Thermos thermophilus and yeast.

(Example 6)
[Coculture method of yeast and thermophilic bacteria (3)]
A specific embodiment of a fermentation product with a combination of Thermus thermophilus and yeast is as follows.

<Preparation of culture solution>
About 20 L of culture solution containing 60 g of peptone, 4 g of wort, 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 , PH value is adjusted and controlled with acid or alkali, water is replenished up to 20L, and different amounts of culture solution increase or decrease according to the above formula.

1. Bacterial species expansion culture <Propagation of yeast>
One loop of Saccharomyces cerevisiae CICC1596 on a test tube slope was taken in a clean bench, inoculated into a 250 ml Erlenmeyer flask containing 50 ml of a culture solution (pH 4.8 ± 0.2), and cultured at 200 rpm at 30 ° C. for 10 hours. The cells are in a logarithmic growth phase, inoculated into a 2.5 L Erlenmeyer flask containing 2 L (pH 4.8 ± 0.2), and the inoculum is 10% (by volume) at 200 rpm and 30 ° C. After culturing for about 10 hours, 3 L of the bacterial species was inoculated into a 50 L tank of a first-class species containing a 30 L fermentation broth (pH 4.8 ± 0.2), the aeration was maintained at 5 m ³ / h / 50 L, and 200 rpm, Incubate at 30 ° C for about 10 hours. 30 L of the bacterial solution is transferred to a second-class 500 L tank containing 300 L, the aeration is maintained at 5 m ³ / h / 50 L, and the culture is performed at 200 rpm and 30 ° C. for about 10 h.

<Propagation of Thermos thermophilus>
Take 2 ml of a frozen solution of Thermos thermophilus HB27 in a clean bench, inoculate it into a 2.5 L Erlenmeyer flask containing 2 L of culture solution (pH 7.8 ± 0.2), and adjust 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. 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. Furthermore, the temperature of the fermentation liquor was raised to 65 ° C., the aeration rate was maintained at 5 m ³ / h / 50 L, the mixture was stirred at 150 rpm, and the pH value was adjusted to 7.8 ± 0.2 with an alkaline solution. And cultured at 65 ° C. for about 18 h.

2. Fermentation The yeast in the 300L secondary seed tank and the Thermus thermophilus culture in the 300L secondary seed tank were simultaneously transferred to a 5000L fermentation tank containing 2400L culture (pH 7.8 ± 0.2), The aeration rate is maintained at 5 m ³ / h / 50 L, and the cells are cultured at 150 rpm at 65 ° C. for about 18 hours. The temperature of the fermentation liquor 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 rotation was performed at 200 rpm and 30 ° C. For about 14 h.

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

<Purification>
The fermentation supernatant is added to activated carbon to adsorb impurities, remove odors, and filter synda using multiple layers of defatted gauze and diatomaceous earth. (The activated carbon addition ratio is 6%. Purchase).

<Filtration>
The clarified fermentation liquor is filtered through a 0.22 um filtration membrane to obtain a fermentation product of the combination of Thermos thermophilus and yeast.

(Example 7)
[Fermentation method by combination of Thermos thermophilus and yeast (4)]
The difference from Example 1 is that, in the secondary fermentation, after the temperature of the culture solution of the primary fermentation (step 2) was raised from 30 ° C. to 65 ° C., one day after reaching 65 ° C., 300 L The Thermus thermophilus growth solution was transferred to this fermentation tank, the aeration 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, 65 About 18 hours of culturing at ℃.

(Example 8)
[Fermentation method using a combination of Thermos thermophilus and yeast (5)]
The difference from Example 1 is that, in the secondary fermentation, after the temperature of the culture solution of the primary fermentation (step 2) was raised from 30 ° C. to 65 ° C., two days after reaching 65 ° C., 300 L The Thermus thermophilus growth solution was transferred to this fermentation tank, the aeration 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, 65 About 18 hours of culturing at ℃.

(Example 9)
[Fermentation method (6) using a combination of Thermos thermophilus and yeast]
The difference from Example 1 is that, in the secondary fermentation, after the temperature of the culture solution of the primary fermentation (step 2) was raised from 30 ° C. to 75 ° C., and three days after reaching 75 ° C., 300 L The Thermus thermophilus growth solution was transferred to this fermentation tank, the aeration 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, 65 About 18 hours of culturing at ℃.

(Example 10)
<Cytotoxicity test>
Log phase cells (human dermal fibroblasts and human keratinocytes) were collected, a single cell suspension was prepared in culture, the final cell concentration was adjusted to 40000 cells / mL, and the cells were added to a 96-well plate. After adding 100 uL of the cell suspension, incubating at 37 ° C., 5% CO ₂ and attaching the cells to the wall, a test substance was added (cell culture solution 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, hatching is continued for 3 h, and the culture is terminated. Carefully aspirate the medium in the wells, add 100 uL DMSO to each well, and shake for 10 min to allow the crystals to thaw sufficiently. The absorbance value of each well is measured with a microplate reader, a wavelength of 490 nm is selected, and 630 nm is set as a reference wavelength. Each process is repeated five times.

  A high saccharide DMEM cell culture containing each fermentation product of Examples 1-9 was added, and the cell culture was a high saccharide DMEM cell culture containing 3% fermentation product and 3% The growth rate of human dermal fibroblasts of Example 1 (a fermentation product of the combination of Thermos thermophilus and yeast) is 131%, compared to 3% of Example 3 (a single Thermos thermophilus fermentation product). For example, see Table 1 below.

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

  According to analysis of variance, the difference between Examples 1 and 7 and Examples 2-6 and 8-9 was P <0.01, respectively, and reached a very significant difference, and Examples 2-6 and 8-9 There is no noticeable difference between the two. This is because the culture method according to the combination of Example 1 of the present invention can remarkably promote the growth rate of human dermal fibroblasts, and also has a remarkable promoting effect on human keratinocytes. It is explained that the effect of (1) is the best, and the effect of the mixing action of the embodiment 1 or 7 is the best. At the same time, when performing mixed culture, the timing of mixing is also very important. Mixing immediately after the temperature is raised or mixing one day later is the best, but the effect of mixing beyond 2 days is the fiber. It also explains that the effect on blasts declines sharply. Similar effects are similar to the effects of cells on the growth rate of human cutaneous keratinocytes.

(Example 11)
<Anti-inflammatory effect test>
Harvest logarithmic phase of human keratinocytes, make single cell suspension in culture, adjust final cell concentration to 40000 cells / mL, add 100 uL cell suspension to 96 well plate Hatch at 37 ° C., 5% CO ₂ for 24 hours, aspirate the culture solution, wash once with PBS, aspirate, add 20 uL of PBS, irradiate with UVB, and control the radiation amount to 60 mJ / cm ^2. The test substance was replaced (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%), and cultured for 24 hours. Collect the supernatant. The content of inflammatory factors (IL-1a, IL-6, IL-8, TNF-a) is detected (detected according to the instructions of the finished reagent kit). Repeat the test 5 times.

  The results show that when adding a high-sugar DMEM cell culture containing 10% of the fermentation product of the combination of Thermus thermophilus and yeast (Example 1), the content of inflammatory factor IL-1α is 110.12 pg / IL, IL-6 is 16.23 pg / mL, IL-8 is 1300.20 pg / mL and TNF-α is 42.25 pg / mL, and the secreted amount is 10% of a single thermus. The anti-inflammatory effect is clearly reduced compared to the Thermophilus fermentation product, and the anti-inflammatory effect is evident (analysis shows a significant reduction in the content of inflammatory factors), corresponding to the anti-inflammatory effect of 100 ppm dexamethasone.

  This is because the active product obtained by the cultivation method of the present invention can significantly reduce the production of inflammatory factor, and according to the analysis of variance according to Example 1, compared to a single fermentation or a mixture of fermentations, It will be explained that all of 7 and other processes have reached a high significant difference (specific test data is omitted). This is because the co-culture of yeast and Thermus thermophilus is a primary culture using yeast, and then a secondary mixed culture is performed, both of which act on the bioactive substances of the two types of bacteria, Explain that the activity is also significantly improved. Conversely, single culture, mixing after single culture, and mixed culture by other methods cannot obtain such an effect. This is thought to be due to the inclusion of the B vitamins in Thermus thermophilus, but the main effect of the B vitamins is probably skin inflammation, which occurs after co-culturing two different bacteria. Can explain the increased activity of the B vitamins in Thermus thermophilus, or explain that other unknown substances make the B vitamins more active.

  The present invention also found that there was no apparent change in the vitamin B group of the fermented product per 100 grams in each treatment as a result of simultaneously measuring the vitamin B groups in the nine treatments by the usual method. This explains that fermentation by the combination increases the activity of the vitamin B group by producing a factor having further activity in some vitamin B groups, and has an excellent anti-inflammatory effect. The mechanism of production of these specific activators requires further investigation.

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

<Test method>
Select 20 volunteers, 22-55 years old, who have a skinless disease or have a serious allergy to some past drugs, cosmetics or some chemicals. Two consecutive mornings and evenings, a serum containing the control group and 10% of the fermentation products of Examples 1 to 9 in the half of the face (the main components of serum include deionized water, Tween-80, propylene glycol, preservatives) Agent and 10% of the fermentation products of Examples 1 to 9 of the present invention, the solvent employed for the 10% fermentation product was prepared using PBS as solvent and the pH is 7.1) After using the product for about 7 days, a TEWL test is performed, and the rate of change of the TEWL is calculated. The calculation method of the change rate of TEWL is [(TEWL value after 7 days−TEWL value before 7 days) / TEWL value before 7 days] * 100%. The lower the change rate of TEWL, the better the barrier effect on the skin. (Repeated 5 times in each example). Table 3 shows the results.

  According to the significant difference analysis, Examples 1 and 7 to 8 and Examples 2 to 6 and 9 reached a significant difference in which the repair effect on the barrier was high (specific test data is omitted). The co-cultured product obtained by culturing according to the method of Example 1 can have a remarkable repair function on the stratum corneum of the skin, and the activity or output of the physiologically active substance for repairing the cells of the stratum corneum of the skin, Or it may have increased the yield 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 constituted by one luminance (L) and two hue (a, b) axes. Luminance is a measure for displaying gray scale, and its value is between 0 and 100, where 0 displays black and 100 displays white. a * is the saturation between red and green, and its change range is +60 to -60, and a positive value indicates a change in the intensity of red. b * indicates the saturation between yellow and indigo, and its change range is +60 to -60, and a positive value indicates a change in yellow intensity. The skin color is also indicated by L * a * b *, and the lower the b * value, the clearer the effect of yellowing elimination, and the lower the Δb *, the better the yellowing effect.

<Test method>
Twenty volunteers aged 22 to 55 years were selected, and two consecutive mornings and evenings, a serum containing the control group and 10% of the fermentation product of Examples 1 to 6 in half of the face (a major 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 device was a LAB test device spectrophotometer CM2600d (Minolta, Osaka, Japan). Before the test, the subject thoroughly washed his face, wiped it off with a tissue, and was heated at a temperature of 22 ° C. ± 1 ° C. and a humidity of 50%. Sit in a ± 5% room for 20 minutes.

  According to our analysis of variance, the reagent of Example 1 and the reagent of Example 7 showed a significant difference from each of the other treatments, and the co-culture of the present invention showed an increase in bioactive substances that cancel aging and pigmentation. Explain that it is useful. It is also relatively important that there is an appropriate addition time, especially for the co-culture. No significant difference is observed between Examples 2 to 6 and 8 to 9 (FIG. 1).

  We have shown from human skin cell tests that cell viability increased (concentration 1.25% to 12%) with increasing amounts of the raw material (Examples 1 or 7), thus the raw material was It has been found that the reproduction ability is improved. Conversely, adding the same concentration to the raw materials of similar Examples 2-6, 8-9 does not increase the viability of the cells, and some are, for example, reversed in Examples 8-9. Decreases cell viability. The reasonable interpretation for this is as follows. The normal renewal cycle of the skin is 3-4 weeks, and promoting cell proliferation and renewal promotes metabolism of the epidermal layer of the skin, eliminating keratin of aging and pigmentation and yellowing the appearance of the skin. Only the effect of erasure can be provided. According to analysis and tests, the raw materials contain large amounts of cellular nutrients such as polysaccharides, polyhydric phenols, amino acids, etc. Helps with growth and regeneration, improves cell viability. This is further because in a co-culture fermentation process, the timing of the mixing of the two bacteria is particularly important, and the selection of the appropriate timing helps to produce some beneficial bioactive substances, and vice versa. Of harmful substances may be produced, or may produce harmful substances for certain symptoms.

(Example 14)
(Anti-inflammatory test of skin preparation)
<Anti-inflammatory effect test>
3 x 160 = 480 patients with acne (acne levels 3-4) aged 15 to 35 years old (80 men and women in each group) were selected and tested, and the fermentation products obtained in Examples 1 and 8 Were tested.

<How to use>
After washing the face, the fermented products of Examples 1 and 8 were used twice a day every morning and evening (a skin preparation of a 15% fermented product prepared with sterilized water), and the sterilized water was used as a control for 10 consecutive days. Use and observe the degree of redness, swelling and skin damage of the affected area. Based on the scoring by a professional (0 to 4 points, the degree of acne is serious, 0- acne is not recognized, 1- light acne Yes, sporadic and frequent blackheads, with sporadic inflammatory acne; 2-moderate acne, superficial pustules, many inflammatory acne spots, limited to face; 3-severe, (Deep inflammatory acne is observed; 4-severe, cysts, scars are likely to form)).

  Specific data is as follows. The fermented product of Example 1 can significantly reduce the level of acne, where 75 women are converted from level 3 or 4 before use to levels 0-1 after 10 days of use. , 50 women changed to level 0 and 68 male patients changed from level 4 before use to level 1 after use. Those who have no apparent change are acne due to other causes or ineffective. However, when the same method is adopted, there is no apparent change in the 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 using Examples 3 and 4 Showed that only six women changed from level 3 to level 1 and only four men changed from level 4 to level 1. In view of the anti-inflammatory effect employing the fermentation product of Example 8, it has a certain anti-inflammatory effect, but the effect is not clear, 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 body experiments, Example 1 has better suppression and anti-inflammatory effects on acne than Comparative Examples 2 to 6 and 8 to 9, and the effect is more apparent, and is compatible with the anti-inflammatory effect in Example 8, It was found that there was no apparent change in the sterilized water.

  The terms and expressions used to describe the methods herein are not unique and specific, and it is intended that these terms and expressions be used to exclude any equivalent expression that describes the invention or features. The present invention recognizes various expression schemes within a specified range without intending to do so. Therefore, while we believe that the invention has been elucidated herein with an explanation of various specific solutions and optional features, further modifications to the presentation of the design disclosed herein are required. Relying on an experienced professional should make these changes consistent with the statement accompanying the present invention. The text, patent, and application of the patent are combined with all other textual content and useful electronic information referred to and substantiated herein, and should be referred to as complete content only, This point must be clearly indicated when any part is announced. Applicant reserves the right to incorporate any or all of these texts, patents, patent applications or other textual information and materials into the application and make this specification part of the disclosure.

Claims (7)

Fermentation method of the combination of thermus thermophilus and yeast,
First, a step (1) of fermenting Saccharomyces cerevisiae once or plural times under acidic conditions at 20 to 30 ° C. to grow yeast cells;
0 or 1 day after the yeast was further raised to a temperature of 55 to 65 ° C. after the late logarithmic growth phase, inoculated with Thermos thermophilus, fermented a second time under alkaline conditions, and fermented. Obtaining a thing (2);
A fermentation method for a combination of Thermos thermophilus and a yeast, comprising:   The method according to claim 1, wherein in step (1), the yeast is subjected to at least two times, at least three times, at least four times, five times, or six times of growth culture, whereby the yeast is brought to a logarithmic growth phase. The described method.   The method according to claim 1 or 2, wherein in step (2), one or more growth cultures are performed on the Thermos thermophilus before inoculating the yeast culture with the Thermos thermophilus.   The method further comprises the step of removing impurities by removing bacteria, decolorizing and deodorizing, and filtering the fermented liquor of step (2) to obtain a fermentation product by a combination of Thermos thermophilus and yeast. Item 4. The method according to any one of Items 1 to 3. The strain of Saccharomyces cerevisiae is CICC1596, which was purchased from the China Industrial Microbial Species Preservation and Management Center, and the storage number is CICC1596;
The method according to any one of claims 1 to 4, wherein the Thermus thermophilus HB27 used is purchased from the American Type Culture Collection (ATCC) and has a storage number BAA-163. The described method.   The yeast is subjected to low-temperature culturing, the temperature is 20 ° C to 30 ° C, the yeast is subjected to acidic culturing, and the pH value is 4.0 to 5.0. Or the method of claim 1.   0 or 1 day after the temperature is raised, the thermophilic bacteria are inoculated, 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 7.0 to 8.5. The method according to claim 1, wherein: