Thermus thermophilus fermentation product as well as preparation method, application and oil control product thereof

Abstract

The invention discloses a thermophilic thermophilic bacteria fermentation product, a preparation method and application thereof and an oil control product, and belongs to the technical field of cosmetics. Thermophilic thermophilic bacteria are cultured in a first-stage seed solution culture medium and a second-stage seed solution culture medium in sequence, the thermophilic thermophilic bacteria are promoted to the logarithmic phase, then a mulberry leaf extracting solution is added for fermentation, and the oil control product is obtained. According to the method, the growth stability of thalli is effectively prolonged, the stability of a fermentation microenvironment is maintained, the metabolic activity of the thermophilic thermophilic bacteria is improved, normal replication of thallus DNA at high temperature can be guaranteed, and the thermophilic thermophilic bacteria can grow at a high speed in the whole growth period. According to the method disclosed by the invention, the accumulation of byproducts can be avoided, active ingredients with an oil control effect in the thermophilic thermophilic bacteria are effectively enriched, and a thermophilic thermophilic bacteria fermentation product which has the effects of improving the mRNA expression quantity of AMPK, effectively inhibiting the activity of 5 alpha-reductase, inhibiting sebaceous gland cells from synthesizing lipid droplets and reducing hormone-induced lipid synthesis is obtained; the oil control activity is excellent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cosmetics, in particular to a Thermus thermophilus fermentation product, a preparation method, application and oil control product thereof. BACKGROUND

[0002] Thermus thermophilus is a representative group of extremophilic microorganisms, belonging to the domain Archaea, order Thermi, genus Thermus. It was first isolated from hot springs in Yellowstone National Park, USA (temperature 70-80℃, pH neutral to slightly alkaline), and has been found in high-temperature environments (such as volcanic hot springs, deep-sea hot springs, and industrial high-temperature wastewater pools) in many places around the world. It is adapted to a temperature range of 50-85℃, with an optimal growth temperature of about 70-75℃, and belongs to the "extreme thermophilic bacteria" (optimal growth temperature > 60℃). Thermus thermophilus is short rod-shaped or filamentous, with a diameter of about 0.5-0.8μm and a length of about 2-5μm. The cell wall contains unique pseudopeptide glycan, and the cell membrane phospholipid is mainly saturated fatty acid, with longer fatty acid chains and fewer branches, which can maintain the fluidity and integrity of the cell membrane at high temperatures and avoid thermal denaturation.

[0003] The secretion of oil (produced by sebaceous glands) on the skin is a normal physiological function. Healthy oil can form a natural protective film on the skin surface, thereby locking in moisture, resisting external dust pollution, and maintaining skin barrier stability. However, when oil secretion is excessive or unevenly distributed, it can cause a series of problems, such as clogged pores, reduced acne and blackhead growth, and sensitive symptoms such as redness and stinging. The current oil control effect of cosmetics still has deficiencies.

[0004] Thermus thermophilus fermentation product refers to various substances synthesized, secreted or transformed by the strain under artificial controlled fermentation conditions through its own metabolic activity. Patent CN115252649A discloses the use, composition and preparation of a thermophilic bacteria fermentation product in preventing hair loss and / or promoting hair growth, which has a hair loss prevention and / or hair growth effect, but it does not disclose the effect of the thermophilic bacteria fermentation product in oil control. Patent CN118931973A discloses a thermophilic bacteria fermentation product and its preparation method and application. The thermophilic bacteria fermentation product obtained by the specific fermentation method contains relatively rich soluble lipid substances, which is beneficial to improve cell vitality and ATP generation capacity when used in skin care products, and has the effects of anti-inflammatory, photodamage repair and skin antioxidant, but it does not disclose the effect of the thermophilic bacteria fermentation product in oil control. Patent CN116497077A discloses a method for preparing a thermophilic bacteria fermentation product using tremella extract. The method for preparing a thermophilic bacteria fermentation product using tremella extract, through the method of organic acid combined with high pressure cooking, makes the active ingredients in tremella further dissolve, creating a good growth environment for microorganisms. The tremella extract is directly used as a fermentation substrate, and thermophilic bacteria and saccharomyces cerevisiae are co-fermented to effectively improve the antioxidant performance of thermophilic bacteria fermentation, but it does not disclose the effect of the thermophilic bacteria fermentation product in oil control. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide a thermophilic bacteria fermentation product, its preparation method, application and oil control product. The thermophilic bacteria fermentation product has excellent oil control effect.

[0006] To achieve the above-mentioned purpose, the first aspect of the present application provides a preparation method of a thermophilic bacteria fermentation product, comprising the following steps:

[0007] Inoculating the thermophilic bacteria into the first-stage seed liquid culture medium for culture to obtain a first-stage seed liquid;

[0008] Inoculating the first-stage seed liquid into the second-stage seed liquid culture medium for culture to obtain a second-stage seed liquid;

[0009] Inoculating the second-stage seed liquid into the fermentation culture medium for fermentation to obtain a fermentation liquid, adding the mulberry leaf extract into the fermentation liquid for fermentation, centrifuging, filtering to obtain the thermophilic bacteria fermentation product.

[0010] The present application promotes the logarithmic growth of the thermophilic strain by successively culturing the thermophilic strain in a first seed liquid medium and a second seed liquid medium, so as to rapidly propagate the thermophilic strain and improve the biological activity thereof. Then, the thermophilic strain is fermented in a fermentation medium, and then fermented again by adding mulberry leaf extract, so as to effectively prolong the growth stability of the bacterial body, maintain the stability of the fermentation microenvironment, improve the metabolic activity of the thermophilic strain, ensure the normal replication of the bacterial DNA under high temperature, and maintain the high-speed growth of the thermophilic strain throughout the growth cycle. At the same time, the accumulation of by-products (such as palmitic acid, oleic acid and other fatty acids) can be avoided, the active ingredients with oil control effect in the thermophilic strain can be effectively enriched, and a thermophilic strain fermentation product with improved mRNA expression of AMPK (AMP-activated protein kinase), effectively inhibited 5α-reductase activity, inhibited the synthesis of lipid droplets in sebaceous gland cells, and reduced hormone-induced lipid synthesis is obtained, which has excellent oil control activity.

[0011] The inventors of the present application have found that the performance of the thermophilic strain fermentation product is closely related to the fermentation method, and different fermentation methods have a significant impact on the active ingredients of the thermophilic strain fermentation product. The thermophilic strain fermentation product prepared by the preparation method of the present application can directionally enrich the active ingredients with oil control effect in the thermophilic strain, and the obtained thermophilic strain fermentation product has various oil control active ingredients (such as polysaccharides, polypeptides, and organic acids such as propionic acid and lactic acid), which can achieve oil control effect in multiple ways and has wide application prospects.

[0012] As an embodiment of the present application, when the thermophilic strain is inoculated in the first seed liquid medium for culture, the culture temperature is 70-75℃, and the culture time is 12-16h. By controlling the temperature and time during the first culture within this range, the bacterial body can be rapidly propagated and the content of active ingredients can be improved.

[0013] As an embodiment of the present application, when the first seed liquid is inoculated in the second seed liquid medium for culture, the culture temperature is 70-75℃, and the culture time is 12-24h. By controlling the second culture parameters within this range, the thermophilic strain can be promoted to the logarithmic growth phase, so as to rapidly propagate the thermophilic strain and improve the biological activity thereof.

[0014] As an embodiment of the present application, when the second seed liquid is inoculated in the fermentation medium for fermentation, the fermentation temperature is 70-75℃, and the fermentation time is 12-16h. By controlling the fermentation temperature and time within this range, DNA synthesis can be efficiently catalyzed, the enrichment of active ingredients with oil control effect can be promoted, the stability of the fermentation microenvironment can be maintained, and the metabolic activity of the thermophilic strain can be improved.

[0015] As an implementation scheme of this application, when mulberry leaf extract is added to fermentation liquid for fermentation, the fermentation temperature is 60-65℃ and the fermentation time is 24-48h.

[0016] By controlling the fermentation temperature and time of thermophilic bacteria at each stage, it is possible to control the growth state of thermophilic bacteria, promote their fermentation and reproduction at the most suitable temperature, control the cell state of thermophilic bacteria, effectively enrich the active ingredients with oil-controlling effects, and reduce the content of by-products.

[0017] After adding mulberry leaf extract, the temperature was adjusted to 60-65℃. At the time of addition, the bacteria had already completed significant proliferation, and their metabolic focus gradually shifted from "rapid growth" to "product synthesis." 70-75℃ is the optimal temperature for the rapid proliferation of thermophilic bacteria. Lowering the temperature moderately slows down the basal metabolic rate of the bacteria, preventing them from excessively consuming nutrients (including the newly added mulberry leaf substrate) for their own growth. Instead, they allocate more metabolic resources to the synthesis of target products (such as extracellular polysaccharides, polypeptides, and propionic and lactic acids), thus improving product accumulation efficiency. During the feeding process, the addition of mulberry leaf solution causes slight changes in the overall composition and viscosity of the fermentation broth. Maintaining a high temperature of 70-75℃ may exacerbate water evaporation in the fermentation broth, leading to an abnormally high concentration of the culture medium. Simultaneously, at high temperatures, the bacteria's tolerance to environmental fluctuations (such as changes in pH and substrate concentration) decreases, making them prone to metabolic disorders. Lowering the temperature to 60-65℃ can reduce the rate of water evaporation, mitigate the impact of environmental fluctuations on the cells, and, together with the pH stabilizing effect of HEPES buffer, maintain the stability of the fermentation system and ensure the smooth progress of subsequent culture processes.

[0018] As an embodiment of this application, the primary seed culture medium comprises the following components: 0.2–2 g / L tryptone, 0.1–2 g / L yeast extract, 0.2–1 g / L sodium chloride, 50–200 mL / L water, and basal culture medium; and / or

[0019] The secondary seed culture medium comprises the following components: 0.2–2 g / L tryptone, 0.1–2 g / L yeast extract, 0.2–1 g / L sodium chloride, 50–200 mL / L water, 0.5–2 g / L glucose, 0.2–0.1 g / L glutamate, and basal culture medium;

[0020] The fermentation medium comprises the following components: 20-30 g / L tryptone, 10-15 g / L yeast extract, 2-6 g / L sodium chloride, 5-12 mmol / L HEPES buffer, and basal medium.

[0021] As an embodiment of this application, the basal culture medium in the primary seed culture medium includes TM culture medium.

[0022] As an embodiment of this application, the basal culture medium in the secondary seed culture medium includes TM culture medium.

[0023] As an embodiment of this application, the basal culture medium in the fermentation medium includes TM culture medium.

[0024] As an embodiment of this application, the volume ratio of the primary seed culture medium to the secondary seed culture medium is (0.05-0.2):1.

[0025] As an embodiment of this application, the volume ratio of the secondary seed liquid to the fermentation culture medium is (0.01~0.05):1.

[0026] As an implementation scheme of this application, when adding mulberry leaf extract to the fermentation liquid for fermentation, mulberry leaf extract is added every 6 to 8 hours.

[0027] As an embodiment of this application, the initial addition amount of the mulberry leaf extract is 5-10% of the total volume of the fermentation liquid.

[0028] As an embodiment of this application, the amount of mulberry leaf extract added each time is 3 to 5% of the total volume of the fermentation liquid.

[0029] As an embodiment of this application, the secondary seed culture is inoculated into a fermentation medium for fermentation, and air is introduced at a rate of 0.5–0.8 vvm (volume air / liquid volume / min); and / or

[0030] When adding mulberry leaf extract to the fermentation liquid for fermentation, air is introduced at a rate of 1-2 vvm.

[0031] The facultative anaerobic nature of thermophilic bacteria necessitates adjusting aeration as needed to balance growth and product synthesis. As facultative anaerobic microorganisms, thermophilic bacteria grow best under aerobic conditions. This means that while the bacteria can survive in low-oxygen environments, sufficient oxygen is crucial for their efficient growth and product synthesis. The bacteria utilize mulberry leaf components to synthesize the target product, a process that consumes more oxygen for metabolic reactions. Therefore, the aeration rate needs to be increased to 1–2 vvm to ensure a sufficient oxygen supply and prevent metabolic disorders or product synthesis inhibition due to hypoxia. After adding mulberry leaf extract, the composition of the fermentation broth changes. The viscosity of the fermentation broth may increase slightly due to solid particles in the mulberry leaves (even after processing, trace amounts may still exist) or soluble substances. Increased viscosity reduces oxygen transfer efficiency in the liquid. Increasing the aeration rate to 1–2 vvm at this time compensates for the reduced oxygen transfer efficiency by increasing the amount of air introduced per unit time, ensuring that the dissolved oxygen concentration in the fermentation broth remains at a suitable level required by the bacteria.

[0032] As an embodiment of this application, the preparation method of the mulberry leaf extract is as follows: mulberry leaf powder is mixed with water and sterilized by steam to obtain mulberry leaf extract.

[0033] As an embodiment of this application, the solid-liquid ratio of the mulberry leaf powder and water is 1g:(8-20)mL.

[0034] As an embodiment of this application, the steam sterilization temperature is 115-130°C and the time is 5-30 minutes.

[0035] As an embodiment of this application, the thermophilic bacteria are activated before being inoculated into the primary seed culture medium.

[0036] In a clean bench, take a small amount of thermophilic thermophilic bacteria in glycerol tubes or slant culture using a sterile inoculation loop and streak it onto a TM solid medium plate. Place the inoculated plate in a constant temperature incubator at 70-75℃ for 24-48 hours until a clear single colony appears on the plate, at which point the strain is activated.

[0037] This application does not limit the source of thermophilic bacteria. Those skilled in the art can obtain thermophilic bacteria through conventional commercial purchases.

[0038] For example, those skilled in the art can purchase the thermophilic bacteria described in this application from manufacturers such as Guangdong Provincial Microbial Culture Collection Center (GDMCC), China Industrial Microbial Culture Collection Center (CICC), China General Microbial Culture Collection Center (CGMCC), and Beina Biotechnology.

[0039] The thermophilic bacteria mentioned include at least one of the following: thermophilic bacteria GDMCC NO.1.3378, thermophilic bacteria GDMCC NO.1.2605, thermophilic bacteria BNCC 186418, thermophilic bacteria BNCC 387182, thermophilic bacteria BNCC 387183, and thermophilic bacteria CGMCC No.6186.

[0040] The second aspect of this application provides a thermophilic bacteria fermentation product prepared using the preparation method described above.

[0041] A third aspect of this application provides the use of *Thermophilicus thermophilus* fermentation products in the preparation of products that increase AMPK mRNA expression levels. The AMPK gene encodes AMP-activated protein kinase, playing a central role in cellular energy balance and metabolic regulation. AMPK is involved in reducing fat production and promoting fatty acid oxidation. The *Thermophilicus thermophilus* fermentation products described in this application can effectively increase AMPK mRNA expression levels, effectively activate AMPK, effectively block the signal transduction of sebum synthesis, and reduce lipid accumulation in sebaceous glands.

[0042] As an embodiment of this application, the product includes cosmetics or pharmaceuticals.

[0043] The fourth aspect of this application provides the use of a thermophilic bacteria fermentation product as a 5α-reductase inhibitor. 5α-reductase is a membrane protein located on the microsomes of target cells that relies on reduced coenzyme II (NADPH) to provide electrons, reducing the unsaturated double bond at the 4,5 positions of testosterone to convert it into the more active dihydrotestosterone, thereby promoting the proliferation and differentiation of sebaceous gland cells. The thermophilic bacteria fermentation product described in this application effectively inhibits the proliferation and differentiation of sebaceous gland cells by inhibiting 5α-reductase activity and reducing the synthesis of dihydrotestosterone.

[0044] The fifth aspect of this application provides the role of thermophilic bacteria fermentation products in the preparation of products with 5α-reductase inhibitory activity.

[0045] As an embodiment of this application, the product includes cosmetics or pharmaceuticals.

[0046] The sixth aspect of this application provides the use of thermophilic bacteria fermentation products in the preparation of products that inhibit the synthesis of lipid droplets by sebaceous gland cells.

[0047] Sebaceous gland cells synthesize lipid droplets, which are the source of skin oil production. The rate, total amount, and maturity of lipid droplet synthesis directly determine the level of sebum secretion. By inhibiting the synthesis of lipid droplets by sebaceous gland cells, the level of sebum secretion can be effectively reduced.

[0048] As an embodiment of this application, the product includes cosmetics or pharmaceuticals.

[0049] The seventh aspect of this application provides the use of thermophilic bacteria fermentation products in the preparation of products that reduce hormone-induced lipid synthesis.

[0050] Hormone levels are significantly correlated with lipid secretion and synthesis. For example, androgens (such as testosterone) are converted to dihydrotestosterone (DHT) by 5α-reductase. DHT activates androgen receptors, promoting lipid synthesis in sebaceous gland cells and leading to oily skin. The thermophilic bacteria fermentation product described in this application can significantly reduce hormone-induced lipid synthesis, especially reducing cortisol and androgen-induced lipid synthesis, effectively reducing lipid synthesis.

[0051] As an embodiment of this application, the hormone includes at least one of cortisol and androgens.

[0052] As an embodiment of this application, the product includes cosmetics or pharmaceuticals.

[0053] The eighth aspect of this application provides the use of thermophilic bacteria fermentation products in the preparation of products with oil-controlling effects.

[0054] The thermophilic bacteria fermentation product described in this application can effectively increase the mRNA expression level of AMPK and effectively inhibit 5α-reductase activity. It also inhibits the synthesis of lipid droplets by sebaceous gland cells and reduces hormone-induced lipid synthesis, exhibiting excellent oil-controlling activity. The thermophilic bacteria fermentation product contains multiple oil-controlling active ingredients, which can synergistically achieve oil-controlling effects through multiple pathways.

[0055] As an embodiment of this application, the product includes cosmetics or pharmaceuticals.

[0056] The ninth aspect of this application provides an oil-controlling product comprising the above-described thermophilic bacteria fermentation product, the product including cosmetics or pharmaceuticals.

[0057] As an embodiment of this application, the dosage form of the cosmetic is an aqueous solution, emulsion, cream, gel, mask, or serum.

[0058] As an embodiment of this application, the thermophilic bacteria fermentation product has a mass percentage content of 0.01-10% in cosmetics.

[0059] As an embodiment of this application, the thermophilic bacteria fermentation product has a mass percentage content of 0.1% to 5% in cosmetics.

[0060] As an embodiment of this application, the cosmetic also includes excipients acceptable in the cosmetic field.

[0061] As an embodiment of this application, the excipients acceptable in the cosmetic field include, but are not limited to, at least one of moisturizers, emollients, emulsifiers, preservatives, antioxidants, pH adjusters, penetration enhancers, chelating agents, surfactants, fragrances, and pigments.

[0062] As an embodiment of this application, suitable moisturizers include, but are not limited to, at least one of sodium hyaluronate, panthenol, hydroxyethyl urea, polyethylene glycol, xylitol, maltose, sodium polyglutamate, erythritol, sorbitol, mannitol, glucose, lactose, betaine, inositol, trehalose, and hydrogenated starch hydrolysate.

[0063] As an embodiment of this application, suitable emollients include, but are not limited to, at least one of the following: isododecane, isohexadecane, caprylic / capric triglyceride, pentaerythritol tetraisostearate, polydimethylsiloxane, isononyl isononanoate, diisostearate malate, phytosterol malate oleate, squalane, hexyl laurate, castor oil, hydrogenated polyisobutylene, octyldodecyl alcohol, shea butter, dioctyl carbonate, jojoba oil, lanolin, sweet almond oil, dioctyl adipate, and cocoyl caprylate / capric ester.

[0064] As an embodiment of this application, suitable emulsifiers include, but are not limited to, at least one of glyceryl stearate, polyglycerol-10 stearate, polyglycerol-10 laurate, polyglycerol-10 myristate, polyglycerol-5 trioleate, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PPG-13-decyltetradecyl alcohol polyether-24, sodium stearoyl glutamate, stearyl alcohol polyether-2, stearyl alcohol polyether-21, and hydrogenated lecithin.

[0065] As an embodiment of this application, suitable antioxidants include, but are not limited to, ascorbic acid and its derivatives, resveratrol, pentaerythritol tetra(bis-tert-butylhydroxyhydrocinnamic acid) ester, arbutin, tocopherol (vitamin E), sodium metabisulfite, and combinations thereof.

[0066] As an embodiment of this application, suitable pH adjusters include, but are not limited to, arginine, tromethamine, aminomethylpropanol, tetrahydroxypropyl ethylenediamine, triethanolamine, citric acid, sodium citrate, sodium hydroxide, potassium hydroxide, and combinations thereof.

[0067] As an embodiment of this application, suitable colorants include, but are not limited to, white, black, yellow, blue, green, pink, red, orange, purple, indigo, brown, and combinations thereof.

[0068] As an embodiment of this application, suitable preservatives include, but are not limited to, p-hydroxyacetophenone, 1,2-hexanediol, 1,2-pentanediol, isopentanediol, ethylhexylglycerin, caprylyl glycol, chlorphenesin, methylparaben, potassium sorbate, sodium benzoate, phenoxyethanol, caprylyl hydroxamic acid, glyceryl caprylate, cetylpyridinium chloride, cetylpyridinium chloride, and combinations thereof.

[0069] The beneficial effects of this application are as follows: By successively culturing *Thermophilus thermophilus* in primary and secondary seed culture media, the application promotes *Thermophilus thermophilus* to the logarithmic growth phase, enabling rapid reproduction and enhancing its biological activity. Then, it is fermented in a fermentation medium, followed by the addition of mulberry leaf extract. This effectively prolongs the growth stability of the bacteria, maintains the stability of the fermentation microenvironment, and improves the metabolic activity of *Thermophilus thermophilus*. It ensures normal DNA replication at high temperatures, allowing *Thermophilus thermophilus* to maintain high-speed growth throughout its entire growth cycle. Simultaneously, it avoids the accumulation of byproducts and effectively enriches the active components in *Thermophilus thermophilus* with oil-controlling effects. The resulting *Thermophilus thermophilus* fermentation product exhibits excellent oil-controlling activity, increasing AMPK mRNA expression, effectively inhibiting 5α-reductase activity, inhibiting sebaceous gland cell lipid droplet synthesis, and reducing hormone-induced lipid synthesis. Attached Figure Description

[0070] Figure 1 This is a staining image of lipid droplets in group BC of test example 3.

[0071] Figure 2 This is a lipid droplet staining image of the NC group in test example 3.

[0072] Figure 3 The image shows the lipid droplet staining of 0.1% salicylic acid in test example 3.

[0073] Figure 4 This is a lipid droplet staining image from Example 1 in Test Example 3.

[0074] Figure 5 The image shows a fluorescence microscope observation of the BC group in the cortisol stimulation experiment of test example 4.

[0075] Figure 6 The image shows a fluorescence microscope observation of the NC group in the cortisol stimulation experiment of test example 4.

[0076] Figure 7 This is a fluorescence microscope image of an example of the cortisol stimulation experiment in Test Example 4.

[0077] Figure 8 The image shows a fluorescence microscope observation of the BC group in the testosterone stimulation experiment of Example 4.

[0078] Figure 9The image shows a fluorescence microscope observation of the NC group in the testosterone stimulation experiment of Example 4.

[0079] Figure 10 This is a fluorescence micrograph of Example 1, which is the testosterone stimulation experiment in Example 4. Detailed Implementation

[0080] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0081] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0082] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0083] The following embodiments are provided to facilitate understanding of this application. These embodiments are provided not to limit the scope of the claims.

[0084] Example 1

[0085] A method for preparing a fermentation product of thermophilic bacteria includes the following steps:

[0086] (1) Preparation of mulberry leaf extract: Select fresh, disease-free mulberry leaves, rinse them with deionized water, and dry them in a 70℃ oven until constant weight. Crush the dried mulberry leaves through a 100-mesh sieve to obtain mulberry leaf powder; mix the mulberry leaf powder and water at a solid-liquid ratio of 1g:10mL evenly, sterilize by steam (121℃, 20min), and cool for later use.

[0087] (2) The thermophilic thermophilic bacteria slant culture was used and stored at 4℃. The thermophilic thermophilic bacteria was GDMCC NO.1.2605.

[0088] In a clean bench, a loopful of bacteria is picked up with a sterile inoculation loop and streaked onto a TM medium plate. After incubation at 72°C for 36 hours, a distinct single colony appears on the plate, thus obtaining an activated single colony.

[0089] (3) In a clean bench, add 60 mL of primary seed culture medium to a 250 mL Erlenmeyer flask, pick up a single activated colony with a sterile inoculation loop, inoculate it into the primary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 15 h to obtain the primary seed solution.

[0090] The primary seed culture medium consists of the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, and the remainder being TM culture medium.

[0091] (4) In a clean bench, add 120 mL of secondary seed culture medium to a 500 mL Erlenmeyer flask, inoculate the primary seed solution into the secondary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 20 h to obtain the secondary seed solution.

[0092] The secondary seed culture medium includes the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, 1 g / L glucose, 0.05 g / L glutamate, and the remainder TM medium.

[0093] The volume ratio of primary seed culture medium to secondary seed culture medium is 0.1:1.

[0094] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0095] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.6 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 15h to obtain the fermentation broth.

[0096] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0097] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0098] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.08:1, introduce air at a rate of 1.5 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0099] The amount of mulberry leaf extract added each time is 5% of the total volume of the fermentation liquid.

[0100] Example 2

[0101] The difference between Example 2 and Example 1 is that the preparation parameters in step (6) are different, but everything else is the same.

[0102] Step (6) in this embodiment is specifically as follows:

[0103] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.05:1, introduce air at a rate of 1.5 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0104] The amount of mulberry leaf extract added each time is 6% of the total volume of the fermentation liquid.

[0105] Example 3

[0106] The difference between Example 3 and Example 1 is that the preparation parameters in step (6) are different, but everything else is the same.

[0107] Step (6) in this embodiment is specifically as follows:

[0108] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.1:1, introduce air at a rate of 1.5 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0109] The amount of mulberry leaf extract added each time is 3% of the total volume of the fermentation liquid.

[0110] Example 4

[0111] The difference between Example 4 and Example 1 is that the preparation parameters in steps (5) and (6) are different, but everything else is the same.

[0112] Steps (5) and (6) in this embodiment are specifically as follows:

[0113] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0114] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.5vvm (volume air / liquid volume / min). The mixture was stirred at 300rpm and fermented at 0.04MPa and 72℃ for 15h to obtain the fermentation broth.

[0115] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0116] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0117] (6) Add mulberry leaf extract to fermentation liquid at a volume ratio of 0.08:1, introduce air at a rate of 2 vvm (volume air / liquid volume / min), ferment at 0.04 MPa and 62℃ for 40 h, add mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove solid particles remaining in mulberry leaves, and obtain thermophilic bacteria fermentation product.

[0118] The amount of mulberry leaf extract added each time is 5% of the total volume of the fermentation liquid.

[0119] Example 5

[0120] The difference between Example 5 and Example 1 is that the preparation parameters in steps (5) and (6) are different, but everything else is the same.

[0121] Steps (5) and (6) in this embodiment are specifically as follows:

[0122] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0123] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.8vvm (volume air / liquid volume / min). The mixture was stirred at 300rpm and fermented at 0.04MPa and 72℃ for 15h to obtain the fermentation broth.

[0124] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0125] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0126] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.08:1, introduce air at a rate of 1 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0127] The amount of mulberry leaf extract added each time is 5% of the total volume of the fermentation liquid.

[0128] Example 6

[0129] The difference between Example 6 and Example 1 is that the preparation parameters in steps (5) and (6) are different, but everything else is the same.

[0130] Steps (5) and (6) in this embodiment are specifically as follows:

[0131] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0132] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 1.5 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 15 h to obtain the fermentation broth.

[0133] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0134] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0135] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.08:1, introduce air at a rate of 0.6 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0136] The amount of mulberry leaf extract added each time is 5% of the total volume of the fermentation liquid.

[0137] Example 7

[0138] The difference between Example 7 and Example 1 is that the preparation parameters in steps (5) and (6) are different, but everything else is the same.

[0139] Steps (5) and (6) in this embodiment are specifically as follows:

[0140] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0141] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30 min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.3 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 15 h to obtain the fermentation broth.

[0142] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0143] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0144] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.08:1, introduce air at a rate of 0.5 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0145] The amount of mulberry leaf extract added each time is 5% of the total volume of the fermentation liquid.

[0146] Comparative Example 1

[0147] The difference between Comparative Example 1 and Example 1 is that no mulberry leaf extract was added to Comparative Example 1 (the total fermentation time was the same as that of Example 1).

[0148] A method for preparing a fermentation product of thermophilic bacteria includes the following steps:

[0149] (1) Preparation of mulberry leaf extract: Select fresh, disease-free mulberry leaves, rinse them with deionized water, and dry them in a 70℃ oven until constant weight. Crush the dried mulberry leaves through a 100-mesh sieve to obtain mulberry leaf powder; mix the mulberry leaf powder and water at a solid-liquid ratio of 1g:10mL evenly, sterilize by steam (121℃, 20min), and cool for later use.

[0150] (2) The thermophilic thermophilic bacteria slant culture was used and stored at 4℃. The thermophilic thermophilic bacteria was GDMCC NO.1.2605.

[0151] In a clean bench, a loopful of bacteria is picked up with a sterile inoculation loop and streaked onto a TM medium plate. After incubation at 72°C for 36 hours, a distinct single colony appears on the plate, thus obtaining an activated single colony.

[0152] (3) In a clean bench, add 60 mL of primary seed culture medium to a 250 mL Erlenmeyer flask, pick up a single activated colony with a sterile inoculation loop, inoculate it into the primary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 15 h to obtain the primary seed solution.

[0153] The primary seed culture medium consists of the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, and the remainder being TM culture medium.

[0154] (4) In a clean bench, add 120 mL of secondary seed culture medium to a 500 mL Erlenmeyer flask, inoculate the primary seed solution into the secondary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 20 h to obtain the secondary seed solution.

[0155] The secondary seed culture medium includes the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, 1 g / L glucose, 0.05 g / L glutamate, and the remainder TM medium.

[0156] The volume ratio of primary seed culture medium to secondary seed culture medium is 0.1:1.

[0157] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0158] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30 min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed culture was added, and air was introduced at a rate of 0.6 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 55 h. The mixture was then centrifuged at 8000 rpm for 10 min and filtered to obtain the thermophilic bacteria fermentation product.

[0159] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0160] Comparative Example 2

[0161] The difference between Comparative Example 2 and Example 1 is that the preparation parameters in step (6) are different.

[0162] Step (6) of this comparative example is as follows:

[0163] (6) Add the mulberry leaf extract to the fermentation liquid at a volume ratio of 0.02:1, introduce air at a rate of 1.5 vvm (volume air / liquid volume / min), and ferment at 0.04 MPa and 62℃ for 40 h. During this period, add the mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove the solid particles remaining in the mulberry leaves, and obtain the thermophilic bacteria fermentation product.

[0164] The amount of mulberry leaf extract added each time is 2% of the total volume of the fermentation liquid.

[0165] Comparative Example 3

[0166] The difference between Comparative Example 3 and Example 1 is that the preparation parameters in step (6) are different.

[0167] Step (6) of this comparative example is as follows:

[0168] (6) Add mulberry leaf extract to fermentation liquid at a volume ratio of 0.12:1, introduce air at a rate of 1.5 vvm (volume air / liquid volume / min), ferment at 0.04 MPa and 62℃ for 40 h, add mulberry leaf extract every 8 h, centrifuge at 8000 rpm for 10 min, filter to remove solid particles remaining in mulberry leaves, and obtain thermophilic bacteria fermentation product.

[0169] The amount of mulberry leaf extract added each time is 10% of the total volume of the fermentation liquid.

[0170] Comparative Example 4

[0171] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 uses an equal amount of aloe vera extract to replace mulberry leaf extract, while everything else is the same.

[0172] Preparation of aloe vera extract in this comparative example: Dry aloe vera was pulverized and passed through a 100-mesh sieve. Aloe vera powder and water were mixed evenly at a solid-liquid ratio of 1g:10mL. The mixture was then steam-sterilized (121℃, 20min) and cooled to obtain aloe vera extract.

[0173] Comparative Example 5

[0174] The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 directly mixed the fermentation product obtained without adding mulberry leaf extract (the total fermentation time was the same as in Example 1) with mulberry leaf extract.

[0175] A method for preparing a fermentation product of thermophilic bacteria includes the following steps:

[0176] (1) Preparation of mulberry leaf extract: Select fresh, disease-free mulberry leaves, rinse them with deionized water, and dry them in a 70℃ oven until constant weight. Crush the dried mulberry leaves through a 100-mesh sieve to obtain mulberry leaf powder; mix the mulberry leaf powder and water at a solid-liquid ratio of 1g:10mL evenly, sterilize by steam (121℃, 20min), and cool for later use.

[0177] (2) The thermophilic thermophilic bacteria slant culture was used and stored at 4℃. The thermophilic thermophilic bacteria was GDMCC NO.1.2605.

[0178] In a clean bench, a loopful of bacteria is picked up with a sterile inoculation loop and streaked onto a TM medium plate. After incubation at 72°C for 36 hours, a distinct single colony appears on the plate, thus obtaining an activated single colony.

[0179] (3) In a clean bench, add 60 mL of primary seed culture medium to a 250 mL Erlenmeyer flask, pick up a single activated colony with a sterile inoculation loop, inoculate it into the primary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 15 h to obtain the primary seed solution.

[0180] The primary seed culture medium consists of the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, and the remainder being TM culture medium.

[0181] (4) In a clean bench, add 120 mL of secondary seed culture medium to a 500 mL Erlenmeyer flask, inoculate the primary seed solution into the secondary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 20 h to obtain the secondary seed solution.

[0182] The secondary seed culture medium includes the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, 1 g / L glucose, 0.05 g / L glutamate, and the remainder TM medium.

[0183] The volume ratio of primary seed culture medium to secondary seed culture medium is 0.1:1.

[0184] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0185] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.6 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 15 h. The volume of the fermentation liquid was measured at this time. Fermentation was carried out at 0.04 MPa and 62℃ for 40 h. After fermentation, the mixture was centrifuged at 8000 rpm for 10 min, filtered, and the fermentation product was obtained. The fermentation product was mixed evenly with mulberry leaf extract to obtain the thermophilic bacteria fermentation product. The volume ratio of mulberry leaf extract to fermentation liquid after 15 h of fermentation was 0.08:1.

[0186] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0187] Comparative Example 6

[0188] The difference between Comparative Example 6 and Example 1 is that the preparation methods are different.

[0189] A method for preparing a fermentation product of thermophilic bacteria includes the following steps:

[0190] (1) Preparation of mulberry leaf extract: Select fresh, disease-free mulberry leaves, rinse them with deionized water, and dry them in a 70℃ oven until constant weight. Crush the dried mulberry leaves through a 100-mesh sieve to obtain mulberry leaf powder; mix the mulberry leaf powder and water at a solid-liquid ratio of 1g:10mL evenly, sterilize by steam (121℃, 20min), and cool for later use.

[0191] (2) The thermophilic thermophilic bacteria slant culture was used and stored at 4℃. The thermophilic thermophilic bacteria was GDMCC NO.1.2605.

[0192] In a clean bench, a loopful of bacteria is picked up with a sterile inoculation loop and streaked onto a TM medium plate. After incubation at 72°C for 36 hours, a distinct single colony appears on the plate, thus obtaining an activated single colony.

[0193] (3) In a clean bench, add 60 mL of primary seed culture medium to a 250 mL Erlenmeyer flask, pick up a single activated colony with a sterile inoculation loop, inoculate it into the primary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 15 h to obtain the primary seed solution.

[0194] The primary seed culture medium consists of the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, and the remainder being TM culture medium.

[0195] (4) In a clean bench, add 120 mL of secondary seed culture medium to a 500 mL Erlenmeyer flask, inoculate the primary seed solution into the secondary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 20 h to obtain the secondary seed solution.

[0196] The secondary seed culture medium includes the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, 1 g / L glucose, 0.05 g / L glutamate, and the remainder TM medium.

[0197] The volume ratio of primary seed culture medium to secondary seed culture medium is 0.1:1.

[0198] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0199] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid and mulberry leaf extract were added. Air was introduced at a rate of 0.6 vvm (volume air / liquid volume / min) and stirred at 300 rpm. Fermentation was carried out at 0.04 MPa and 72℃ for 55 h. The mixture was centrifuged at 8000 rpm for 10 min and filtered to remove the solid particles remaining from the mulberry leaves, yielding the thermophilic bacteria fermentation product.

[0200] The fermentation medium consists of the following components: 1.5 g / L tryptone, 0.8 g / L yeast extract, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0201] The volume ratio of the secondary seed culture to the fermentation medium is 0.02:1.

[0202] Comparative Example 7

[0203] The difference between Comparative Example 7 and Example 1 is that mulberry leaf extract was added to the seed culture medium in Comparative Example 7.

[0204] A method for preparing a fermentation product of thermophilic bacteria includes the following steps:

[0205] (1) Preparation of mulberry leaf extract: Select fresh, disease-free mulberry leaves, rinse them with deionized water, and dry them in a 70℃ oven until constant weight. Crush the dried mulberry leaves through a 100-mesh sieve to obtain mulberry leaf powder; mix the mulberry leaf powder and water at a solid-liquid ratio of 1g:10mL evenly, sterilize by steam (121℃, 20min), and cool for later use.

[0206] (2) The thermophilic thermophilic bacteria slant culture was used and stored at 4℃. The thermophilic thermophilic bacteria was GDMCC NO.1.2605.

[0207] In a clean bench, a loopful of bacteria is picked up with a sterile inoculation loop and streaked onto a TM medium plate. After incubation at 72°C for 36 hours, a distinct single colony appears on the plate, thus obtaining an activated single colony.

[0208] (3) In a clean bench, add 60 mL of primary seed culture medium to a 250 mL Erlenmeyer flask, pick up a single activated colony with a sterile inoculation loop, inoculate it into the primary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 15 h to obtain the primary seed solution.

[0209] The primary seed culture medium consists of the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 100 mL / L water, and the remainder being TM culture medium.

[0210] (4) In a clean bench, add 120 mL of secondary seed culture medium to a 500 mL Erlenmeyer flask, inoculate the primary seed solution into the secondary seed culture medium, and place the Erlenmeyer flask in a constant temperature shaker at 72℃ and 200 rpm for 20 h to obtain the secondary seed solution.

[0211] The secondary seed culture medium includes the following components: 1 g / L tryptone, 0.5 g / L yeast extract, 0.5 g / L sodium chloride, 10 g / L mulberry leaf extract, 100 mL / L water, 1 g / L glucose, 0.05 g / L glutamate, and the remainder TM medium.

[0212] The volume ratio of primary seed culture medium to secondary seed culture medium is 0.1:1.

[0213] (5) Clean the fermenter and sterilize it with steam (121℃, 30min).

[0214] The fermentation medium was added to the fermenter and steam sterilized again (121℃, 30min). After sterilization, sterile air was introduced until the temperature inside the fermenter was 55℃±1℃. Secondary seed liquid was added, and air was introduced at a rate of 0.6 vvm (volume air / liquid volume / min). The mixture was stirred at 300 rpm and fermented at 0.04 MPa and 72℃ for 55 h. The mixture was then centrifuged at 8000 rpm for 10 min and filtered to remove the solid particles remaining from the mulberry leaves, yielding the thermophilic bacteria fermentation product.

[0215] The fermentation medium consists of the following components: 25 g / L tryptone, 13 g / L yeast extract, 5 g / L sodium chloride, 10 mmol / L HEPES buffer, and the remainder is TM medium.

[0216] Test Example 1

[0217] 1.5α-Reductase Inhibition Assay

[0218] 1.1 Experimental Principles and Methods

[0219] 1.1.1 Experimental materials: 5α-reductase preparation solution (extracted from the liver of male mice, protein concentration 0.08 mg / mL, Guangzhou Analysis and Testing Center, China), BCA protein concentration assay kit (Beyotime), testosterone (Sinopharm Chemical Reagent Co., Ltd.), reduced coenzyme II NADPH (Beijing Bailingwei Technology Development Co., Ltd.), anhydrous ethanol (Tianjin Kangkede Technology Co., Ltd.), sucrose (Zhejiang Tianyi Food Additives Co., Ltd.), dutasteride (DTS) (Tianjin Kangkede Technology Co., Ltd.), sodium nitrate (Sinopharm Chemical Reagent Co., Ltd.).

[0220] 1.1.2 Experimental instruments: microplate reader (Perkinelmer, Singapore, EnSight), mixer (SCILOGEX, USA, MX-S), water bath (Thermo Fisher Scientific, GP10), analytical balance (Sartorius, MCA224S-2CCN-I-QP99).

[0221] 1.2 Experimental Principle:

[0222] 5α-Reductase catalyzes the conversion of testosterone to dihydrotestosterone, a process that requires the coenzyme NADPH. Reduced NADPH exhibits a characteristic absorption at 340 nm, and as the reaction proceeds, NADPH is converted to oxidized NADP. +The characteristic absorption at 340 nm will then disappear. The change in NADPH absorbance in the reaction system at 340 nm reflects the change in 5α-reductase activity. Inhibiting 5α-reductase to reduce dihydrotestosterone levels is an important way to alleviate excessive sebum secretion. This experiment evaluates the oil-controlling efficacy of the samples by testing their effect on 5α-reductase.

[0223] 1.3 Test Methods

[0224] 1.3.1 The reaction system is shown in Table 1.

[0225] Table 1

[0226]

[0227] 1.3.2 Blank group determination: Following the above reaction system, 12 μL of 0.08 mg / mL liver homogenate, 108 μL of enzyme diluent, 60 μL of 2 mg / mL NADPH tetrasodium salt, 10 μL of 1 mg / mL testosterone, and 10 μL of phosphate buffer (pH 7.2 ± 0.2) were added to each well of a 96-well plate. After thorough mixing, the absorbance A0 was measured. After incubation at 37℃ for 20 min, the absorbance A1 was measured, yielding the NADPH absorbance change value ΔA0.

[0228] 1.3.3 Sample / Positive Group (0.5 mg / mL DTS) Determination: The sample was prepared into a 1 mg / mL test solution. Liver tissue homogenate, enzyme diluent, test solution, testosterone, and NADPH were added sequentially to a 96-well plate according to the above reaction system. After thorough mixing, the absorbance An was measured. After incubation at 37℃ for 20 min, the absorbance was measured again, and the change in NADPH ΔAn was determined.

[0229] 1.3.4 Calculation of 5α-reductase inhibition rate

[0230] △A0=(A0-A1) / A0

[0231] △An=(A0-An) / A0

[0232] 5α-Reductase inhibition rate (%) = 100% * (△A0 - △An) / △A0

[0233] Where: A0: absorbance of the reaction system after mixing; A1: absorbance of the blank group reaction system after 20 min; An: absorbance of the sample group reaction system after 20 min.

[0234] 1.4 Result Interpretation

[0235] Compared with the blank control group, the positive control group showed a significantly increased 5α-reductase inhibition rate of 0.5 mg / mL DTS, and the difference between the groups was statistically significant (P<0.05), thus the experimental system was considered effective.

[0236] Compared with the blank control group, the 5α-reductase inhibition rate of the sample at a certain detection concentration was significantly increased, and the difference between groups was statistically significant (P<0.05). Therefore, the sample has the effect of inhibiting 5α-reductase at this concentration and has the effect of controlling oil production.

[0237] Table 2

[0238]

[0239]

[0240] Note: Dunnett-t test: *P<0.05 / **P<0.01 / ***P<0.001 indicates that the difference between the sample group and the negative control is statistically significant.

[0241] As can be seen from Table 1, the thermophilic bacteria fermentation product described in this application can effectively inhibit 5α-reductase activity.

[0242] Test Example 2

[0243] 2. AMPK mRNA expression assay

[0244] 2.1 The AMPK gene encodes AMP-activated protein kinase (AMPK), a key metabolic regulatory enzyme that plays a central role in cellular energy balance and metabolic regulation. Studies have shown that AMPK is involved in reducing fat production and promoting fatty acid oxidation.

[0245] 2.2 Instruments and equipment: microbalance, clean bench, carbon dioxide incubator, microplate reader, water bath, ultra-micro spectrophotometer, real-time fluorescence PCR instrument.

[0246] 2.3 Reagents and Materials

[0247] 2.3.1 Reagents: Cell Counting Kit-8 reagent; Sebomed basal medium; 0.25% Trypsin-EDTA; EGF; 5α-DHT; Linoleic acid; FBS; FastPure Cell / Tissue Total RNA Isolation Kit; OneStep RT-qPCR Kit; Chloroform; Anhydrous ethanol; Primers.

[0248] 2.3.2 Cell line SZ95.

[0249] 2.4 Test Methods

[0250] 2.4.1 mRNA expression assay

[0251] 2.4.1.1 Pretreatment of test substance

[0252] The original sample solution was sterilized by filtration using a 0.22 μm filter, and then the sample was prepared to the required test concentration using culture medium according to the table.

[0253] 2.4.1.2 Digesting Cells

[0254] Cells reaching the logarithmic growth phase can be seeded in 96-well plates for assays. The procedure is as follows: When keratinocytes in the cell culture flask reach 50-70% confluence, remove the culture medium and wash twice with 5-10 mL of sterile PBS, gently agitating to ensure no significant foaming occurs. Remove the washing solution. Add trypsin / EDTA solution to the culture flask (1 mL for T25 flasks, 2 mL for T75 flasks), aspirate after 30 seconds, and incubate at room temperature for 3-7 minutes, observing the cell status periodically. When more than 50% of the cells are floating, gently tap the culture flask to detach the cells. When the vast majority of cells have detached, add 2 volumes of room temperature complete culture medium containing FBS and gently pipette the cells. Transfer the cell suspension to centrifuge tubes; centrifuge for 5 minutes and remove the supernatant.

[0255] 2.4.1.3 Cell counting and seeding / plating

[0256] Gently pipette the cells into complete culture medium to resuspend the pellet, forming a uniformly distributed single-cell suspension. Use a cell counter to count the cells and calculate the number of viable cells and cell viability. After cell counting, use a multichannel pipette to plate the cells to achieve a culture medium volume of 100 μL / well and a viable cell count of 1–2 × 10⁻⁶ cells / well. 4 cell / well.

[0257] 2.4.1.4 Cell Plating

[0258] After cell counting, dilute to the desired concentration and add cell suspension to cell culture dishes to achieve a culture volume of 4 mL / well and a viable cell count of 5 × 10⁶ cells / well. 5 cell / well.

[0259] 2.4.1.5 Sample Addition Processing

[0260] After 24 hours of plating, the cell fusion rate was 40-60%. Different concentrations of the test substance were added to achieve the final concentration required for testing, and the test wells were set to be parallel (n≥3). The test groups were set as follows: blank group, negative group, and sample group.

[0261] Table 3

[0262] Grouping Concentration BC / NC: 5aDHT + oleic acid / Sample: Thermus thermophilus fermentation product 1％

[0263] 2.4.1.6 RNA extraction:

[0264] 48 hours after sample loading, waste liquid was removed, cells were collected, and mRNA was extracted using a cell / tissue mRNA extraction kit.

[0265] 2.4.1.7 RNA quality testing

[0266] RNA purity detection: RNA purity was detected using an ultra-micro spectrophotometer, with a detection standard OD(260 / 280) = 1.8-2.1.

[0267] 2.4.1.8 Primer Design

[0268] Specialized primer synthesis software is used to synthesize specific primers for the gene (AMPK) that needs to be detected.

[0269] 2.1.1.9 RT-PCR detection of target gene expression

[0270] Add 10 μL of 2×One Step SYBR Master Mix, 0.65 μL of RT Enzyme Mix, and specific primers and template RNA according to the specified ratio. Make up the volume with water, mix well, and use for testing. The reaction program for the real-time quantitative RNA instrument is shown in Table 4.

[0271] Table 4

[0272] Stage 1 Reverse transcription Rep: 1 50℃ 5 min Stage 2 Pre-denaturation Rep: 1 95℃ 3 min Stage 3 Cycling reaction Rep: 45 95℃ / 60℃ 10 sec / 30 sec Stage 4 Melting curve / According to instrument requirements

[0273] After the reaction is complete, confirm the amplification curve and melting curve of Real-Time PCR, and prepare the standard curve, etc.

[0274] 2.5 Data Statistics

[0275] The average Ct value of three replicates for each gene in each cDNA sample was used as the amplification result, and the amplification amount of the ACTB gene was used as the internal reference gene to calculate the relative expression level of the gene.

[0276] Relative gene expression level: 2 -△△CT

[0277] In the formula: ①CT processing - CT internal reference (processing) = △CT processing

[0278] ②CT control - CT internal reference (control) = △CT control

[0279] ③△CT processing - △CT control = △△CT

[0280] The statistical analysis software used was GraphPadPrism 8.0 to perform one-way ANOVA between groups. p<0.05 was considered statistically significant.

[0281] 2.6 Judgment Criteria: If the mRNA expression level of AMPK in human SZ95 cells in the sample group is significantly higher than that in the control group, then the sample can be judged to effectively upregulate the mRNA expression of AMPK in human SZ95 cells.

[0282] Table 5

[0283]

[0284]

[0285] *P<0.05 / *P<0.01 indicates that the difference between the sample group and the negative control is statistically significant.

[0286] As can be seen from Table 5, the thermophilic bacteria fermentation product described in this application can effectively increase the mRNA expression level of AMPK.

[0287] Test Example 3

[0288] 3. Assay for inhibiting lipid droplet synthesis in sebaceous gland cells

[0289] 3.1 Principle

[0290] The evaluation was conducted using human immortalized sebaceous gland cells SZ95. An in vitro sebum secretion model was established, and after drug treatment, the oil-controlling efficacy of the test samples was evaluated by assessing the lipid droplet synthesis trend.

[0291] 3.2 Instruments and Equipment

[0292] Microbalance, biosafety cabinet, CO2 incubator, microplate reader, water bath, fluorescence microscope.

[0293] 3.3 Reagents and Materials

[0294] 3.3.1 Reagents: Cell Counting Kit-8 reagent; Sebomed basal medium; fetal bovine serum; 0.25% Trypsin-EDTA; EGF; 5α-DHT; linoleic acid; Lile-red staining kit.

[0295] 3.3.2 Cell lines

[0296] SZ95 human sebaceous gland cells

[0297] 3.4 Test Methods

[0298] 3.4.1 Pretreatment of test substance

[0299] The original sample solution was sterilized by filtration using a 0.22 μm filter, and then the sample was prepared to the required test concentration using culture medium according to the table.

[0300] 3.4.2 Lipid droplet staining

[0301] Cell seeding: After diluting cells to the desired seeding density with cell culture medium, cells were seeded into 96-well plates and incubated in a CO2 incubator for 24 h ± 2 h. For drug administration, once the cells in the 96-well plates reached a suitable confluence, drug administration was performed according to the experimental groups. The culture medium in the wells was discarded. The test wells were filled with cell culture medium containing the test substance and 5α-DHT and oleic acid; the control wells were filled with cell induction medium containing only 5α-DHT and oleic acid. After drug administration, the 96-well plates were incubated in a CO2 incubator for 24 h ± 2 h.

[0302] Staining: Discard the old solution from the well plate, wash the well plate twice with buffer solution, add fixative to each well to fix the cells; discard the fixative, add Lile-Red solution and stain at 37℃ for 15 min; then discard the staining solution, add an appropriate amount of ultrapure water and wash for 5 min; repeat 2-3 times and observe under a microscope.

[0303] 3.4.3 Observation and content calculation of lipid droplets by staining

[0304] Under a microscope, Lile-red staining was observed to bind to intracellular lipid droplets, emitting red fluorescence. The staining results were photographed and recorded. Lipid secretion was determined based on the red fluorescence in the images, with the amount of lipid droplets secreted being directly proportional to the fluorescence intensity. ImageJ was used to calculate the percentage of stained oil droplet area. The lipid droplet staining images for groups BC, NC, 0.01% salicylic acid, and Example 1 are shown below. Figures 1-4 As shown.

[0305] Table 6

[0306]

[0307]

[0308] Compared with the control group: *, p<0.05; **, p<0.01.

[0309] As can be seen from Table 6, the fermentation products of thermophilic bacteria described in this application can effectively inhibit the synthesis of lipid droplets by sebaceous gland cells.

[0310] Test Example 4

[0311] 4. Hormone (testosterone, cortisol) induced lipid synthesis assay

[0312] 4.1 Cortisol-induced lipid synthesis assay

[0313] 4.1.1 Purpose

[0314] An in vitro oil model was established by inducing human sebaceous gland cells (SZ95) with cortisol to determine the oil-controlling effect.

[0315] 4.1.2 Test Materials

[0316] 4.1.2.1 Main Reagents

[0317] PBS (1× Corning), SZ95 (ATCC), DMEM cell culture medium (Gibco), fetal bovine serum (Gibco), trypsin (Gibco), penicillin-streptomycin solution (100×, Gibco), linoleic acid (Maclean), testosterone (Titan), Nile red (Aladdin).

[0318] Main equipment: Biosafety cabinet (ESCO, AC2-4S1), inverted microscope (OLYMPUS, CKX53), cell counter (THERMO countess3), refrigerated centrifuge (Xiangyi, CHT210R), cell incubator (PHCbi, MCO-18AIC), motorized inverted fluorescence microscope (Nikon).

[0319] Table 7 shows the experimental design for cortisol-induced lipid content reduction.

[0320] Table 7

[0321]

[0322] 4.1.3 Experimental Methods The experiments were conducted according to Tables 7 and 8, with the specific steps as follows:

[0323] 4.1.3.1 Plating: Collect cells in the logarithmic growth phase, count the cells, and then plate them at a cell density of 2.5 × 10⁻⁶. 5 Inoculate one well per cell into a 6-well culture plate.

[0324] 4.1.3.2 Grouping: After culturing in an incubator (37℃, 5% CO2) for 24 hours, the supernatant was discarded, and the cells were divided into a blank group, a model group, and a sample group.

[0325] 4.1.3.3 Sample addition: 1 mL of DMEM basal medium was added to the blank group, 200 μM cortisol was added to the model group, and 1 mL of 200 μM cortisol was added to the sample group and cultured for 3 h, followed by the addition of 10 μL of thermophilic bacteria fermentation product.

[0326] 4.1.3.4 Incubation: After the sample is added, place it in an incubator (37℃, 5% CO2) and incubate for 24 hours.

[0327] 4.1.3.5 Staining and photography: Discard the culture medium, wash with PBS, then fix with 4% formaldehyde for 15 minutes at room temperature, wash with PBS, stain the fixed cells with 1 μg / ml Nile Red solution for 15 minutes, discard the solution, wash with PBS, observe and photograph with an electric inverted fluorescence microscope.

[0328] The fluorescence microscopy images of the BC group, NC group, and Example 1 in the cortisol stimulation test are shown below. Figures 5-7 As shown.

[0329] 4.1.3.6 Data Processing: The average fluorescence intensity per unit area was obtained using ImageJ software. Statistical analysis was performed using the t-test two-tailed method.

[0330] 4.1.3.7 Calculation Method

[0331] Relative fluorescence intensity = sample fluorescence intensity / blank group fluorescence intensity.

[0332] Lipid content decrease rate = 100 * (relative fluorescence intensity of model group - relative fluorescence intensity of sample group) / relative fluorescence intensity of model group.

[0333] 4.2 Testosterone-induced lipid synthesis assay

[0334] 4.2.1 Purpose

[0335] An in vitro oil model was established by inducing human sebaceous gland cells (SZ95) with testosterone to determine the oil-controlling effect.

[0336] 4.2.2 Test Materials

[0337] 4.2.2.1 Main Reagents

[0338] PBS (1× Corning), SZ95 (ATCC), DMEM cell culture medium (Gibco), fetal bovine serum (Gibco), trypsin (Gibco), penicillin-streptomycin solution (100×, Gibco), cortisol (Aladdin), Nile red (Aladdin).

[0339] Main equipment: Biosafety cabinet (ESCO, AC2-4S1), inverted microscope (OLYMPUS, CKX53), cell counter (THERMO countess3), refrigerated centrifuge (Xiangyi, CHT210R), cell incubator (PHCbi, MCO-18AIC), motorized inverted fluorescence microscope (Nikon).

[0340] Table 8 shows the experimental design for testosterone-induced lipid content reduction.

[0341] Table 8

[0342]

[0343] 4.2.3 Experimental Methods The experiments were conducted according to Tables 7 and 8, with the specific steps as follows:

[0344] 4.2.3.1 Plating: Collect cells in the logarithmic growth phase, count the cells, and then plate them at a cell density of 2.5 × 10⁻⁶. 5 Inoculate one well per cell into a 6-well culture plate.

[0345] 4.2.3.2 Grouping: After culturing in an incubator (37℃, 5% CO2) for 24h, the supernatant was discarded, and the cells were divided into a blank group, a model group, and a sample group.

[0346] 4.2.3.3 Sample addition: 1 mL of DMEM basal medium was added to the blank group, 1 mL of 0.313 mM linoleic acid + 62.5 μM testosterone was added to the model group, and 1 mL of 0.313 mM linoleic acid + 62.5 μM testosterone was added to the sample group and cultured for 3 h. Then, 10 μL of thermophilic bacteria fermentation product was added to each group.

[0347] 4.2.3.4 Incubation: After the sample is added, place it in an incubator (37℃, 5% CO2) and incubate for 24 hours.

[0348] 4.2.3.5 Staining and photography: Discard the culture medium, wash with PBS, then fix with 4% formaldehyde for 15 minutes at room temperature, wash with PBS, stain the fixed cells with 1 μg / ml Nile Red solution for 15 minutes, discard the solution, wash with PBS, observe and photograph with an electric inverted fluorescence microscope.

[0349] Among them, the fluorescence microscopic observation images of the BC group, NC group, and Example 1 in the testosterone stimulation test are as follows: Figures 8-10 As shown.

[0350] 4.2.3.6 Data Processing: The average fluorescence intensity per unit area was obtained using ImageJ software. Statistical analysis was performed using the t-test two-tailed method.

[0351] 4.2.3.7 Calculation Method

[0352] Relative fluorescence intensity = sample fluorescence intensity / blank group fluorescence intensity.

[0353] Lipid content decrease rate = 100 * (relative fluorescence intensity of model group - relative fluorescence intensity of sample group) / relative fluorescence intensity of model group.

[0354] Table 9

[0355]

[0356] As can be seen from Table 9, the thermophilic bacteria fermentation products described in this application have the effect of reducing hormone (cortisol, androgen)-induced lipid synthesis.

[0357] In summary, the thermophilic bacteria fermentation product described in this application has the effects of increasing AMPK mRNA expression, effectively inhibiting 5α-reductase activity, inhibiting sebaceous gland cell lipid droplet synthesis, and reducing hormone-induced lipid synthesis, thus exhibiting excellent oil-controlling activity.

[0358] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for preparing fermentation products of thermophilic bacteria, characterized in that, Includes the following steps: The thermophilic bacteria were inoculated into a primary seed culture medium and cultured to obtain a primary seed culture. The primary seed culture was inoculated into the secondary seed culture medium and cultured to obtain the secondary seed culture; The secondary seed culture was inoculated into a fermentation medium for fermentation to obtain a fermentation broth. Mulberry leaf extract was added to the fermentation broth for fermentation, centrifuged, and filtered to obtain the thermophilic bacteria fermentation product.

2. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, Satisfy at least one of the following (a) to (d): (a) When inoculating Thermophilic Bacillus into a primary seed culture medium, the culture temperature is 70-75°C and the culture time is 12-16 h; (b) When inoculating the primary seed culture into the secondary seed culture medium, the culture temperature is 70-75℃ and the culture time is 12-24h; (c) When the secondary seed liquid is inoculated into the fermentation medium for fermentation, the fermentation temperature is 70-75℃ and the fermentation time is 12-16h. (d) When adding mulberry leaf extract to fermentation liquid, the fermentation temperature is 60-65℃ and the fermentation time is 24-48h.

3. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, The primary seed culture medium comprises the following components: 0.2–2 g / L tryptone, 0.1–2 g / L yeast extract, 0.2–1 g / L sodium chloride, 50–200 mL / L water, and basal culture medium; and / or The secondary seed culture medium comprises the following components: 0.2–2 g / L tryptone, 0.1–2 g / L yeast extract, 0.2–1 g / L sodium chloride, 50–200 mL / L water, 0.5–2 g / L glucose, 0.2–0.1 g / L glutamate, and basal medium; and / or The fermentation medium comprises the following components: 20–30 g / L tryptone, 10–15 g / L yeast extract, 2–6 g / L sodium chloride, 5–12 mmol / L HEPES buffer, and basal medium.

4. The method for preparing the fermentation product of thermophilic bacteria according to claim 3, characterized in that, The basal culture medium in the primary seed culture medium includes TM medium; and / or The basal culture medium in the secondary seed culture medium includes TM medium; and / or The basal culture medium in the fermentation medium includes TM medium.

5. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, The volume ratio of the primary seed culture medium to the secondary seed culture medium is (0.05–0.2):1; and / or The volume ratio of the secondary seed liquid to the fermentation medium is (0.01-0.05):

1.

6. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, When adding mulberry leaf extract to the fermentation liquid for fermentation, add mulberry leaf extract every 6 to 8 hours.

7. The method for preparing the fermentation product of thermophilic bacteria according to claim 6, characterized in that, The initial addition of the mulberry leaf extract is 5-10% of the total volume of the fermentation broth; and / or The amount of mulberry leaf extract added each time is 3 to 5% of the total volume of the fermentation liquid.

8. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, The secondary seed culture was inoculated into the fermentation medium for fermentation, with air introduced at a rate of 0.5–0.8 vvm; and / or When adding mulberry leaf extract to the fermentation liquid for fermentation, air is introduced at a rate of 1-2 vvm.

9. The method for preparing the fermentation product of thermophilic bacteria according to claim 1, characterized in that, The method for preparing the mulberry leaf extract is as follows: mulberry leaf powder is mixed with water and sterilized by steam to obtain the mulberry leaf extract.

10. The method for preparing the fermentation product of thermophilic bacteria according to claim 9, characterized in that, The solid-liquid ratio of the mulberry leaf powder to water is 1g:(8-20)mL; and / or The steam sterilization temperature is 115–130°C, and the time is 5–30 minutes.

11. A fermentation product of a thermophilic bacterium, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 10.

12. The use of the thermophilic bacteria fermentation product of claim 11 in the preparation of products that enhance the mRNA expression level of AMPK.

13. The application according to claim 12, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

14. The use of the thermophilic bacteria fermentation product of claim 11 as a 5α-reductase inhibitor.

15. The use of the thermophilic bacteria fermentation product of claim 11 in the preparation of products having 5α-reductase inhibitory activity and / or inhibiting sebaceous gland cell lipid droplet synthesis and / or reducing hormone-induced lipid synthesis.

16. The application according to claim 15, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

17. The application according to claim 15, characterized in that, The hormones include at least one of cortisol and androgens.

18. The use of the thermophilic bacteria fermentation product according to claim 11 in the preparation of a product with oil-controlling effect.

19. The application according to claim 18, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

20. An oil-controlling product, characterized in that, The product includes the thermophilic bacteria fermentation product of claim 11, and the product includes cosmetics or pharmaceuticals.

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