Thermus thermophilus fermentation broth, and preparation method and use thereof

Abstract

The application discloses a thermus thermophilus fermentation liquor and a preparation method and application thereof. The thermus thermophilus fermentation liquor is obtained by fermenting fermentation raw materials by thermus thermophilus, and the fermentation raw materials comprise ectoine and alkaline amino acids. The thermus thermophilus fermentation liquor disclosed by the application has high total protein content, especially heat shock proteins, and the total protein content is high. In addition, the fermentation liquor disclosed by the application can achieve the clinical efficacy effect of ectoine at a low concentration, and can be widely applied to the cosmetic field to promote skin keratin exfoliation, skin moisturizing, skin tightening, skin anti-wrinkle, skin moisturizing, skin anti-inflammation, skin barrier repair, antioxidation and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a thermus thermophilus fermentation liquor and a preparation method and application thereof. BACKGROUND

[0002] Bioactive proteins are proteins or fragments that affect the physiological and functional activities of the body, and have a wide range of functions, nutrients and biological functions. These proteins have antioxidant, anti-allergic, antimicrobial and antithrombotic effects. For a long time, people have obtained protein sources mainly through meat, eggs, milk and other products. However, in recent years, the resource, climate, environment, ethics and other factors have restricted the upgrading of protein raw materials, and consumers have pursued healthy and nutritious food. Microbial fermentation protein and cell culture protein have important strategic significance and have become the future development trend of the industry.

[0003] Under different conditions or modifications, a variety of functional proteins such as cytokines, hormones, growth factors, enzymes and other active substances can be produced by microbial fermentation. These substances have multiple functions, such as promoting cell viability, skin repair, antioxidant, and can be used in food, health food, cosmetics, medicine, biological carriers and other multiple purposes.

[0004] Ectoine, the chemical name of which is tetrahydro-methyl pyrimidine carboxylic acid, is an important osmotic pressure compensation solute in microorganisms. Initially, scientists discovered ectoine from a high-salt-tolerant bacterium that lived in an environment with high salt concentration, so it is also called "salt-tolerant bacterium extract". Ectoine not only balances osmotic pressure, but also has good protective effect on DNA, enzymes, proteins, nucleic acids, cell membranes and whole cells under adverse conditions such as cold, high temperature, high ultraviolet radiation, dryness, high salt, extreme pH, high pressure and high radiation.

[0005] CN115537428 discloses a heat shock protein-ectoine composition and a preparation method thereof. Thermus thermophilus is inoculated into seed culture medium A to obtain seed culture solution, and then the seed culture solution is inoculated into fermentation culture medium A containing ectoine extract solution for fermentation. The fermentation broth is subjected to ultrasonic disruption treatment to promote the release of the lysate of thermus thermophilus, and a heat shock protein-ectoine composition is obtained. However, the total protein and heat shock protein content obtained by the patent is very low.

[0006] In recent years, a large number of studies have shown that heat shock proteins (HSPs) have protective effects on different organs under different stress conditions, and cell proliferation and cell cycle maintenance are related to heat shock proteins. In addition, its important role is to act as a molecular chaperone to promote protein synthesis and folding in cells. According to the size of the protein, heat shock proteins (HSPs) are divided into HSP110, HSP90, HSP70, HSP60 and small molecular heat shock proteins (sHSPs). In the skin, the cells in the dermis and epidermis layers express heat shock proteins, which can resist damage caused by stress sources such as sunlight. For example, HSP27 stabilizes the actin cytoskeleton and participates in keratinocyte differentiation; HSP32 can protect cells from oxidative damage; HSP47 is related to collagen synthesis in fibroblasts; HSP72 can reduce the accumulation of oxidized and glycosylated proteins in skin cells and can also reduce UVB-induced cell damage.

[0007] Since heat shock proteins have significant protective and preventive capabilities, there is a need for a fermentation broth with high total protein and heat shock proteins to play a role in protecting the skin. SUMMARY

[0008] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a Thermus thermophilus fermentation broth and a preparation method and use thereof, in order to solve the above-mentioned problems.

[0009] To achieve the above-mentioned purpose, the present application specifically adopts the following technical solutions.

[0010] The first aspect of the present application protects a Thermus thermophilus fermentation broth, which is obtained by fermentation of a fermentation substrate by Thermus thermophilus, wherein the fermentation substrate comprises ectoine and basic amino acids.

[0011] The second aspect of the present application protects a preparation method of the fermentation broth as described above, comprising the following steps: fermenting a fermentation substrate by Thermus thermophilus to obtain the fermentation broth.

[0012] The third aspect of the present application protects the use of the fermentation broth as described above as a raw material in the preparation of cosmetics.

[0013] The fourth aspect of the present application protects a cosmetic containing the fermentation broth as described above.

[0014] The fifth aspect of the present application protects the use of the fermentation broth as described above or the cosmetic as described above, wherein the use is selected from one or more of skin anti-inflammatory, skin barrier repair, skin moisturizing, skin tightening, skin anti-wrinkle, reduction of skin keratosis and skin antioxidant.

[0015] The thermus fermentation broth of the present application can obtain high expression of bioactive proteins, especially heat shock proteins, by adding ectoine and basic amino acids in the fermentation substrate, and also improves the total protein content in the fermentation broth.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1) The thermus fermentation broth of the present application uses ectoine and basic amino acids as the fermentation substrate, which improves the total protein and total heat shock protein content in the fermentation broth.

[0018] 2) The present application uses fermentation under the conditions of pulse high temperature stimulation and reduced dissolved oxygen stimulation to further obtain high expression of bioactive proteins, especially heat shock proteins. Compared with the traditional 68℃ constant temperature and constant aeration fermentation method, the method of the present application makes the total protein in the fermentation product increase by 2-20 times, and the heat shock protein increase by 3-30 times.

[0019] 3) The thermus fermentation broth of the present application can reduce the effective concentration of ectoine, and has better skin care effect compared with the same concentration of ectoine aqueous solution.

[0020] 4) The thermus fermentation broth of the present application or the cosmetic formed by the fermentation broth of the present application has obvious effects of reducing skin keratin exfoliation, skin moisturizing, skin tightening, skin anti-wrinkle, antioxidant, repairing skin barrier, and skin anti-inflammatory effect.

[0021] 5) The thermus fermentation broth of the present application is easy to operate, easy to scale up, and has good stability, which is suitable for large-scale industrial production, and provides a new method for promoting active protein induction or high expression by microbial fermentation. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A schematic diagram showing four fermentation stages in the preparation method of the thermus fermentation broth of the present application.

[0023] Figure 2 An electrophoresis diagram showing the heat shock proteins in the four fermentation stages in Example 2 of the present application.

[0024] Figure 3 A 0h scratch chart showing Example 2 of the application.

[0025] Figure 4 A 24h scratch chart showing Example 2 of the application.

[0026] Figure 5 A chart showing the results of the skin keratin layer moisture content of consumers using the fermentation broth diluent and 2% ectoine aqueous solution respectively in Example 3 of the present application.

[0027] Figure 6 A result chart of TEWL after the consumers respectively use the fermentation liquid diluent and 2% ectoine aqueous solution in the application example 3 of the present application is shown.

[0028] Figure 7 A result chart of skin elasticity parameters after the consumers respectively use the fermentation liquid diluent product and 2% ectoine aqueous solution in the application example 3 of the present application is shown.

[0029] Figure 8 A result chart of eye tail wrinkle wrinkle area after the consumers respectively use the fermentation liquid diluent product and 2% ectoine aqueous solution in the application example 3 of the present application is shown.

[0030] Figure 9 A result chart of skin smoothness parameter SEsm after the consumers respectively use the fermentation liquid diluent and 2% ectoine aqueous solution in the application example 3 of the present application is shown.

[0031] Figure 10 A result chart of eye tail wrinkle wrinkle proportion after the consumers respectively use the fermentation liquid diluent 2% ectoine aqueous solution in the application example 3 of the present application is shown.

[0032] Figure 11 A result chart of skin keratolysis parameter SEsc after the consumers respectively use the fermentation liquid diluent and 2% ectoine aqueous solution in the application example 3 of the present application is shown. DETAILED DESCRIPTION

[0033] It is occasionally found in the present application that the addition of basic amino acids in the fermentation substrate of thermophilic thermus improves the content of total protein and heat shock protein, and the obtained fermentation liquid also has obvious effects of reducing keratolysis, improving skin moisturizing, improving skin firmness, antioxidant and anti-wrinkle, in addition, it also has the ability of anti-inflammatory repair and scar repair on skin cells, and reduces the effective concentration of the fermentation liquid. On this basis, the present application is completed.

[0034] The first aspect of the present application protects a thermophilic thermus fermentation liquid, which is obtained by fermenting a fermentation substrate by thermophilic thermus, wherein the fermentation substrate comprises ectoine and basic amino acids.

[0035] Heat shock proteins have anti-stress function and wound healing function, and are now widely used in cosmetics, and can be used as molecular chaperones involved in collagen biosynthesis, and have the functions of whitening promotion, anti-stain, anti-wrinkle, anti-stress, anti-cell death, anti-inflammatory, cell protection, gastric mucosa protection and anti-DNA damage.

[0036] The total protein content of the Thermus thermophilus fermentation broth of the present application is above 287 mg / L, the heat shock protein is above 1657 μg / L, and the proportion of total heat shock protein to total protein is above 0.45%. Heat shock proteins are a class of heat stress proteins widely existing from bacteria to mammals. According to the size of the protein, heat shock proteins are divided into HSP110, HSP90, HSP70, HSP60 and small molecular heat shock proteins (sHSPs). The present application finds that the effect of the Thermus thermophilus fermentation broth containing 1% ectoine on skin keratin exfoliation, skin moisturizing, skin tightening, antioxidant and anti-wrinkle is far superior to that of 2% ectoine aqueous solution. In addition, the Thermus thermophilus fermentation broth of the present application also has the abilities of anti-inflammatory repair and scar repair.

[0037] In some embodiments, the Thermus thermophilus can be obtained through a commercial channel, such as one or both of Thermus thermophilus ATCC 33923 and Thermus thermophilus JL-18.

[0038] In some embodiments, the basic amino acid is selected from one or more of arginine, lysine and histidine. Preferably, the basic amino acid is a combination of arginine and lysine. Further, the mass ratio of arginine to lysine can generally be (0.5-3):1, (0.5-1.1):1, (0.8-2.5):1, or (2.1-3):1. In some specific embodiments, it is 1:1.

[0039] In some embodiments, the concentration of ectoine, based on the total mass of the fermentation substrate, can generally be 2-30%, 2-15%, 10-22%, or 21-30%. In some specific embodiments, it is 20%, 11%, 2% or 30%.

[0040] In some embodiments, the concentration of basic amino acid, based on the total mass of the fermentation substrate, can generally be 0.5-5%, 0.5-1.5%, 1.0-3.8%, or 3.2-5%. In some specific embodiments, it is 4.8%, 3%, 3.2% or 1%.

[0041] In some embodiments, the fermentation substrate further comprises a culture medium. The culture medium used for the fermentation of Thermus thermophilus in the present application is a commonly used culture medium that can be selected by those skilled in the fermentation field according to common knowledge, as long as the culture medium can meet the requirement of protein peptone content > 0.1 wt%.

[0042] Preferably, the culture medium comprises peptone and further comprises one or more of beef extract, peptone, NaCl, FeCl3, CaSO4, MgSO4, KNO3, NaNO3, Na2HPO4, MnSO4 and ZnSO4. In some embodiments, the culture medium comprises beef extract 0.3%, peptone 1%, NaCl 0.5%, FeCl3 0.005%, CaSO4 0.05%, MgSO4 0.03%, KNO3 0.05%, NaNO3 0.1%, Na2HPO4 0.04%, MnSO4 0.1%, ZnSO4 0.1%, and the balance is water.

[0043] The second aspect of the present application protects a method for preparing the fermentation liquor as described above, comprising the following steps: fermenting a fermentation substrate by Thermus thermophilus to obtain the fermentation liquor.

[0044] In some embodiments, the method further comprises, before the fermenting, inoculating the Thermus thermophilus into a seed culture medium to culture and obtain a seed culture liquor, and inoculating the seed culture liquor into the fermentation substrate to expand the Thermus thermophilus to the logarithmic growth phase. The temperature of the culture can be 65-80°C, 65-75°C, 68-72°C, 71-80°C, or 65°C, 68°C, 70°C. The time of the culture can be 10-26h, 10-18h, 15-24h, 20-26h, or 15h, 18h, 20h. The seed culture medium comprises beef extract, peptone, NaCl, FeCl3, CaSO4, MgSO4, KNO3, NaNO3, Na2HPO4, MnSO4 and ZnSO4. The concentrations of the raw materials in the seed culture medium can be obtained by experiments, for example, beef extract 0.1-0.5%, peptone 0.5-1.5%, NaCl 0.3-0.4%, FeCl3 0.003-0.007%, CaSO4 0.03-0.07%, MgSO4 0.01-0.03%, KNO3 0.03-0.07%, NaNO3 0.05-0.15%, Na2HPO4 0.02-0.06%, MnSO4 0.5-1.5%, and ZnSO4 0.05-0.15%. In some embodiments, the seed culture medium comprises beef extract 0.3%, peptone 1%, NaCl 0.5%, FeCl3 0.005%, CaSO4 0.05%, MgSO4 0.03%, KNO3 0.05%, NaNO3 0.1%, Na2HPO4 0.04%, MnSO4 0.1%, and ZnSO4 0.1%. The above raw materials are calculated by weight.

[0045] In some embodiments, the volume ratio of the seed culture solution to the fermentation substrate is typically 1:(50-150), 1:(50-90), 1:(80-120), 1:(110-150), or 1:100.

[0046] In some embodiments, the fermentation is carried out at a temperature of 65-80°C, for a time period of 8-24h, at a pH of 7.0-8.5, and at a ventilation rate of 5-100m 3 / h, based on a 100L fermenter. In the present application, the ventilation rate of fermentation refers to the volume of air passing through a 100L fermenter per hour, unless otherwise specified. Figure 1 As shown in FIG. 1, in some embodiments, the fermentation comprises a first stage fermentation, a second stage fermentation, a third stage fermentation, and a fourth stage fermentation. In some more specific embodiments, the first stage fermentation is carried out at a temperature of 63-67°C for a time period of 4-8h, at a ventilation rate of 80-100m 3 / h. In some specific examples, the first stage fermentation is carried out at a temperature of 65°C for a time period of 6h, at a ventilation rate of 100m 3 / h.

[0047] In some more specific embodiments, the second stage fermentation is carried out at a temperature of 68-72°C for a time period of 1-5h, at a ventilation rate of 20-80m 3 / h. In some specific examples, the second stage fermentation is carried out at a temperature of 70°C for a time period of 2h, at a ventilation rate of 50m 3 / h.

[0048] In some more specific embodiments, the third stage fermentation is carried out at a temperature of 73-77°C for a time period of 1-5h, at a ventilation rate of 5-10m 3 / h. In some specific examples, the third stage fermentation is carried out at a temperature of 75°C for a time period of 2h, at a ventilation rate of 5m 3 / h.

[0049] In some more specific embodiments, the fourth stage fermentation is carried out at a temperature of 78-82°C for a time period of 2-6h, at a ventilation rate of 5-10m 3 / h. In some specific examples, the fourth stage fermentation is carried out at a temperature of 80°C for a time period of 4h, at a ventilation rate of 5m 3 / h.

[0050] In some more specific embodiments, the temperature increasing rate in the first stage fermentation, the second stage fermentation, the third stage fermentation, and the fourth stage fermentation is typically (1-10)°C / 2h, (1-5)°C / 2h, (4-8)°C / 2h, (6-10)°C / 2h, or 5°C / 2h.

[0051] The present application stimulates Thermus thermophilus by four-stage fermentation with temperature rising and air volume reducing, and increases the content of heat shock protein and total protein. Compared with the fermentation method without adding ectoine and basic amino acid and with constant temperature and constant air volume, the four-stage fermentation increases the total protein in the fermentation liquor by 2-20 times and the heat shock protein by 3-30 times.

[0052] In some embodiments, the preparation method further comprises post-treatment of the fermentation product after fermentation. The post-treatment is: solid-liquid separation of the fermentation product to obtain a first supernatant and a first precipitate, lysis of the first precipitate, solid-liquid separation to obtain a second precipitate; acid extraction of the second precipitate, solid-liquid separation to obtain a second supernatant, mixing of the second supernatant with a base to neutralize, solid-liquid separation to obtain a third supernatant; mixing of the third supernatant with the first supernatant to obtain the fermentation liquor.

[0053] Preferably, the lysis method is: mixing of the first precipitate with a PBS solution, cell breakage at 4-10℃ and 80MPa ultra-high pressure to obtain a cell lysate. The mass ratio of the first precipitate to the PBS is 1: (1-3), preferably 1:2; the concentration of the PBS is 0.1M. In some specific embodiments, the first precipitate is mixed with 1-fold amount of 0.1M PBS solution, and the cell is broken at 8℃ and 80MPa high pressure.

[0054] Preferably, the solid-liquid separation is centrifugation, filtration, and more preferably centrifugation. The centrifugation speed is 4000-4500rpm / min.

[0055] Preferably, the acid is hydrochloric acid, and the base is sodium hydroxide. The concentration of the hydrochloric acid is 0.02M, and the concentration of the sodium hydroxide is 0.02M.

[0056] The third aspect of the present application protects the use of the aforementioned fermentation liquor as a raw material in the preparation of cosmetics.

[0057] The fourth aspect of the present application protects a cosmetic containing the aforementioned fermentation liquor.

[0058] In some embodiments, the cosmetic is a skin care preparation, and the skin care preparation is one or more of a solution, a suspension, a mask, a lotion, a cream, a paste, a gel, a dry powder, a wet powder, and a spray. Preferably, it is a solution.

[0059] In some embodiments, the addition amount of the fermentation liquor is not more than 10% based on the total mass of the cosmetic.

[0060] In some embodiments, the pH of the cosmetic is generally 3.5-8.5, 3.5-5.5, 4.5-6.5, 5.5-7.5, 6.5-8.5, or 5.5-7.0.

[0061] The fifth aspect of the present application protects the use of the fermentation broth as described above or the cosmetic as described above, which is selected from one or more of skin anti-inflammation, skin barrier repair, skin moisturization, skin tightening, skin anti-wrinkle, skin exfoliation, and skin anti-oxidation.

[0062] In some embodiments, the skin anti-inflammation refers to down-regulating the level of inflammatory factors secreted by skin cells, which are human skin keratinocytes. The present application finds that the Thermus thermophilus fermentation broth can significantly reduce the secretion of inflammatory factors such as IL-1α, IL-6, and IL-8 by cells, indicating that it has an anti-inflammatory repair effect.

[0063] In some embodiments, the skin barrier repair refers to repairing skin cell scratches, which are human embryonic lung fibroblasts. The present application finds that the Thermus thermophilus fermentation broth can reduce the scratch area by 21.35%, indicating that it has a significant skin barrier repair effect.

[0064] In some embodiments, the skin moisturization refers to increasing the water content of the stratum corneum or reducing the trans-epidermal water loss rate (TEWL). The present application finds that after diluting the Thermus thermophilus fermentation broth, the water content of the stratum corneum in the cheek area is increased by 50.92% with extremely significant difference; the trans-epidermal water loss rate (TEWL) in the cheek area is reduced by 28.26% with extremely significant difference.

[0065] In some embodiments, the skin tightening refers to increasing the skin elasticity parameter ratio. The present application finds that after diluting the Thermus thermophilus fermentation broth, the skin elasticity parameter in the cheek area is increased by 26.03% with extremely significant difference.

[0066] In some embodiments, the skin anti-wrinkle refers to reducing the wrinkle area of the crow's feet, or reducing the skin smoothness parameter, or reducing the proportion of the crow's feet wrinkle. The present application finds that after diluting the Thermus thermophilus fermentation broth, the wrinkle area of the crow's feet is decreased by 13.50% with extremely significant difference; the skin smoothness parameter SEsm is decreased by 26.19% with extremely significant difference; the proportion of the crow's feet wrinkle is decreased by 12.75% with extremely significant difference.

[0067] In some embodiments, the skin exfoliation refers to reducing the skin exfoliation parameter SEsc. The present application finds that after diluting the Thermus thermophilus fermentation broth, the skin exfoliation parameter SEsc in the cheek area is decreased by 85.32% with extremely significant difference.

[0068] In some embodiments, the antioxidant refers to improving total antioxidant capacity or scavenging DPPH free radicals. The present application finds that after dilution of the Thermus thermophilus fermentation broth, the total antioxidant capacity can be improved, and DPPH free radicals can be scavenged.

[0069] The fermentation broth of the present application can improve skin moisturizing properties, improve skin firmness, anti-wrinkle, reduce skin keratin stripping, promote skin cell repair and cell proliferation, while inhibiting skin oil secretion, anti-inflammatory and very superficial and superficial exfoliation ability, reducing abnormal keratinization of hair follicle infundibulum and accelerating keratinocyte turnover, which can potentially be used as an adjuvant therapy for mild to moderate acne, lightening spots, and treatment of acute or chronic dermatitis, skin photoaging, and keratosis pilaris.

[0070] The sixth aspect of the present application protects the use of a mixture of ectoine and basic amino acids as an effective ingredient for increasing the content of total protein and heat shock protein in the final product of Thermus thermophilus fermentation.

[0071] Applicants have found through long-term research that the combination of ectoine and basic amino acids as key ingredients for fermentation substrates can significantly increase the content of total protein and heat shock protein in the final product of Thermus thermophilus fermentation.

[0072] Compared with fermentation substrates containing ectoine and acidic amino acids, fermentation substrates containing ectoine and basic amino acids under the action of Thermus thermophilus increased total protein by 48.7-149.2% and heat shock protein by 64.4-211.7%.

[0073] The following specific examples illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure.

[0074] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present application are for the purpose of describing the specific embodiments, but not for limiting the scope of protection of the present application. The test methods in the following examples are not specified, and are usually carried out under conventional conditions, or under the conditions recommended by the manufacturers.

[0075] When the embodiments give numerical ranges, it is understood that every numerical range is a continuum of values and that only discrete values are selected from the continuum by the endpoints of the ranges. Unless otherwise specified, all technical and scientific terms and any acronyms used in the present disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. In addition, any method, device, material, or apparatus that is reasonably

[0076] Thermus thermophilus ATCC 3392, JL-18 used in the following examples were purchased from American Type Culture Collection and were available commercially.

[0077] Example 1-10

[0078] According to the formula of Table 1, the Thermus thermophilus fermentation broth was prepared by fermentation. It included the following:

[0079] 1.1, Inoculation

[0080] Thermus thermophilus was recovered and cultured at 65°C in the seed broth medium for 18h to become the seed broth.

[0081] Seed broth medium: beef extract 0.3%, peptone 1%, NaCl 0.5%, FeCl3 0.005%, CaSO40.05%, MgSO4 0.03%, KNO3 0.05%, NaNO30.1%, Na2HPO4 0.04%, MnSO4 0.1%, ZnSO4 0.1%, the rest was water, pH 7.2, 121°C high temperature sterilization for 30 min.

[0082] 1.2, Fermentation culture

[0083] The seed broth obtained in step 1.1 was inoculated into the fermentation substrate of the fermenter at a ratio of 1:100 for fermentation. The fermentation substrate in the fermenter was a mixture of seed broth medium, ectoine and basic amino acids. The concentrations of basic amino acids and ectoine in the mixture were shown in Table 1, and the rest was supplemented with seed broth medium.

[0084] Under stirring at 150 rpm, the pH value was adjusted to 7.7±0.5 by PBS, and the first stage fermentation culture was carried out at a temperature rising rate of 5°C / 2h to 65°C for 6h, with a ventilation amount of 100m 3 / h;

[0085] The second stage fermentation culture was continued at a temperature rising rate of 5°C / 2h to 70°C for 2h, with a ventilation amount of 50m3 / h;

[0086] Then, the third stage fermentation culture was carried out at a temperature of 75°C with a temperature increasing rate of 5°C / 2h for 2h, and the ventilation amount was 5m 3 / h;

[0087] Then, the fourth stage fermentation culture was carried out at a temperature of 80°C with a temperature increasing rate of 5°C / 2h for 4h, and the ventilation amount was 5m 3 / h, to obtain the fermentation product.

[0088] 1.3, Lysis

[0089] The fermentation product obtained in step 1.2 was centrifuged at 4000-4500 rpm / min to obtain a first supernatant and a first precipitate, and the first supernatant was stored for later use after being filtered with a 0.22 μL filter. The first precipitate was taken and dispersed with 1 times the amount of 0.1M PBS solution, and then lysed at 80MPa high pressure at 4-10°C. The second precipitate was obtained by centrifugation at 4000-4500 rpm / min and discarding the supernatant.

[0090] 1.4, Extraction and purification

[0091] The second precipitate obtained in step 1.3 was extracted with 0.02M dilute hydrochloric acid solution, and then centrifuged at 4000-4500 rpm / min to discard the precipitate and obtain a second supernatant. The second supernatant was neutralized with 0.02M NaOH, and then centrifuged at 4000-4500 rpm / min to discard the precipitate. The filtered first supernatant obtained in step 1.3 was combined with the second supernatant to obtain the Thermus thermophilus fermentation broth.

[0092] 1.5, Detection of total protein and heat shock protein content

[0093] 1 mL of the Thermus thermophilus fermentation broth obtained in step 1.4 was taken, 5.0 mL of Coomassie Brilliant Blue G-250 reagent was added, and the mixture was mixed on a vortex mixer. The light absorption value of each sample at 595 nm was measured on a spectrophotometer, and the total protein mass of the sample was calculated according to the standard curve. The results are shown in Table 1.

[0094] The sample with detected total protein content was precipitated and enriched with 60% ammonium sulfate, then the precipitate was collected by centrifugation, dissolved with buffer A (buffer A contains 25 mM Tris-HCl (pH=7.5), 5 mM MgCl2 and 0.1 mM DTT), dialyzed, collected with DEAE filler after gradient elution, directly dried after dialysis, dissolved with 1 mL water, and then subjected to SDS-PAGE electrophoresis, with 5% concentrated gel and 8% separation gel. The target protein was detected by Coomassie brilliant blue (G250) method, and the content of each heat shock protein was calculated by gray method test combined with the corresponding Marker chart of identified HSP60, HSP70 and small molecule sHSPs. The sum of the above heat shock proteins was calculated. The results are shown in Table 1.

[0095] Table 1

[0096]

[0097] As can be seen from Table 1, the total protein and heat shock protein in the fermentation broth obtained by using basic amino acids and ectoine as fermentation substrates in Examples 1-10 are higher than those in Comparative Example 1 using acidic amino acids and ectoine as fermentation substrates, and the proportion of heat shock protein in total protein is also significantly higher than that in Comparative Example 1.

[0098] After the first stage fermentation, the second stage fermentation, the third stage fermentation and the fourth stage fermentation in Example 1 were completed, 1 mL of supernatant was taken from each stage and detected by the same method as the total protein and heat shock protein in Example 1. The electrophoretogram of heat shock protein in each stage is shown in Figure 2 . The content of total protein and heat shock protein in each stage is shown in Table 2.

[0099] Example 11

[0100] The difference between this Example 11 and Example 1 is that the preparation method of the fermentation broth is different, and four-stage fermentation is not used, that is, the Thermus thermophilus ATCC 33923 is inoculated into the fermentation medium containing L-arginine and ectoine, and then fermented at 68°C for 14 h, and the aeration amount during the fermentation is constant at 100 mL / h. 3

[0101] The results of total protein, heat shock protein and heat shock protein in the fermentation broth obtained in this Example 11 are shown in Table 2.

[0102] Comparative Example 1

[0103] The formula of Comparative Example 1 is shown in Table 1.

[0104] ​From Table 1, it can be seen that the total protein content in the fermentation broth obtained by using the acidic amino acid and ectoine as the fermentation substrate in Comparative Example 1 is only 193 mg / L, the heat shock protein is 787 µg / L, and the proportion of the heat shock protein in the total protein is 0.408%; compared with Example 1, the total protein content and the heat shock protein content are reduced by nearly half.

[0105] Comparative Example 2

[0106] The difference between Comparative Example 2 and Example 1 is that no ectoine and basic amino acid is added, only the fermentation medium is used as the fermentation substrate, and the fermentation is carried out at a constant temperature of 68℃ for 14h, the aeration amount is constant at 100m 3 / h during the fermentation, and the other conditions are the same as those in Example 1.

[0107] The fermentation broth of Comparative Example 2 is detected for total protein and heat shock protein, and the results are shown in Table 2.

[0108] Table 2

[0109]

[0110] From Table 2, it can be seen that the total protein content, total heat shock protein yield and the proportion of heat shock protein in the total protein in the supernatant obtained by the second stage fermentation culture, the third stage fermentation culture and the fourth stage fermentation culture of Example 1 are much higher than those obtained by the constant temperature fermentation and the constant aeration fermentation method; the total protein and heat shock protein content in the fermentation broth obtained by using the fermentation substrate of Example 11 under the constant temperature and constant aeration are lower than those obtained by the four-stage fermentation method of Example 1.

[0111] Application Example 1 Study on Anti-inflammatory Ability of Thermus thermophilus Fermentation Broth

[0112] In this application example 1, HaCAT cells (human skin keratinocytes) are inoculated in the culture medium containing the Thermus thermophilus fermentation broth of Example 1 to study the anti-inflammatory ability.

[0113] The experiment is divided into an experimental group, a positive control group and a blank control group.

[0114] The specific operation of the experimental group is as follows:

[0115] 1) HaCAT cells are inoculated in a 96-well plate at a ratio of 0.8×10 4 cells / well, and 200µL of complete culture medium is added to each well.

[0116] 2) After the cells are cultured for 24h, the culture medium is aspirated, washed once with PBS, aspirated, and then 20μL of PBS is added, with the radiation dose controlled at 30mJ / cm 2UVB irradiation was performed, 200 μL of total culture solution was added, and the culture was continued for 24 hours. After 24 hours of culture, centrifugation was performed at 2500 rpm for 20 min, and the supernatant was taken to determine the inflammatory factors. The total culture solution was composed of the Thermus thermophilus fermentation liquor of Example 1 and complete culture medium, and the volume ratio of the Thermus thermophilus fermentation liquor and the complete culture medium was 3:97.

[0117] Meanwhile, 60 ppm dexamethasone was used to replace the Thermus thermophilus fermentation liquor in the experimental group as a positive control group; and no ultraviolet irradiation and no addition of the Thermus thermophilus fermentation liquor were used as a blank control. The rest was the same as in Example 1, and the supernatant was taken to determine the inflammatory factors.

[0118] The inflammatory factors included IL-1α, IL-6 and IL-8, which were all detected by using a kit.

[0119] The experimental results are shown in Table 3.

[0120] Table 3

[0121]

[0122] As can be seen from Table 3, after the HaCAT cells were cultured with the Thermus thermophilus fermentation liquor of the application, the inflammatory factors IL-1α, IL-6 and IL-8 were all significantly reduced, and were better than the positive control group, indicating that the Thermus thermophilus fermentation liquor of the application had a strong anti-inflammatory repair ability on the HaCAT cells.

[0123] Application Example 2: Study on the Scratch Repairing Ability of Thermus thermophilus Fermentation Liquor

[0124] In this application example 2, HSF cells (human embryonic lung fibroblasts) were inoculated in a culture medium containing the Thermus thermophilus fermentation liquor of Example 1 to study the scratch repair ability. The following was included:

[0125] The HSF cells were inoculated in a 6-well plate at 2×10 5 cells per well, and were cultured in 1640 containing 10% fetal bovine serum (containing double antibodies) at 37°C, 5% CO2 and saturated humidity until the logarithmic growth phase. The culture solution was discarded, and a sterile pipette gun head was used to vertically scratch the cell layer to establish an in vitro cell wound model. The scratch result is shown in Figure 3 .

[0126] Then, the in vitro cell wound model was inoculated in a mixed culture solution, the mixed culture solution was formed by mixing 1640 culture medium and the Thermus thermophilus fermentation liquor of Example 1 at a volume ratio of 95:5, and the culture was continued for 24 hours. The scratch repair test was performed to study the repair area, and the result is shown in Figure 4 .

[0127] The principle of the cell scratch assay is to simulate the migration speed of cells in vivo. A "scratch" is made on the cell growth plane to cause cell damage. After adding the sample, images are captured periodically by a time-lapse microscope to study the cell migration movement and repair ability.

[0128] From Figure 3 and 4 It can be seen that the scratch area is reduced by 21.35% after 24 hours, indicating that the Thermus thermophilus fermentation broth of the present application has a significant repair effect.

[0129] Application Example 3: Study of Thermus thermophilus Fermentation Broth on Skin Moisturizing, Firming, Anti-wrinkle, and Reducing Stratum Corneum Exfoliation

[0130] A 28-day randomized half-face control test was conducted on 24 women and 6 men to study the effect of the Thermus thermophilus fermentation broth obtained in Example 1 on skin moisturizing, firming, anti-wrinkle, and reducing stratum corneum exfoliation. This includes the following:

[0131] 1) Method of use

[0132] Each time, half of the face, i.e. the left side of the face, uses 0.5 mL of the fermentation broth diluent (mixing Thermus thermophilus of Example 1 with water at a volume ratio of 5:95), which is detected to have a concentration of 1% of ectoin, as the experimental group.

[0133] At the same time, the right side of the face uses 0.5 mL of 2% ectoin aqueous solution as the control group. Ectoin water can be obtained through commercial channels.

[0134] 2) Measurement indicators

[0135] Stratum corneum water content: The Delfin MoistureMeterSC instrument is used to detect skin moisture before use (D0) and 28 days after use (D28). The lower the value, the lower the stratum corneum water content.

[0136] Trans-epidermal water loss rate (TEWL): The Delfin VapoMeter is used to detect skin moisture before use (D0) and 28 days after use (D28). The lower the value, the less the skin surface water loss.

[0137] Face image collection + IPP analysis of wrinkle area and wrinkle area ratio: The Visia-CR instrument is used to detect the periorbital wrinkles before use (D0) and 28 days after use (D28). The lower the value, the fewer the skin wrinkles.

[0138] Skin tightness parameter: The facial skin tightness was measured by Delfin ElastiMeter instrument before use (D0) and 28 days after use (D28). The lower the value, the worse the skin tightness.

[0139] Skin exfoliation parameter SEsc in cheek area: The degree of skin exfoliation was measured by Visioscan VC 20 plus instrument before use (D0) and 28 days after use (D28). The lower the value, the lower the degree of skin exfoliation.

[0140] Skin smoothness parameter SEsm: The skin smoothness was measured by Visioscan VC20 plus instrument before use (D0) and 28 days after use (D28). The lower the value, the smoother the skin.

[0141] 3) Statistical analysis

[0142] Δ difference = X time node - X initial node

[0143] Note: "X" - instrument measurement value

[0144] Change rate% = (x time node - x initial node) / x initial node x 100%

[0145] Note: "X" - instrument measurement value (positive value indicates increase, negative value indicates decrease).

[0146] The statistical method adopts two-tailed test, and the test level a = 0.05.

[0147] SPSS 26.0 software was used for data statistical analysis. When there is a significant difference in the results, the significant level a = 0.05, p > 0.05 is not significant, "*" indicates significant difference (0.01 ≤ p < 0.05), "**" indicates very significant difference (0.001 ≤ p < 0.01), and "***" indicates extremely significant difference (p < 0.001).

[0148] 4) Results

[0149] From Figure 5 it can be seen that after using the fermentation broth diluent for 28 days, the moisture content of the stratum corneum in the cheek area increased by 50.92% compared with D0 day; after using 2% aqueous solution of ekdin for 28 days, the moisture content of the stratum corneum in the cheek area increased by 26.85% compared with D0 day; after using the product for 28 days (D28), the moisture content of the stratum corneum using the thermophilic aquifer obtained in Example 1 was significantly higher than that of the 2% aqueous solution of ekdine.

[0150] From Figure 6It can be seen that, compared with D0 day, after using the fermentation broth diluent for 28 days, the TEWL of the cheek area was extremely significantly reduced by 28.26%; compared with D0 day, after using 2% aqueous solution of ikkinko for 28 days, the TEWL of the cheek area was extremely significantly increased by 15.64%; after using the product for 28 days (D28), the TEWL of the cheek area using the thermus thermophilus fermentation broth obtained in Example 1 was extremely significantly reduced compared with using 2% aqueous solution of ikkinko.

[0151] From Figure 7 It can be seen that, compared with D0 day, after using the fermentation broth diluent for 28 days, the skin elasticity parameter of the cheek area was extremely significantly increased by 26.03%; compared with D0 day, after using 2% aqueous solution of ikkinko for 28 days, the skin elasticity parameter of the cheek area was extremely significantly increased by 12.09%; after using the product for 28 days (D28), the skin elasticity parameter of the cheek area using the thermus thermophilus fermentation broth obtained in Example 1 was extremely significantly increased compared with using 2% aqueous solution of ikkinko.

[0152] From Figure 8 It can be seen that, compared with D0 day, after using the fermentation broth diluent for 28 days, the wrinkle area of the tail of the eye was extremely significantly decreased by 13.50%; compared with D0 day, after using 2% aqueous solution of ikkinko for 28 days, the wrinkle area of the tail of the eye was significantly decreased by 6.18%; after using the product for 28 days, the wrinkle area of the tail of the eye using the thermus thermophilus fermentation broth obtained in Example 1 was very significantly decreased compared with using 2% aqueous solution of ikkinko.

[0153] From Figure 9 It can be seen that, compared with D0 day, after using the fermentation broth diluent for 28 days, the skin smoothness parameter SEsm was extremely significantly decreased by 26.19%; compared with D0 day, after using 2% aqueous solution of ikkinko for 28 days, the skin smoothness parameter SEsm was significantly decreased by 10.38%; after using the product for 28 days, the skin smoothness parameter SEsm using the thermus thermophilus fermentation broth obtained in Example 1 was very significantly decreased compared with using 2% aqueous solution of ikkinko.

[0154] From Figure 10 It can be seen that, compared with D0 day, after using the fermentation broth diluent for 28 days, the proportion of the wrinkle of the tail of the eye was extremely significantly decreased by 12.75%; compared with D0 day, after using 2% aqueous solution of ikkinko for 28 days, the proportion of the wrinkle of the tail of the eye was significantly decreased by 5.98%; after using the product for 28 days, the proportion of the wrinkle of the tail of the eye using the thermus thermophilus fermentation broth obtained in Example 1 was very significantly decreased compared with using 2% aqueous solution of ikkinko.

[0155] From Figure 11It can be seen that, compared with D0 day, the skin exfoliation parameter SEsc of the cheek area is significantly decreased by 85.32% after 28 days of using the fermentation broth diluent; compared with D0 day, the skin exfoliation parameter SEsc of the cheek area is significantly decreased by 30.12% after 28 days of using the 2% ikkduin water solution; after 28 days (D28) of using the product, the skin exfoliation parameter SEsc of the cheek area using the thermophilic meihermicus fermentation broth obtained in Example 1 is significantly decreased compared with the 2% ikkduin water solution.

[0156] It is shown comprehensively that the thermophilic meihermicus fermentation broth has the effects of moisturizing, tightening, anti-wrinkle and exfoliation, and is significantly better than the 2% ikkduin water solution.

[0157] Application Example 4: Study on the antioxidant effect of the thermophilic meihermicus fermentation broth on the skin

[0158] In this application example 4, the total antioxidant capacity and DPPH free radical scavenging capacity of the fermentation broth diluent obtained in application example 3 (i.e. the thermophilic meihermicus fermentation broth of Example 1 mixed with water at a volume ratio of 5:95) are determined.

[0159] At the same time, the 2% ikkduin water solution prepared in application example 3 is used as a control group.

[0160] The operation steps for determining the total antioxidant capacity are as follows: 1 mL of the sample to be tested (fermentation broth diluent or 2% ikkduin water solution), 1 mL of phosphate buffer solution (pH 6.6, 0.2 M), 1 mL of potassium ferricyanide solution (w / v: 1%), are reacted at 50°C for 20 min, after the reaction is completed, 1 mL of trichloroacetic acid solution (w / v: 10%) is added, and then centrifuged at 3000g for 10 min, 2.5 mL of supernatant is taken, 0.5 mL of ferric chloride solution (w / v: 0.1%) is added for 10 min, and then distilled water is added to 10 mL, and the absorbance is detected at 700 nm.

[0161] The operation steps for determining the DPPH free radical scavenging capacity are as follows: 2 mL of DPPH ethanol solution and 2 mL of the sample to be tested (fermentation broth diluent or 2% ikkduin water solution) are added to a test tube, shaken and mixed, and then reacted in the dark for 30 min, the absorbance A is measured at 517 nm, the absorbance A0 of the mixture of 2 mL of DPPH ethanol solution and 2 mL of anhydrous ethanol, and the absorbance Ab of the mixture of 2 mL of anhydrous ethanol and 2 mL of the sample to be tested are also measured, and the DPPH· free radical scavenging rate is calculated.

[0162] DPPH· free radical scavenging rate (%) = (A0- (A-Ab)) / A0 x 100

[0163] The total antioxidant capacity and DPPH free radical scavenging rate are shown in Tables 4 and 5.

[0164] Table 4

[0165]

[0166] Table 5

[0167]

[0168] From Table 4 and Table 5, it can be seen that the total antioxidant capacity and DPPH free radical scavenging capacity of the Thermus thermophilus fermentation liquor of the present application are both superior to 2% ectoine aqueous solution.

[0169] The above examples only illustrate the principles of the present application and its effects, and are not intended to limit the present application. Any modification or change made by any person skilled in the art without departing from the spirit and scope of the present application shall be covered by the claims of the present application.

Claims

1. A method for preparing a Thermus thermophilus fermentation broth, characterized by, comprising the steps of: fermenting a fermentation substrate by a thermus thermophilus to obtain the fermentation broth; the thermus thermophilus is selected from one or both of thermus thermophilus ATCC 33923 and thermus thermophilus JL-18; the fermentation substrate is composed of ectoine, basic amino acid and culture medium, the basic amino acid is selected from one or more of arginine, lysine and histidine; the fermentation comprises first stage fermentation, second stage fermentation, third stage fermentation and fourth stage fermentation; wherein, The conditions of the first stage fermentation are: 63-67℃ for 4-8h, and the ventilation is 80-100m 3 / h. The second stage fermentation is carried out at 68-72°C for 1-5h with aeration of 20-80m 3 / h. The third stage fermentation is carried out at 73-77°C for 1-5h with aeration of 5-10m 3 / h; The fourth stage fermentation is carried out at 78-82°C for 2-6 hours with aeration of 5-10 m 3 / h.

2. The production method according to claim 1, wherein the concentration of the ectoine is 2% to 30% based on the total mass of the fermentation substrate; and / or, the concentration of the basic amino acid is 0.5% to 5% based on the total mass of the fermentation substrate; and / or, the culture medium comprises peptone.

3. The production method according to claim 1, wherein the temperature of the fermentation is 65 to 80℃; and / or, the time of the fermentation is 8 to 24h; and / or, the pH value of the fermentation is 7.0 to 8.5; and / or, before the fermentation, further comprising inoculating the thermus thermophilus in a seed culture medium to culture and obtain a seed culture broth, the seed culture broth is inoculated to the fermentation substrate.

4. The production method according to claim 3, wherein the volume ratio of the seed culture broth to the fermentation substrate is 1: (50 to 150).

5. The thermus thermophilus fermentation broth prepared by the method of any one of claims 1 to 4.

6. The use of the fermentation broth of claim 5, the use is selected from one or more of skin moisturizing, skin tightening, skin anti-wrinkling, reducing skin keratin exfoliation and skin antioxidant, the use is for non-disease diagnosis and / or treatment.

7. The use of the fermentation broth of claim 5 as a raw material in the preparation of cosmetics.

8. A cosmetic product, characterized by, containing the fermentation broth of claim 5.

9. The use of the cosmetic of claim 8, the use is selected from one or more of skin moisturizing, skin tightening, skin anti-wrinkling and reducing skin keratin exfoliation.

10. The use of a mixture of ectoine, basic amino acid and culture medium as a fermentation substrate for increasing the content of total protein and heat shock protein in the final product of thermus thermophilus fermentation, the thermus thermophilus is selected from one or both of thermus thermophilus ATCC 33923 and thermus thermophilus JL-18, the basic amino acid is selected from one or more of arginine, lysine and histidine.

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