Bacillus fermentation product and lysate as well as preparation method and application thereof

Abstract

The invention relates to a bacillus fermentation product and lysate as well as a preparation method and application thereof, and the scheme comprises the following steps: inoculating bacillus subtilis as a strain into a seed culture medium for activation to obtain a seed solution; inoculating the seed solution on a flat plate, simulating the ultraviolet radiation condition of the desert abdominal land, and screening out a radiation-resistant strain with genetic stability; inoculating the radiation-resistant strain into a desert culture medium, and further screening out a hypertonic-resistant strain with genetic stability; the method comprises the following steps: inoculating a high-permeability-resistant strain into a fermentation tank, carrying out fermentation culture, and separating to obtain a fermentation clear liquid and a thallus precipitate; degerming the fermentation clear liquid to obtain fermentation liquid, and performing resuspension, crushing and high-speed centrifugation on thallus precipitate to obtain a thallus crushing product; and carrying out decoloration and deodorization treatment on the fermentation liquor and the thallus crushing product, and adding a stabilizer to obtain a fermentation product and a lysate product. The obtained product can play a role in the aspects of moisturizing, water locking and light aging resistance.

Description

Technical Field

[0001] The present application relates to the fields of skin care products and fermentation technology, and specifically to Bacillus fermentation products and lysate products, and preparation methods and applications thereof. Background Art

[0002] Skin aging is a complex and inevitable biological process, which is mainly driven by two mechanisms: endogenous natural aging and exogenous photoaging. Endogenous natural aging is due to the fact that over time, the body's cell renewal rate slows down, and the synthesis of collagen and elastic fibers decreases, causing the skin to gradually lose elasticity and firmness, and develop problems such as wrinkles, sagging, dull color, and texture changes. Exogenous aging, especially photoaging, is caused by long-term ultraviolet radiation, and manifests as darker skin pigmentation, dryness and dehydration, frequent inflammatory reactions, and a significant increase in wrinkles, which seriously affects skin health and beauty.

[0003] At the same time, there is a class of microorganisms in nature that are highly resistant to adverse environments, among which Bacillus bacteria are particularly noteworthy. Take Bacillus subtilis as an example. This Gram-positive bacterium can adapt and survive in extreme environments such as drought, high heat, high acidity and alkali, and high osmotic pressure by forming stable spore dormant bodies. Once the environment improves, it can quickly resume growth. Bacillus subtilis has been widely used in the food industry, pharmaceutical industry, and cosmetics industry due to its fast growth, strong tolerance, and diverse and safe metabolites, showing great potential.

[0004] In addition, microorganisms in extreme environments, especially those that can survive under conditions of high radiation, high temperature, high salinity, strong acid and alkali, have significant research value for their unique biological characteristics and metabolites. In order to adapt to harsh environments, these microorganisms can produce special biologically active substances that resist adverse conditions such as ultraviolet radiation, high temperature, and extreme dryness. For example, some bacteria living in extreme environments such as deserts have evolved mechanisms that can resist UV radiation, maintain water balance, and resist heat.

[0005] In view of this, in-depth research and development and utilization of these extreme environment microorganisms, especially the active substances produced by them with anti-aging, anti-oxidation, anti-ultraviolet damage and other functions, are extremely important for developing a new generation of cosmetics and health products that can effectively delay and improve skin aging. The present invention is based on such a background, aiming to explore the application potential of extreme environment microbial resources, especially Bacillus microorganisms in the field of skin anti-aging, and develop products with unique anti-aging effects. Therefore, the development and utilization of extreme environment microorganisms are of great significance in the field of cosmetics and health products. Summary of the invention

[0006] The purpose of the present application is to address the above-mentioned problems existing in the prior art and to provide a Bacillus fermentation product and a lysate and a preparation method and application thereof.

[0007] In order to achieve the above application purpose, the present application adopts the following technical scheme: The preparation method of Bacillus fermentation product and lysate comprises the following steps:

[0008] S00, inoculating Bacillus subtilis as a bacterial species into a seed culture medium for activation to obtain a seed solution;

[0009] S10, inoculating the seed solution on a plate, simulating ultraviolet radiation conditions in the heart of the desert, and screening out radiation-resistant strains with genetic stability;

[0010] S20, inoculating the radiation-resistant strains into desert culture medium to further screen out genetically stable hyperosmotic-resistant strains;

[0011] Among them, the desert culture medium is based on the seed culture medium, and salt ions are added according to the soluble salt ratio of shallow groundwater in the heart of the desert, and some promoting factors are added;

[0012] S30, inoculating the hyperosmotic-resistant strain into a fermentation tank for fermentation culture, and obtaining a fermentation clear liquid and bacterial precipitate by separation;

[0013] After the fermentation liquid is sterilized, the fermentation liquid is obtained, and the bacterial precipitate is resuspended, crushed, and centrifuged at high speed to obtain a bacterial crushing product;

[0014] S40, decolorizing and deodorizing the fermentation liquid and the bacterial cell crushing product, and adding a stabilizer to obtain a fermentation product and a lysate.

[0015] Further, in step S00, the seed culture medium is a nutrient broth culture medium or a brain heart infusion broth culture medium. Among them, the nutrient broth (NB) culture medium (containing 10.0 g / L peptone, 3.0 g / L beef extract powder, 5.0 g / L sodium chloride), the brain heart infusion broth (BHI) culture medium (containing 10.0 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5.0 g / L dehydrated beef heart extract powder, 5.0 g / L sodium chloride, 5.0 g / L glucose, 2.5 g / L disodium hydrogen phosphate), and inoculated in the seed culture medium for activation by adopting a step-by-step expansion culture method, the preserved strain is inoculated in a suitable culture medium for culture, fermented for 2-3 generations for rejuvenation, and a vigorous seed liquid with sufficient inoculation quantity is obtained.

[0016] Furthermore, in step S10, the method for simulating the ultraviolet radiation situation in the heart of the desert specifically includes:

[0017] According to the average daily ultraviolet radiation in the heart of the desert, the cells were treated for 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours, and then placed in a dark environment for 16-24 hours;

[0018] The bacterial count of the UV-treated group at 0 h was used as the control. The bacterial counts of the groups treated with different treatment times were calculated. The colonies with a lethality rate of 70%-90% were selected for expansion culture. After culturing for 2-3 generations, they were placed in 0.87-1.05 MJ·m -2 UV treatment for the same time;

[0019] If the survival rate is below 70%, the surviving strains are extracted and expanded for a set generation until the survival rate remains above 70% and several colonies are screened.

[0020] Furthermore, in step S30, the fermentation temperature of the fermentation culture is 30-40°C, the inoculation amount is 3-10%, the fermentation speed is 100-300r / min, and the ventilation ratio is 1.0-2.0VVM.

[0021] Furthermore, in step S10, the promoting factors include vitamin E, Fe 2+ 、Zn 2+ , Mn 2+ One or more of the .

[0022] Furthermore, in step S30, the fermentation medium formula is 10.0-20.0 g / L peptone, 3.0-5.0 g / L beef extract powder, 3.0-10.0 g / L sodium chloride, 0.1-30.0 g / L other inorganic salts, and the rest is water, and the pH is 6.7-7.2.

[0023] Furthermore, in step S40, the stabilizer is one or more of 2,6-di-tert-butyl-4-methylphenol, tocopherol, potassium dihydrogen phosphate, Tween-80, and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

[0024] The lysate is prepared by the above-mentioned Bacillus fermentation product and lysate preparation method.

[0025] The fermentation product is prepared by the above-mentioned method for preparing the Bacillus fermentation product and lysate.

[0026] Biopharmaceuticals and health products include fermentation products and / or lysates prepared by the above-mentioned preparation methods of Bacillus fermentation products and lysates. Biopharmaceuticals and health products include cosmetics, health products and medicines.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] 1. Different from the traditional UV breeding method of using short-term high-intensity UV radiation to cause bacterial gene mutation, the UV radiation used in this application to simulate the extreme environment of the desert is continuous high-intensity UV radiation for a long time. This method can screen out strains that synthesize and secrete metabolites to resist high UV radiation;

[0029] 2. Unlike traditional hypertonic screening, which is only a change in osmotic pressure, the hypertonic environment created by simulating the desert in this application is to add salt ions according to the soluble salt ratio of shallow groundwater in the hinterland of the desert, with the purpose of specifically screening strains that can synthesize and secrete metabolites to resist the negative effects of hypertonic water shortage;

[0030] 3. This application uses ultraviolet and hyperosmotic conditions in simulated desert extreme environments to screen Bacillus subtilis. This technology combines ultraviolet mutagenesis with natural selection, and can screen out a strain of Bacillus subtilis that can independently synthesize and secrete metabolites to resist high ultraviolet radiation and high osmotic conditions. The fermentation products and lysates of this strain are rich in proteins, peptides, amino acids, polyphenols, polysaccharides and other active substances, and have excellent moisturizing and water-locking, and anti-photoaging activities;

[0031] 4. This application adds a certain proportion of Zn to the simulated desert culture medium 2+ , Mn 2+ , vitamin E and other promoting factors. These substances can enhance the vitality of the strain, enter the bacteria to activate the spore receptor protein, transmit the signal of spore formation, and promote the strain to produce spores to resist the hypertonic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the results of whether there is toxicity to human fibroblasts in the cytotoxicity study of the effect embodiment of the present application;

[0033] Figure 2 This is a schematic diagram of the results of whether there is toxicity to human keratinocytes in the cytotoxicity study of the effect embodiment of the present application;

[0034] Figure 3 This is a schematic diagram of the results of the moisturizing and water-locking effect evaluation of the present application embodiment. Figure 1 ;

[0035] Figure 4 This is a schematic diagram of the results of the moisturizing and water-locking effect evaluation of the present application embodiment. Figure 2 ;

[0036] Figure 5 This is a schematic diagram of the results of the moisturizing and water-locking effect evaluation of the present application embodiment. Figure 3 ;

[0037] Figure 6This is a schematic diagram of the results of the anti-photoaging efficacy evaluation of the embodiment of the present application. Figure 1 ;

[0038] Figure 7 This is a schematic diagram of the results of the anti-photoaging efficacy evaluation of the embodiment of the present application. Figure 2 ;

[0039] Figure 8 This is a schematic diagram of the results of the anti-photoaging efficacy evaluation of the embodiment of the present application. Figure 3 ;

[0040] Fig. 9 It is a flow chart of the preparation method of the embodiment of the present application. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0042] Example 1

[0043] like Fig. 9 As shown, the preparation method of the Bacillus fermentation product and the lysate comprises:

[0044] 1) Activation of strains: Bacillus subtilis was purchased from the China Industrial Microbiological Culture Collection Administration Center, numbered CICC 20030. Use a sterile pipette to absorb 0.5 mL of NB culture medium to completely dissolve the strain, transfer the dissolved bacterial solution to a sterile test tube containing 5 mL of liquid culture medium and mix well. After static culture at 37°C for 24 hours, the strain was inoculated into a 250 mL conical flask containing 50 mL of NB culture medium, placed in a constant temperature shaking box and shaken for 18 hours at 37°C and 220 r / min for standby use.

[0045] 2) Screening in simulated desert ultraviolet environment: The activated strain was diluted 10 times, inoculated into a culture dish containing NB medium, and placed in a 37°C incubator. A UV lamp was placed in the incubator to make the UV radiation reach the daily average UV radiation in the hinterland of the Taklimakan Desert (0.87 MJ·m -2 ) after treatment for 0, 1, 2, 4, 6, 8, 12, and 24 hours respectively. Turn off the UV lamp and incubate at 37°C for 24 hours. Take the bacterial count of the UV treatment group for 0 hours as the control, calculate the bacterial count of the groups with different treatment time, select the colonies with a lethality rate of 70%-90% for expansion culture, and incubate for 3 generations at 0.87MJ·m -2After the same period of treatment in the ultraviolet irradiation environment, if the survival rate is below 70%, the surviving strains are extracted and expanded for 3 generations until the survival rate remains above 70%, and several colonies are selected for expansion.

[0046] 3) Simulated desert hypertonic environment screening: Add soluble salts to the NB medium to prepare the ion concentration to 6342.90 mg / L (Na + )、54.22mg / L(K + )、100.31mg / L(Ca 2+ )、162.46mg / L(Mg 2+ )、6883.94mg / L(Cl - )、1140.44mg / L(SO 4 2+ )、117.62mg / L(HCO 3- )、3.02mg / L(F - ) desert hypertonic medium, and then add 0.01 mg / L vitamin E and 0.5 mg / L Mn 2+ , and sterilized under high pressure. The UV-screened stable strains were placed in desert hyperosmotic medium and cultured at 37°C. The OD values ​​of the fermentation liquid at different fermentation times were detected to draw growth curves. Several strains of Bacillus subtilis resistant to hyperosmosis were screened out. After being inoculated in normal liquid medium for three generations, they were inoculated in desert hyperosmotic medium to detect the growth curve again. The strains resistant to hyperosmosis with genetic stability were screened out for expanded culture.

[0047] 4) Preparation of fermentation product: The cultured stable Bacillus subtilis was inoculated in a fermentation tank for fermentation culture. The fermentation medium formula was 10.0 g / L peptone, 5.0 g / L beef extract powder, 3.0 g / L sodium chloride, 3.0 g / L other inorganic salts, and the rest was water. The pH was 7.2. The culture conditions were 37° C., the inoculation amount was 4%, the fermentation speed was 180 r / min, and the fermentation liquid obtained by culturing for 48 hours was centrifuged at 4000 r / min for 30 minutes to obtain the supernatant. The supernatant was sterilized by a 0.22 μm filter membrane to obtain the fermentation product.

[0048] 5) Separation and purification of fermentation products: Soak the wood activated carbon in anhydrous ethanol and deionized water for 12 hours respectively, and then place it in a vacuum dryer for drying to remove impurities on the surface of the activated carbon. Then add the fermentation product solution to a container, add activated carbon in an amount of 4 mg / mL, heat at 45°C and mechanically stir for 30 minutes, filter the solution through filter paper to remove activated carbon impurities, obtain a decolorized and deodorized fermentation product, and add 0.02% BHT, 0.01mg / L disodium hydrogen phosphate, 0.01mg / L sodium dihydrogen phosphate, and 0.1% PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol as stabilizers to the solution.

[0049] Example 2

[0050] like Fig. 9 As shown, specifically including:

[0051] 1) Bacteria activation: Same as Example 1.

[0052] 2) Screening in simulated desert ultraviolet environment: The activated strain was diluted 10 times, inoculated into a culture dish containing BHI medium, and placed in a 37°C incubator. A UV lamp was placed in the incubator to make the UV radiation reach the daily average UV radiation in the hinterland of the Taklimakan Desert (0.95 MJ·m -2 ) after treatment for 0, 1, 2, 4, 6, 8, 12, and 24 hours respectively. Turn off the UV light and incubate at 37°C for 24 hours. Take the bacterial count at 0 hour as the control group, calculate the bacterial counts of the groups with different treatment time, select the colonies with a lethality rate of 70%-90% for expansion culture, and incubate for 3 generations at 0.95 MJ·m -2 After the same period of treatment in the ultraviolet irradiation environment, if the survival rate is below 70%, the surviving strains are extracted and expanded for 3 generations until the survival rate remains above 70%, and several colonies are selected for expansion.

[0053] 3) Simulated desert hypertonic environment screening: Add soluble salts to the BHI medium to prepare the ion concentration to 8123.54 mg / L (Na + )、127.15mg / L(K + )、100.30mg / L(Ca 2+ )、336.13mg / L(Mg 2+ )、8859.75mg / L(Cl - )、991.22mg / L(HCO 3- )、2297.22mg / L(SO 4 2- )、5.391mg / L(F - ) desert hypertonic medium, and then add 0.01 mg / L vitamin E, 0.2 mg / L Zn 2+ and 0.1mg / L Mn 2+ , and sterilized under high pressure. The UV-screened stable strains were placed in desert hyperosmotic medium and cultured at 37°C. The OD values ​​of the fermentation liquid at different fermentation times were detected to draw growth curves. Several strains of Bacillus subtilis resistant to hyperosmosis were screened out. After being inoculated in normal liquid medium for three generations, they were inoculated in desert hyperosmotic medium to detect the growth curve again. The strains resistant to hyperosmosis with genetic stability were screened out for expanded culture.

[0054] 4) Preparation of lysate: The cultured and stable Bacillus subtilis was inoculated in a fermenter for fermentation. The fermentation medium formula was 15.0 g / L peptone, 3.0 g / L beef extract powder, 5.0 g / L sodium chloride, 0.5 g / L other inorganic salts, and the rest was water. The pH was 6.9, the culture conditions were 37 ° C, the inoculation amount was 5%, the fermentation speed was 200 r / min, and the fermentation liquid obtained after 48 hours of culture was filtered through a 100 nm porous ceramic membrane. The feed temperature was 40 ° C, and the pressure was controlled at 3.0 bar. The bacteria in the fermentation liquid were trapped. After the bacteria were resuspended in a 1:1 phosphate buffer, they were crushed by a high-pressure homogenizer at a pressure of 40 MPa, the temperature was controlled at 25 ° C, and the cycle was repeated 3 times. The uniformly crushed liquid was centrifuged at a speed of 10000 r / min for 30 minutes, and the supernatant was collected as the lysate.

[0055] 5) Separation and purification of lysate: Soak the macroporous resin D201 in deionized water for 12 hours, then soak it in anhydrous ethanol for 12 hours, wash it with deionized water until it has no alcohol taste, soak it in 4% NaOH and 4% HCl for 12 hours respectively, and then wash it with deionized water until it is neutral. Load the pretreated macroporous resin into the chromatography column, elute the lysate at a flow rate of 200 mL / h to obtain a decolorized and deodorized lysate, and add 1% tocopherol, 0.01 mg / L dipotassium hydrogen phosphate, 0.01 mg / L potassium dihydrogen phosphate, and 0.1% Tween-80 as stabilizers to the solution.

[0056] Example 3

[0057] like Fig. 9 As shown, specifically including:

[0058] 1) Bacteria activation: The difference from Example 1 is that the NB medium is replaced by the BHI medium, and the rest is the same.

[0059] 2) Screening in simulated desert ultraviolet environment: The activated strain was diluted 10 times, inoculated into a culture dish containing BHI medium, and placed in a 37°C incubator. A UV lamp was placed in the incubator to make the UV radiation reach the daily average UV radiation in the hinterland of the Taklimakan Desert (0.95 MJ·m -2 ) after treatment for 0, 1, 2, 4, 6, 8, 12, and 24 hours respectively. Turn off the UV light and incubate at 37°C for 24 hours. Take the bacterial count at 0 hour as the control group, calculate the bacterial counts of the groups with different treatment time, select the colonies with a lethality rate of 70%-90% for expansion culture, and incubate for 3 generations at 0.95 MJ·m -2 After the same period of treatment in the ultraviolet irradiation environment, if the survival rate is below 70%, the surviving strains are extracted and expanded for 3 generations until the survival rate remains above 70%, and several colonies are selected for expansion.

[0060] 3) Simulated desert hypertonic environment screening: Add soluble salts to the NB medium to prepare the ion concentration to 7168.40 mg / L (Na + )、64.90mg / L(K + )、213.75mg / L(Ca 2+ )、265.90mg / L(Mg 2+ )、7229.25mg / L(Cl - )、164.70mg / L(HCO 3- )、921.60mg / L(SO 4 2- )、3.42mg / L(F - ) desert hypertonic medium, and then add 0.01mg / L vitamin E, 0.2mg / L Fe 2+ and 0.1mg / L Mn 2+ , and sterilized under high pressure. The UV-screened stable strains were placed in desert hyperosmotic medium and cultured at 37°C. The OD values ​​of the fermentation liquid at different fermentation times were detected to draw growth curves. Several strains of Bacillus subtilis resistant to hyperosmosis were screened out. After being inoculated in normal liquid medium for three generations, they were inoculated in desert hyperosmotic medium to detect the growth curve again. The strains resistant to hyperosmosis with genetic stability were screened out for expanded culture.

[0061] 4) Preparation of fermentation product and lysate: The cultured and stable Bacillus subtilis was inoculated in a fermentation tank for fermentation culture. The fermentation medium formula was 15.0 g / L peptone, 5.0 g / L beef extract powder, 5.0 g / L sodium chloride, 0.5 g / L other inorganic salts, and the rest was water. The pH was 7.0, the culture conditions were 37°C, the inoculation amount was 5%, the fermentation speed was 200 r / min, and the fermentation liquid obtained by culturing for 48 hours was passed through a high-pressure homogenizer and crushed at a pressure of 40 MPa, the temperature was controlled at 25°C, and the cycle was repeated 3 times. The uniformly crushed liquid was filtered through a 100 nm porous ceramic membrane, the feed temperature was 40°C, the pressure was controlled at 3.0 bar, and the clear liquid was collected as the fermentation product lysate.

[0062] 5) Separation and purification of fermentation products and lysates: Soak the wood activated carbon in anhydrous ethanol and deionized water for 12 hours respectively, and then place it in a vacuum dryer for drying to remove impurities on the surface of the activated carbon. Then add the fermentation product lysate solution to a container, add activated carbon in an amount of 4 mg / mL, heat at 45°C and mechanically stir for 30 minutes, filter the solution through filter paper to remove activated carbon impurities, obtain a decolorized and deodorized fermentation product lysate, and add 0.02% BHT, 0.01mg / L disodium hydrogen phosphate, 0.01mg / L sodium dihydrogen phosphate, and 0.1% PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol as stabilizers to the solution.

[0063] In the actual operation, it was found that the culture medium containing only desert soluble salt ions would cause the Bacillus subtilis to degenerate and inactivate. Therefore, in the above three embodiments, a certain proportion of Zn was added to the simulated desert culture medium. 2 +、Mn 2+ , vitamin E and other promoting factors. These substances can enhance the vitality of the strain, enter the bacteria to activate the spore receptor protein, transmit the signal of spore formation, and promote the strain to produce spores to resist the hypertonic environment.

[0064] Among them, commonly used fermentation broth sterilization methods include high-pressure and high-temperature sterilization, chemical sterilization, filtration sterilization, and ultraviolet sterilization. High-temperature and high-pressure sterilization can effectively kill most bacteria, but it cannot completely kill the bacillus that can form in this application, and high temperature and high pressure will cause the loss of effective ingredients in the fermentation broth. Chemical sterilization uses acid, alkali or oxidants to destroy the structure of bacteria, and has a good sterilization effect, but may cause the loss of effective ingredients in the fermentation broth. Ultraviolet sterilization uses ultraviolet irradiation to destroy microbial DNA to play a bactericidal role, but the bactericidal effect is poor for bacillus that are resistant to certain radiation in this application. Therefore, the present application is preferably filtration sterilization, which is to filter the fermentation broth through a high-efficiency filter, which can effectively remove microorganisms, and the method is relatively mild, which can reduce the loss of effective ingredients and ensure the quality of the product.

[0065] Among them, traditional bacterial cell crushing methods (ultrasonic crushing method, chemical method, enzyme method, freezing crushing method, high temperature crushing method) have the problems of time-consuming, low efficiency, high cost, uneven crushing, loss of active ingredients, etc. The high-pressure microfluidic homogenization method used in this application utilizes high pressure to extrude, shear and impact the bacterial solution through special structural channels, which can solve the above problems and obtain a lysed product with stable components. It has the advantages of simple operation, high extraction rate, good repeatability, and protection of active ingredients.

[0066] Among them, fermented cosmetic raw materials often carry special pigment impurities and unique odors, which affect their sense of use. These impurities come from the culture medium itself or the metabolites after fermentation. Decolorization and deodorization are the key steps before fermented cosmetic raw materials are used in cosmetics, and the purpose is to remove pigment impurities and odor impurities in the fermentation products. Decolorization and deodorization methods include enzyme method, oxidative decolorization method, ultrafiltration method, polyamide method, activated carbon adsorption method, and resin decolorization method. In this application, activated carbon adsorption method and column chromatography are used for decolorization and deodorization. Activated carbon has a developed mesoporous structure and a developed specific surface area, a large adsorption capacity, a fast filtration speed, and can adsorb impurities such as pigments. Column chromatography is to use the different adsorption forces and elution speeds of substances on the porous polymer stationary phase to finely separate substances of different polarities. These two methods are relatively mild, and have a higher decolorization and deodorization efficiency on the basis of retaining the effective ingredients, but the specificity is high, and different activated carbons and macroporous resins need to be selected for different substances.

[0067] Wherein, fermentation product refers to metabolites produced by microbial fermentation process, such as amino acids, proteins, polypeptides, minerals, polysaccharides and vitamins. Lysate is the product obtained after the decomposition of microbial cells, cell wall fragment components (such as lactic acid, short-chain fatty acids, bacteriocin) and various amino acids, polypeptides, polysaccharides, minerals, etc. The Bacillus subtilis fermentation product and lysate obtained in this application are rich in various active ingredients such as protein, polypeptide, amino acid, polysaccharide, polyphenol, etc. Polyphenol is a general term for polyhydroxyphenol compounds, which are sensitive to light, heat, strong acid and alkali conditions and are easily oxidized and inactivated by air. The unstable factors of protein and polypeptide substances come from their own easy oxidation reaction, some peptide bonds are easily hydrolyzed and broken, and the interaction and aggregation between proteins and polypeptides leads to changes in spatial structure. Therefore, due to external factors such as pH, air, temperature, etc. during storage, the active ingredients of Bacillus subtilis fermentation products and lysates will appear to have a phenomenon of reduced content and reduced activity. Adding stabilizers helps to improve the stability of the solution system and plays a role in protecting the active ingredients. Antioxidants (such as BHT, tocopherol, etc.) inhibit or delay the oxidation process of polyphenols and other substances by free radical capture, metal ion complexation and peroxide ester destruction. Buffer salts (such as potassium dihydrogen phosphate) effectively regulate and maintain the pH stability of the system based on their dissociation characteristics, hydrogen ion neutralization, buffer capacity and possible ion exchange mechanism. Non-ionic surfactants (such as Tween-80, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol) are surfactants that do not react with aqueous solution ions. They can adsorb on the surface of proteins and polypeptides to reduce their surface tension, thereby preventing interactions and aggregation between proteins and polypeptides. At the same time, they can form a protective layer on the surface of proteins and polypeptides to prevent them from being disturbed by the external environment.

[0068] Comparative Example 1

[0069] The difference from Example 1 is that the strains do not have the steps of screening in a simulated desert ultraviolet environment and a simulated desert hypertonic environment. The remaining steps of strain activation, preparation of fermentation products and lysates, and separation and purification of fermentation products and lysates are the same as those in Example 1.

[0070] Comparative Example 2

[0071] Different from Example 1, stabilizers such as BHT, sodium dihydrogen phosphate, sodium hydrogen phosphate, PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol were not added to the fermentation product and lysate finally obtained.

[0072] In order to demonstrate the technical effect of the present application, the following are examples of the effects:

[0073] 1. Stability analysis:

[0074] The samples of Example 1, Example 2, Example 3 and Comparative Example 2 were placed at 4°C and 40°C under light conditions for stability experiments, and the polyphenol, protein and polypeptide contents were measured at 1, 2, 3 and 4 weeks, and the polyphenol, protein and polypeptide retention rates were calculated based on the contents. The test results are shown in the following table:

[0075]

[0076]

[0077]

[0078] As can be seen from the above table, the retention rates of polyphenols, polypeptides and proteins in Comparative Example 2 change significantly over time. After being placed at 4°C, 40°C and light conditions for 4 weeks, the polyphenol retention rates are only 97.81%, 94.51% and 95.22%, respectively; the polypeptide retention rates are only 98.63%, 93.78% and 93.82%, respectively; and the protein retention rates are only 97.23%, 94.78% and 93.26%, respectively, which are much lower than those in Examples 1, 2 and 3. This indicates that adding a stabilizer to the fermentation product and the lysate can significantly improve the stability of the system and slow down the degradation and inactivation of polyphenols, polypeptides and proteins.

[0079] 2. Cytotoxicity studies:

[0080] The cell density was 1×10 5 / mL human fibroblasts were seeded in a 96-well plate, and a DMEM medium containing 10% fetal bovine serum was added to culture for 24 hours, and the waste liquid was discarded. The plate was washed with PBS three times, and then 100 μL of 10% fetal bovine serum DMEM medium was added to each well of the blank control group, and 100 μL of 10% fetal bovine serum DMEM medium containing different concentrations of the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 was added to each well of the experimental group. After culturing in an incubator for 24 hours, the waste liquid was discarded, 50 μL of MTT solution was added, and after incubation at 37°C for 4 hours, the MTT solution was discarded, 150 μL of DMSO solution was added, and the plate was oscillated in a dark room for 10 minutes. The absorbance was measured at an absorbance of 570 nm, and the survival rate (%) of human fibroblasts was calculated according to the formula using the existing technology. The results are as follows Figure 1 As shown, within the test range, the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 were non-toxic to human fibroblasts.

[0081] The cell density was 1×10 5 / mL human keratinocytes were inoculated into a 96-well plate, and a DMEM medium containing 10% fetal bovine serum was added to culture for 24 hours, and the waste liquid was discarded. The plate was washed with PBS three times, and then 100 μL of 10% fetal bovine serum DMEM medium was added to each well of the blank control group, and 100 μL of 10% fetal bovine serum DMEM medium containing different concentrations of the fermentation products and lysates obtained in Examples 1, 2, 3 and Comparative Examples 1 and 2 was added to each well of the experimental group. After culturing in an incubator for 24 hours, the waste liquid was discarded, 50 μL of MTT solution was added, and after incubation at 37°C for 4 hours, the MTT solution was discarded, 150 μL of DMSO solution was added, and the plate was oscillated in a dark room for 10 minutes. The absorbance was measured at an absorbance of 570 nm, and the survival rate (%) of human keratinocytes was calculated according to the formula using the existing technology. The results are shown in the following table. Figure 2 As shown, within the test range, the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 were non-toxic to human keratinocytes.

[0082] 3. Evaluation of moisturizing and water-locking effects:

[0083] The cell density was 1×10 5 / mL human keratinocytes were inoculated in a 6-well plate, and a DMEM medium containing 10% fetal bovine serum was added to culture for 24 hours, the waste liquid was discarded, and PBS was washed 3 times, and then 2mL of 10% fetal bovine serum DMEM medium was added to each well of the blank control group, and 2mL of 10% fetal bovine serum DMEM medium containing the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 was added to each well of the experimental group. Cultured in a 37°C incubator for 24 hours, the culture solution was discarded, and the cells were digested and collected. Total RNA was extracted according to the instructions of the RNA extraction kit, and the expression of genes related to human epidermal keratinocytes and natural moisturizing factor (Filaggrin, claudin-1, caspase-14) was detected by RT-qPCR.

[0084] The results are as follows Figure 3 As shown, the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 can up-regulate the expression of the Filaggrin gene, wherein the expression levels of Examples 1, 2, 3 and Comparative Example 2 are significantly higher than that of Comparative Example 1. This gene can promote the complete hydrolysis of filaggrin on the stratum corneum by various levels of proteases, and can produce important natural moisturizing factors such as free amino acids and derivatives, which are essential for maintaining the moisture of the stratum corneum.

[0085] like Figure 4 As shown, the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 can upregulate the expression of claudin-1 gene, wherein the expression levels of Examples 1, 2, 3 and Comparative Example 2 are significantly higher than that of Comparative Example 1. This gene is the main component of the tight junction complex that regulates the permeability of epithelial cells and participates in the formation of impermeable barriers.

[0086] like Figure 5 As shown, the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 can upregulate the expression of caspase-14 gene, wherein the expression levels of Examples 1, 2, 3 and Comparative Example 2 are significantly higher than those of Comparative Example 1. This gene can promote terminal differentiation of epidermal cells and play a key catalytic role in hydration of the stratum corneum. These results mean that the fermentation products and lysates produced by Bacillus subtilis screened by ultraviolet and hypertonic screening in simulated desert environments have higher moisturizing activity than those not screened, and the stabilizer has no effect on the moisturizing effect of the product.

[0087] 4. Evaluation of anti-photoaging efficacy:

[0088] B16 cell melanin secretion inhibition ability: the cell density was 1×10 5 / mL of B16 cells were digested and inoculated into 6-well plates, and 10% fetal bovine serum DMEM medium was added to culture for 24h. When the cell density reached a certain level, the model group and the drug-treated group were irradiated with ultraviolet rays of appropriate intensity to establish a light damage model, and then 2mL of 10% fetal bovine serum DMEM medium was added to each well of the blank group and the model group, and 2mL of 10% fetal bovine serum DMEM medium containing the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 was added to each well of the experimental group, and the cells were collected after continued culture for 96h. Prepare a suitable concentration of NaOH solution, add an appropriate amount of NaOH solution to the centrifuge tube, shake well, place at 55°C for 2h to allow the melanin in the cells to fully precipitate and dissolve, and place in an ELISA instrument to detect the OD 490nm value after completion. Use the existing technology to process the calculated data and draw the corresponding bar graph.

[0089] like Figure 6 As shown, after ultraviolet radiation, the melanin secretion ability of B16 cells in the model group is enhanced, and the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 can inhibit the melanin secretion of B16 cells, among which the inhibition effect of Examples 1, 2, 3 and Comparative Example 2 is better than that of Comparative Example 1. This shows that compared with the unscreened ones, the fermentation products and lysates produced by Bacillus subtilis screened by ultraviolet and hypertonic screening in a simulated desert environment have better ability to inhibit melanin secretion, and the stabilizer has no effect on the ability of the product to inhibit melanin secretion.

[0090] The inhibitory capacity of matrix metalloproteinase (MMP-1) and tissue inhibitor of activated metalloproteinase (TIMP) in human fibroblasts was investigated. The cell density was 1×10 5 / mL human fibroblasts were seeded in a 6-well plate, and a DMEM medium containing 10% fetal bovine serum was added and cultured for 24 hours. When the cell density reached a certain level, the model group and the drug-treated group were irradiated with ultraviolet rays of appropriate intensity to establish a light damage model, and the waste liquid was discarded. The cells were washed with PBS for 3 times, and then 2mL of 10% fetal bovine serum DMEM medium was added to each well of the blank group and the model group, and 2mL of 10% fetal bovine serum DMEM medium containing the fermentation products and lysates prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 was added to each well of the experimental group, and the culture was continued for 24 hours. After the culture was completed, the cell supernatant was taken, centrifuged, and the supernatant was taken for detection using MMP-1 and TIMP kits.

[0091] like Figure 7 As shown, after ultraviolet radiation, the MMP-1 enzyme activity of the model group was enhanced, while the MMP-1 enzyme activity of the fermentation products and lysate groups prepared in Examples 1, 2, 3 and Comparative Examples 1 and 2 was reduced, among which Examples (Examples) 1, 2, 3 and Comparative Example 2 had a better effect of inhibiting MMP-1 enzyme activity than Comparative Example 1.

[0092] like Figure 8 As shown, after ultraviolet radiation, TIMP enzyme activity decreases, while the fermentation products and lysates obtained in Examples 1, 2, 3 and Comparative Examples 1 and 2 can enhance TIMP enzyme activity, wherein Examples (Examples) 1, 2, 3 and Comparative Example 2 have a better effect on enhancing TIMP enzyme activity than Comparative Example 1. Ultraviolet irradiation can induce excessive production of MMP-1, thereby degrading collagen and extracellular matrix proteins, playing an important role in skin anti-aging. TIMP is a specific endogenous inhibitor of MMPs, which can balance the synthesis and degradation of extracellular matrix proteins, and plays a balancing and regulating role in skin photoaging. The above results show that compared with the unscreened ones, the fermentation products and lysates produced by Bacillus subtilis screened by ultraviolet and hypertonicity in simulated desert environments have a better effect of resisting photoaging, and the stabilizer has no effect on the anti-photoaging effect of the product.

[0093] In summary, the fermentation products and lysates of the strain of the present application are rich in proteins, peptides, amino acids, polyphenols, polysaccharides and other active substances, and have excellent moisturizing and anti-photoaging activities. In addition, the present application adds vitamin E, Fe 2+ 、Zn 2+ , Mn 2+ And other promoting factors are used to prevent the degradation and inactivation of strains. The present application replaces the traditional ultrasonic crushing method with a high-pressure microfluidization homogenization method, which can improve the extraction efficiency while ensuring the integrity of the active ingredients. The present application uses activated carbon adsorption and column chromatography to decolorize and deodorize, which can improve the color and smell of the product. In the present application, adding a certain proportion of antioxidants, buffer salts, and non-ionic surfactants as stabilizers to the fermentation products and lysates can enhance the stability of the fermentation product and lysate system, and inhibit or slow down the loss of active ingredients such as proteins, polypeptides, and polyphenols.

[0094] The parts not described in detail in this application are prior art, so this application does not describe them in detail.

[0095] It is to be understood that the term "one" should be understood as "at least one" or "one or more", that is, in one embodiment, the number of an element may be one, while in another embodiment, the number of the element may be multiple, and the term "one" should not be understood as a limitation on the quantity.

[0096] Although this article uses more professional terms, it does not exclude the possibility of using other terms. These terms are used only to more conveniently describe and explain the essence of this application; interpreting them as any additional restrictions is contrary to the spirit of this application.

[0097] The present application is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of this application. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present application.

Claims

1. A method for preparing a Bacillus fermentation product and a lysate, characterized in that: The following steps are involved: S00, inoculating Bacillus subtilis as a bacterial species into a seed culture medium for activation to obtain a seed solution; S10, inoculating the seed solution on a plate, treating the plate with the average daily ultraviolet radiation in the desert for 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours, and then culturing the plate in a dark environment for 16-24 hours; The bacterial count of the UV-treated group at 0 h was used as the control. The bacterial counts of the groups treated with different treatment times were calculated. The colonies with a lethality rate of 70%-90% were selected for expansion culture. After culturing for 2-3 generations, they were placed in 0.87-1.05 MJ·m -2 UV treatment for the same time; If the survival rate is below 70%, the surviving strains are extracted and expanded for several generations until the survival rate remains above 70%, and several colonies are screened to obtain radiation-resistant strains; S20, inoculating the radiation-resistant strain into a desert culture medium to further screen out a genetically stable hyperosmotic-resistant strain; The desert culture medium is based on the seed culture medium, and salt ions are added according to the soluble salt ratio of shallow groundwater in the hinterland of the desert, and some promoting factors are added to obtain the above-mentioned culture medium. The promoting factors include vitamin E, Fe 2+ 、Zn 2+ , Mn 2 + One or more of the following; S30, inoculating the hyperosmotic-resistant strain into a fermentation tank for fermentation culture, and obtaining a fermentation clear liquid and a bacterial precipitate by separation; The fermentation liquid is sterilized to obtain a fermentation liquid, and the bacterial cell precipitate is resuspended, crushed, and subjected to high-speed centrifugation to obtain a bacterial cell crushing product; S40, decolorizing and deodorizing the fermentation liquid and the bacterial cell disruption product, and adding a stabilizer to obtain a fermentation product and a lysate.

2. The method for preparing a Bacillus fermentation product and a lysate according to claim 1, characterized in that: In step S00, the seed culture medium is a nutrient broth culture medium or a brain heart infusion broth culture medium.

3. The method for preparing the Bacillus fermentation product and lysate according to claim 1, characterized in that: In step S30, the fermentation temperature of the fermentation culture is 30-40°C, the inoculation amount is 3-10%, the fermentation speed is 100-300r / min, and the ventilation ratio is 1.0-2.0VVM.

4. The method for preparing the Bacillus fermentation product and lysate according to claim 3, characterized in that: In step S30, the fermentation medium formula is 10.0-20.0 g / L peptone, 3.0-5.0 g / L beef extract powder, 3.0-10.0 g / L sodium chloride, 0.1-30.0 g / L other inorganic salts, and the rest is water, and the pH is 6.7-7.

2.

5. The method for preparing a Bacillus fermentation product and a lysate according to any one of claims 1 to 4, characterized in that: In step S40, the stabilizer is one or more of 2,6-di-tert-butyl-4-methylphenol, tocopherol, potassium dihydrogen phosphate, Tween-80, and PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerol.

6. A lysate, characterized in that The product is prepared by the method for preparing a bacillus fermentation product and a lysate according to any one of claims 1 to 5.

7. A fermentation product, characterized in that The product is prepared by the method for preparing a bacillus fermentation product and a lysate according to any one of claims 1 to 5.

8. Biomedicine and health products, characterized in that: The fermentation product and / or lysate prepared by the method for preparing a Bacillus fermentation product and lysate according to any one of claims 1 to 5, and the biopharmaceutical and health products include cosmetics, health products and medicines.

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