Bletilla striata fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to bletilla striata fermentation liquor as well as a preparation method and application thereof. Specifically, the invention discloses bacillus subtilis for preparing the bletilla striata fermentation liquor, and the preservation number of the bacillus subtilis is CGMCC (China General Microbiological Culture Collection Center) No.29154. According to the method, the bletilla striata is fermented by using the bacillus subtilis, so that the polysaccharide activity of the bletilla striata can be improved, the viscosity of fermentation liquor can be reduced, the loss of active ingredients (such as polysaccharide) in the bletilla striata can be reduced, the bletilla striata is fully utilized, and the waste of resources is reduced. It is accidentally found that the obtained bletilla striata fermentation liquor has excellent oxidation resistance and moisturizing performance. The raw materials are natural single raw materials, and the components are purer, safer and more stable; the preparation method is simple, convenient, easy to operate, high in safety and remarkable in effect, and has wide market and application value in preparation of cosmetics with antioxidant and moisturizing functions when being used as an active ingredient.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and more specifically, to a bletilla striata fermented liquid and a preparation method and application thereof. Background Art

[0002] Bletilla striata, also known as "Baiji", is the dried tuber of Bletilla striata (Thunb.) Reichb. f, an orchid plant. It tastes sweet, bitter and astringent, and is cold in nature. It enters the stomach meridian, lung meridian and liver meridian, and has the effects of astringing sores, tonifying the lungs, reducing swelling, stopping bleeding and promoting tissue regeneration. In the past decade, domestic and foreign scholars have conducted extensive research and discussion on the medicinal value of Bletilla striata, and found that Bletilla striata has a variety of pharmacological effects, including antibacterial, antiviral, anti-tumor, antioxidant, hemostatic and promoting wound healing.

[0003] Bletilla striata polysaccharide, also known as Bletilla striata gum, is a water-soluble polysaccharide extracted from the traditional Chinese medicine Bletilla striata. It is mainly composed of β-mannose and β-glucose through 1-4 glycosidic bonds. Due to different determination methods, the relative molar ratio of mannose to glucose in Bletilla striata polysaccharide reported in the literature is different, and its relative molecular mass is between 70,000 and 600,000. In recent years, studies have found that Bletilla striata polysaccharide has good biodegradability and biocompatibility. Bletilla striata polysaccharide has the effects of astringent and regenerating muscles, antibacterial and anti-inflammatory, and promoting wound healing. It has the potential to be used as an antioxidant additive in the field of cosmetics.

[0004] The existing technology uses a method of directly extracting the active ingredients in Bletilla striata with water and then extracting polysaccharides by alcohol precipitation. This method not only has a low extraction rate, but also has many steps, high extraction costs, and harsh reaction conditions. In addition, the Bletilla striata extract produced has high viscosity and poor moisturizing properties, which is not conducive to cosmetic applications.

[0005] Therefore, there is an urgent need in the art to develop a bletilla striata fermentation liquid with a simple preparation process, low viscosity, high polysaccharide content, and excellent antioxidant and moisturizing properties for research in the cosmetics field. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide a bletilla striata fermented liquid with simple preparation process, low viscosity, high polysaccharide content, and excellent antioxidant and moisturizing properties.

[0007] In a first aspect of the present invention, a Bacillus subtilis for preparing Bletilla striata fermentation broth is provided, and its deposit number is: CGMCC No.29154.

[0008] In a second aspect of the present invention, a method for preparing a Bletilla striata fermentation broth is provided, comprising the following steps: (a) using Bletilla striata as a raw material, sequentially performing pre-fermentation treatment, sterilization treatment, and cooling to room temperature to obtain an initial fermentation system; (b) inoculating the Bacillus subtilis described in the first aspect of the present invention into the initial fermentation system to obtain an initial fermentation mixed liquid, and fermenting and culturing to obtain a Bletilla striata fermentation liquid; The volume of deionized water in the initial fermentation system was taken as the reference, and the number of viable Bacillus subtilis in the initial fermentation system was 10 8 CFU / mL~10 12 CFU / mL.

[0009] In another preferred embodiment, the pre-fermentation treatment comprises the following steps: (a1) mixing dried and sieved Bletilla striata tuber powder with deionized water to obtain a Bletilla striata raw material liquid; or (a2) subjecting the Bletilla striata raw material solution to extraction treatment to obtain a Bletilla striata extract solution.

[0010] In another preferred example, the bletilla striata tuber powder and deionized water are mixed at a mass volume ratio of 1:10-30; the particle size of the bletilla striata tuber powder is 50-150 mesh, preferably 60 mesh.

[0011] In another preferred embodiment, the extraction method includes: ultrasound-assisted water extraction, enzyme extraction or physical, chemical and biological combined extraction method.

[0012] In another preferred embodiment, the sterilization temperature is 110° C. to 135° C., the pressure is 0.1 to 0.3 MPa, and the sterilization time is 10 to 35 minutes.

[0013] In another preferred embodiment, the fermentation culture comprises: (f1) inoculating the Bacillus subtilis into LB (Luria-Bertani) liquid culture medium, culturing at 90-180 r / min and 28-37° C. for 16-24 h, and obtaining a viable cell count of 10 8 CFU / mL~10 12 CFU / mL of inoculum; (f2) adding an inoculum solution including Bacillus subtilis to the initial fermentation system to obtain an initial fermentation mixture; and dynamically culturing and fermenting the initial fermentation mixture in a constant temperature incubator at 30° C. to 45° C. at 90 to 180 r / min to obtain a Bletilla striata fermentation solution.

[0014] In another preferred embodiment, the formula of the LB liquid culture medium is as follows: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl are added to every 1 L of deionized water.

[0015] In another preferred embodiment, the step (b) further comprises the following steps: (b1) fermenting the initial fermentation mixture to obtain a Bletilla striata initial fermentation liquid, sterilizing it, and centrifuging it to obtain a supernatant of the Bletilla striata initial fermentation liquid; (b2) The supernatant of the initial fermentation liquid of Bletilla striata obtained in (b1) is subjected to secondary sterilization to obtain Bletilla striata fermentation liquid, wherein the secondary sterilization adopts a high-temperature water bath sterilization method to reduce residual microorganisms.

[0016] In another preferred embodiment, the preparation method further comprises step (c): The adsorbent and the bletilla striata fermented liquid are mixed and stirred evenly at a mass ratio of 1wt% to 95wt%, filtered, and decolorized and deodorized. The adsorbent is selected from the following group: activated carbon, white clay or adsorption resin.

[0017] In another preferred embodiment, the preparation method further comprises step (d): The preservative is mixed with the Bletilla striata fermented liquid filtered in step (c) at a mass volume ratio of 0.5wt% to 2wt% to obtain a Bletilla striata fermented liquid containing the preservative, wherein the preservative is selected from the group consisting of p-hydroxyacetophenone and 1,2-hexanediol.

[0018] In a third aspect of the present invention, a Bletilla striata fermentation broth is provided, which is prepared according to the preparation method described in the second aspect of the present invention.

[0019] In a fourth aspect of the present invention, there is provided a use of the Bletilla striata fermented liquid as described in the third aspect of the present invention for preparing cosmetics.

[0020] In a fifth aspect of the present invention, a cosmetic is provided, comprising, by mass percentage, 1 wt % to 100 wt % of the Bletilla striata fermented liquid described in the fourth aspect of the present invention.

[0021] In another preferred example, the cosmetic comprises 5wt% to 990wt.% of the Bletilla striata fermented broth described in the third aspect of the present invention, and an auxiliary agent selected from a carrier, an emulsifier, a viscosity regulator, a whitening component, a soothing component, a preservative, a fragrance or a combination thereof; the content of the auxiliary agent is 1wt% to 95wt%.

[0022] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0023] Compared with the prior art, the present invention has the following technical effects: The present invention adopts fermentation technology to ferment Bletilla striata powder to obtain fermentation liquid. The present invention uses Bacillus subtilis (CGMCC No.29154) to ferment Bletilla striata, which can improve the activity of Bletilla striata polysaccharide, reduce the viscosity of fermentation liquid, and reduce the loss of active ingredients (such as polysaccharide) in Bletilla striata, so as to make full use of Bletilla striata and reduce the waste of resources. It is unexpectedly found that the Bletilla striata fermentation liquid obtained by the present invention has excellent antioxidant and moisturizing properties. The raw material is a natural single raw material, and the ingredients are simpler, safer and more stable; the preparation method is simple, easy to operate, and has high safety and significant effect. It has a broad market and application value as an active ingredient in the preparation of cosmetics with antioxidant and moisturizing functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram showing the preparation of Bletilla striata fermentation broth in an embodiment of the present invention.

[0025] Figure 2 The result diagram shows the moisturizing performance of the Bletilla striata fermented liquid prepared in the example of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, all the embodiments described are only partial embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Fermentation of Bletilla striata by fermentation technology to obtain fermentation liquid has many advantages. First, microbial fermentation can improve various properties of polysaccharides: after polysaccharide fermentation, the molecular weight is significantly reduced. Fermentation can also change the monosaccharide composition of polysaccharides, affect the branching degree, affect the solubility, viscosity, particle size and other physical and chemical properties of polysaccharides, and improve antioxidant, anti-inflammatory and antibacterial activities. The Bacillus subtilis studied and screened in the present invention is applied to fermentation of Bletilla striata, and then by optimizing the conditions, a cosmetic raw material with good antioxidant activity, viscosity and moisturizing performance is obtained.

[0028] Bacillus subtilis is a species of Bacillus, CAS number 68038-70-0. Single cell is 0.7-0.8×2-3 microns, uniformly colored. No capsule, flagella around, can move. Gram-positive bacteria, can form endogenous stress-resistant spores, spores are 0.6-0.9×1.0-1.5 microns, elliptical to columnar, located in the center of the bacteria or slightly off, the bacteria do not swell after spore formation. Growth and reproduction speed is fast, the surface of the colony is rough and opaque, dirty white or slightly yellow, and when growing in liquid culture medium, wrinkles are often formed. It is an aerobic bacterium. Bacillus subtilis can synthesize enzymes such as α-amylase, protease, lipase, cellulase, etc., play the role of digestive enzymes, and have a strong ability to decompose plant carbohydrates.

[0029] Strain preservation The Bacillus subtilis of the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with a deposit date of November 27, 2023 and a deposit number of CGMCC No. 29154.

[0030] Example 1

[0031] Preparation of Bletilla striata fermentation broth, the specific steps are as follows: S1, preparing a raw material liquid of Bletilla striata, comprising taking 100 ml of deionized water and mixing 3 g of Bletilla striata tuber powder evenly to obtain a raw material liquid of Bletilla striata; wherein the Bletilla striata tuber powder is obtained by crushing and sieving dried Bletilla striata tubers to obtain a powder having a particle size of 60 meshes, to obtain a raw material liquid of Bletilla striata; S2, preparing an initial fermentation system, comprising sterilizing the Bletilla striata raw material liquid in a high pressure sterilizer at 121° C. for 20 min, and cooling to room temperature (25° C.) after sterilization to obtain an initial fermentation system; S3, fermentation culture, including adding fermentation bacteria to the initial fermentation system for culturing and fermentation to obtain Bletilla striata fermentation liquid; the fermentation bacteria is Bacillus subtilis with a preservation number of CGMCC No.29154, which is added to the initial fermentation system in the form of an inoculated bacterial liquid as the fermentation bacteria, wherein the viable bacterial count of the inoculated bacterial liquid of Bacillus subtilis is 1010 CFU / mL, and the amount of the bacterial liquid added is 1.5 mL, and the inoculated bacterial liquid is added and shaken well, and dynamically cultured in a constant temperature incubator at 32° C. for 15 hours to obtain the initial fermentation liquid of Bletilla striata; S4, sterilization and purification. After the fermentation is completed, the obtained initial fermentation liquid of Bletilla striata is sterilized in a high-pressure steam sterilizer at a sterilization condition of 121° C. and 0.13 MPa for 20 min to terminate the fermentation; After sterilization, the initial fermentation liquid of Bletilla striata was cooled to room temperature (25° C.) and centrifuged at a centrifugal speed of 4800 r / min for 30 min. After centrifugation, the supernatant of the initial fermentation liquid of Bletilla striata was obtained. The supernatant of the initial fermentation liquid of Bletilla striata is sterilized for a second time to obtain the sterilized fermentation liquid of Bletilla striata; the conditions of the second sterilization are 121° C. and a pressure of 0.12 MPa for 30 minutes; After sterilization, an activated carbon adsorbent is added to the bletilla striata fermented liquid, and 1 wt.% is added based on the mass of the bletilla striata fermented liquid, and the mixture is stirred and mixed evenly for 30 minutes and then filtered; a preservative is added to the filtered bletilla striata fermented liquid, and during the mixing process of the bletilla striata fermented liquid and the preservative, the mixing temperature is 75° C. Based on the mass of the bletilla striata fermented liquid, 0.5 wt.% of p-hydroxyacetophenone and 0.5 wt.% of 1,2-hexanediol are added to play a preservative role, and the mixture is mixed evenly to obtain a bletilla striata fermented liquid with a preservative role.

[0032] Example 2

[0033] Preparation of Bletilla striata fermentation broth, the specific steps are as follows: S1. Prepare the Bletilla striata extract by ultrasonic-assisted water extraction: take 100 ml of deionized water and 3 g of Bletilla striata tuber powder and mix them evenly in a 300 ml beaker, put it into an ultrasonic cleaner or use an ultrasonic vibrator, extract under the conditions of ultrasonic power of 100 W, ultrasonic temperature of 50° C., and ultrasonic time of 30 min, and filter to obtain the Bletilla striata extract.

[0034] S2, preparing an initial fermentation system, comprising sterilizing the bletilla striata extract in a high pressure sterilizer at 121° C. for 20 min, and cooling to room temperature (25° C.) after sterilization to obtain an initial fermentation system; S3, fermentation culture, including adding fermentation bacteria to the initial fermentation system for culturing and fermentation to obtain Bletilla striata fermentation liquid; the fermentation bacteria include Bacillus subtilis with a preservation number of CGMCC No. 29154, which is added to the initial fermentation system in the form of an inoculated bacterial liquid as a fermentation bacteria, wherein the number of viable bacteria of the inoculated bacterial liquid of Bacillus subtilis is 10 10 CFU / mL, the amount of bacterial solution added was 1.5mL, the inoculated bacterial solution was added and shaken well, and dynamically cultured in a 32℃ constant temperature incubator for 15h to obtain the initial fermentation liquid of Bletilla striata; S4, sterilization and purification. After the fermentation is completed, the obtained initial fermentation liquid of Bletilla striata is sterilized in a high-pressure steam sterilizer at a sterilization condition of 121° C. and 0.13 MPa for 20 min to terminate the fermentation; After sterilization, the initial fermentation liquid of Bletilla striata was cooled to room temperature (25° C.) and centrifuged at a centrifugal speed of 4800 r / min for 30 min. After centrifugation, the supernatant of the initial fermentation liquid of Bletilla striata was obtained. The supernatant of the initial fermentation liquid of Bletilla striata is sterilized for a second time to obtain the sterilized fermentation liquid of Bletilla striata; the conditions of the second sterilization are 121° C. and a pressure of 0.12 MPa for 30 minutes; After sterilization, an activated carbon adsorbent is added to the bletilla striata fermentation liquid. In 100 g of the bletilla striata fermentation liquid, 1 g of the activated carbon adsorbent is added to perform decolorization and deodorization. The mixture is stirred and mixed evenly for 30 minutes and then filtered to obtain the bletilla striata fermentation liquid. A preservative is added to the bletilla striata fermentation liquid. During the mixing of the bletilla striata fermentation liquid and the preservative, the mixing temperature is 75° C. Based on 100 g of the bletilla striata fermentation liquid, 0.5 g of p-hydroxyacetophenone and 0.5 g of 1,2-hexanediol are added to play a preservative role. The mixture is mixed evenly to obtain the bletilla striata fermentation liquid with a preservative role.

[0035] Comparative Example 1 The preparation method of the Bletilla striata fermentation broth in this comparative example is the same as that in Example 1, except that Zygosaccharomyces bisporus with a preservation number of CGMCC No. 29151 is inoculated, the total amount of bacterial broth added remains unchanged, and other condition parameters are the same as those in Example 1.

[0036] Example 3

[0037] In this example, the fermentation broth prepared in the above examples 1-2 and comparative example 1, the Bletilla striata raw material solution in example 1 and the Bletilla striata extract in example 2 were subjected to performance tests. The performance tests included the following items: 1. DPPH free radical scavenging experiment Prepare 500 μL of Bletilla striata sample solutions of different gradient concentrations, including Bletilla striata fermentation broth, Bletilla striata raw material solution or extract, add an equal amount of 500 μL of 0.2 mM DPPH solution, shake evenly, keep it in the dark for 25 min, and measure the absorbance value at a wavelength of 517 nm. The clearance calculation formula is: DPPH free radical scavenging rate (%) = (1-(A1-A2) / A0) × 100; Among them, A0 is the absorbance value of the blank control, A1 is the absorbance value of the Bletilla striata sample solution with different gradient concentrations, and A2 is the absorbance value of the Bletilla striata sample solution measured by using ultrapure water instead of DPPH solution.

[0038] 2. ABTS free radical scavenging experiment Prepare 100 μL of Bletilla striata sample solutions with different gradient concentrations, including Bletilla striata fermentation broth, Bletilla striata raw material solution and extract, add 2.9 mL ABTS solution, and measure the absorbance value at a wavelength of 734 nm. The clearance calculation formula is: ABTS free radical scavenging rate (%) = (1-(A1-A2) / A0) × 100; Among them, A0 is the absorbance value of the blank control, A1 is the absorbance value of the Bletilla striata sample solution with different gradient concentrations, and A2 is the absorbance value of the Bletilla striata sample solution measured by using ultrapure water instead of ABTS solution.

[0039] 3. Hydroxyl radical scavenging experiment Prepare 500 μL of Bletilla striata sample solutions with different gradient concentrations, including Bletilla striata fermentation broth, Bletilla striata raw material solution and extract, add 200 μL of FeSO4 (6 mM), salicylic acid (6 mM) and H2O2 (6 mM), shake well, heat at 37 °C for 30 minutes, and measure the absorbance at 510 nm. The clearance calculation formula is: Hydroxyl free radical scavenging rate (%) = (1-(A1-A2) / A0) × 100; Among them, A0 is the absorbance value of the blank control, A1 is the absorbance value of the Bletilla striata sample solution with different gradient concentrations, and A2 is the absorbance value of the Bletilla striata sample solution measured by using ultrapure water instead of ABTS solution.

[0040] Experimental results analysis: Based on the experimental process of the above Examples 1-2 and the comparative example, the test results of the DPPH radical scavenging rate, ABTS radical scavenging rate, and hydroxyl radical scavenging rate of the Bletilla striata fermented liquid, the Bletilla striata raw material liquid, and the extract were obtained. Each group was repeated three times, and a significance analysis was performed. The results are shown in Table 1: Table 1

[0041] Note: The raw material liquid of Bletilla striata in Group 4 is a mixture of Bletilla striata tuber powder and water before fermentation and after sterilization in Example 1; the extract of Bletilla striata in Group 5 is an extract of Bletilla striata extracted by ultrasonic-assisted water extraction method before fermentation and after sterilization in Example 2. The Duncan test was performed using IBM SPSS Statistics 27 software to analyze the significance of the three free radical scavenging rates of Example 1 and Comparative Example 1, Example 1 and Bletilla striata raw material liquid, Example 2 and Bletilla striata extract, in pairs. Among them, ** indicates that there is a significant difference between Example 1 group and Bletilla striata raw material liquid group (p< 0.01); ## indicates that there is a significant difference between Example 1 group and Bletilla striata extract group (p< 0.01); ^^ indicates that there is a significant difference between Example 2 group and Bletilla striata extract group (p< 0.01).

[0042] As shown in Table 1, the unfermented Bletilla striata raw material solution and the Bletilla striata extract have certain antioxidant activity. The DPPH radical scavenging rate, ABTS radical scavenging rate and hydroxyl radical scavenging rate of the three groups of Example 1, Comparative Example 1 and the Bletilla striata raw material solution are significantly different (p < 0.01), and the three groups of scavenging rates of Example 2 and the Bletilla striata extract are significantly different (p < 0.01).

[0043] The scavenging rate of DPPH free radical, ABTS free radical and hydroxyl free radical of the fermented liquor of Example 1 were the highest (p<0.01), which were 85.21±1.32%, 58.63±2.73 and 59.34±1.97 respectively. This indicates that the fermented liquor of Bacillus subtilis as fermentation bacteria in Example 1 of the present application was fermented in a constant temperature incubator at 32°C for 15h, and had the highest antioxidant activity, which was higher than the three scavenging rates of the fermented liquor of Bacillus bisporus as fermentation bacteria in Comparative Example 1.

[0044] The DPPH free radical scavenging rate, ABTS free radical scavenging rate, and hydroxyl free radical scavenging rate of the bletilla fermented liquid in Example 2 were all improved compared with the bletilla extract (p < 0.01), which were 56.45 ± 1.96, 43.26 ± 1.78, and 45.61 ± 1.35, respectively. The results lower than those shown in Comparative Example 1 are because Example 1 and Comparative Example 1 are directly fermented after mixing bletilla powder with water, and the substrate polysaccharide is not lost by the extraction process. Example 2 is an extract obtained by extracting bletilla powder through extraction technology and then fermented. The extraction process has limited extraction rates of active ingredients and polysaccharides and other substances of bletilla, resulting in less polysaccharides and other substances in the substrate of Example 2 than in the raw material liquid directly fermented.

[0045] The antioxidant effects of the unfermented Bletilla striata raw liquid and the Bletilla striata extract are not much different. This is because the Bletilla striata raw liquid contains liquid and undissolved Bletilla striata powder, the polysaccharide dissolution efficiency is poor, and the viscosity is relatively high. Therefore, when only the liquid in the raw liquid is compared with the extract, the antioxidant effects of the unfermented Bletilla striata raw liquid and the Bletilla striata extract are not much different.

[0046] Example 4 Detection of polysaccharide content Example The polysaccharide content of the fermentation broth prepared in the above Examples 1-2 and Comparative Example 1, the Bletilla striata raw material solution in Example 1, and the Bletilla striata extract in Example 2 was determined by the phenol-sulfuric acid method. The specific steps are as follows: 4.1 Preparation of standard curve: Accurately weigh 20 mg of standard dextran (or glucose) into a 500 ml volumetric flask, add water to the scale, and draw 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 ml respectively, make up to 2.0 ml each with distilled water, then add 1.0 ml of 6% phenol and 5.0 ml of concentrated sulfuric acid, shake well and let stand for 30 minutes, then measure the absorbance at 490 nm, use 2.0 ml of water as blank according to the same color development operation, the horizontal axis is the number of micrograms of polysaccharide, and the vertical axis is the optical density value, and the standard curve is obtained.

[0047] 4.2 Determination of polysaccharide concentration: Take the above-mentioned Bletilla striata fermentation liquid, Bletilla striata raw material liquid and Bletilla striata extract respectively, dilute 200 times, take 1.0ml of the sample after dilution and before dilution, add 1.0ml of distilled water, then add 1.0ml of 6% phenol, quickly add 5.0ml of concentrated sulfuric acid, shake on a vortex instrument, mix thoroughly, let stand for 30 minutes, measure the absorbance at 490nm, and bring the measured absorbance into the standard curve to obtain the polysaccharide concentration. The polysaccharide content detection results are shown in Table 2: Table 2

[0048] Note: The Bletilla striata raw material liquid is a mixture of Bletilla striata tuber powder and water before fermentation and after sterilization in Example 1; the Bletilla striata extract is an extract obtained by extracting Bletilla striata by ultrasound-assisted water extraction method before fermentation and after sterilization in Example 2.

[0049] The results are shown in Table 2. The polysaccharide content of the Bletilla striata fermented broth of Example 1 is the highest, reaching 23.62±1.71 mg / ml, indicating that the polysaccharide content of the Bletilla striata fermented broth dynamically fermented for 15 hours in a constant temperature incubator at 32°C using Bacillus subtilis as the fermentation bacteria in Example 1 of the present application is the highest, which is higher than the polysaccharide content of the Bletilla striata fermented broth fermented with Zygosaccharomyces bisporus as the fermentation bacteria in Comparative Example 1 (20.70±1.43).

[0050] The Bletilla striata raw material liquid is a mixture of sterilized Bletilla striata tuber powder and water, which contains liquid and undissolved tuber powder. After fermentation, its polysaccharide content increases (from 10.47±1.56 mg / ml to 23.62±1.71 mg / ml). The reason is that there is undissolved Bletilla striata tuber powder in the Bletilla striata raw material liquid, which contains a large amount of polysaccharides. After fermentation, the large molecular polysaccharides are decomposed into small molecular polysaccharides, which improves the solubility of the polysaccharides, thereby increasing the polysaccharide content in the Bletilla striata fermentation liquid, which is equivalent to using a mild method to increase the extraction rate of polysaccharides.

[0051] However, the polysaccharide content of the Bletilla striata extract decreased after fermentation (from 15.82±1.67mg / ml to 12.33±1.46mg / ml). The reason is that the extract after filtration through the extraction process does not contain Bletilla striata residue, and the Bletilla striata residue still contains a large amount of unextracted polysaccharides. The polysaccharides in the extract cannot be supplemented by the polysaccharides in the residue during the fermentation process of Bacillus subtilis, so the polysaccharides are decomposed, resulting in a decrease in the polysaccharide content.

[0052] Example 5 Determination of viscosity This example uses a rotational viscometer to measure the viscosity of the fermentation broth prepared in the above examples 1-2, the Bletilla striata raw material liquid in example 1, and the Bletilla striata extract fermentation broth in example 2. A rotational viscometer is an instrument used to measure the viscosity of a liquid, and it mainly uses the resistance encountered by a rotating column when rotating in a liquid to calculate the viscosity value. The formula for calculating the rotational viscometer is: Viscosity = (2πNR^4) / (60V) Where N is the rotation speed of the rotating column (rpm), R is the radius of the rotating column (mm), and V is the speed of the liquid flowing on the rotating column (mm / s). The unit in this formula is Pa·s (Pascal seconds), which can also be expressed in cP (centipoise). The viscosity measurement results are shown in Table 3: Table 3

[0053] The results in Table 3 show that the viscosity of the raw material liquid of Bletilla striata before fermentation is 2737 mPa.S, and the viscosity of the extract of Bletilla striata is 283 mPa.S. The viscosity of the fermented liquid of Bletilla striata after fermentation with Bacillus subtilis and Example 1 is 23 mPa.S. The viscosity of the fermented liquid of Bletilla striata after fermentation with Bacillus subtilis and Example 2 is 17.6 mPa.S. This shows that the viscosity of the raw material liquid of Bletilla striata or the extract of Bletilla striata decreases significantly before and after fermentation due to the action of Bacillus subtilis.

[0054] Example 6 In vitro moisturizing performance evaluation This example measures the moisturizing properties of the fermentation broth prepared in the above examples 1-2 and comparative example 1. The moisturizing property test is to measure the mass change of the sample after being placed for different periods of time under a fixed relative humidity, so as to evaluate the water retention capacity of the moisturizer. This method characterizes the water retention of the moisturizer by calculating the water loss rate of the moisturizer. The smaller the water loss rate, the stronger the water retention of the moisturizer and the better the long-term moisturizing effect. In the experiment, a saturated potassium carbonate solution is often used to maintain a relative humidity of about 43%, and the water loss rate is tested by weighing.

[0055] The specific experimental operations are as follows: Take several glass dishes of the same specifications, dry them in a vacuum drying oven, cool them to room temperature in a desiccator filled with color-changing silica gel, weigh them and record them as H (color-changing silica gel has the function of absorbing moisture and indicating water content. When the color-changing silica gel is colorless, it indicates that there is no water in the desiccator. When the color-changing silica gel is colored, it indicates that the moisture content in the desiccator is high and cannot be used as a desiccator. When using color-changing silica gel, it needs to be placed in a 60°C oven in advance to dry it to colorless before loading it into the desiccator) to prevent moisture in the air from condensing on the surface of the dish and changing its weight.

[0056] Accurately weigh 0.5 g of Bletilla striata fermentation liquid and 0.5% sodium alginate solution from different cases in a plate, with a liquid thickness of 1 mm, and weigh the plate and record it as Ho. Prepare a desiccator with a saturated potassium carbonate solution at the bottom to stabilize its humidity at 43%. Use a porous partition to separate the solution, and place the plate containing the sample on the partition of the desiccator containing saturated potassium carbonate solution. Take out the plate at 2h, 4h, 6h, 8h, 12h, 16h, 24h, and 36h, weigh it and record it as Hn. Calculate the moisture retention rate from the mass difference: Moisture retention rate (%) = (Hn-H) / (Ho-H)×100% Wherein, Hn is the weight of the plate containing the sample after being placed for a period of time, Ho is the mass of the plate containing the sample before being placed, and H is the weight of the plate after drying.

[0057] The test results of the moisturizing properties of the fermented liquor of Bletilla striata in the embodiment and the comparative example are shown in Table 4: Table 4

[0058] like Figure 2 As shown in Table 4, the moisture retention rate of the Bletilla striata fermented liquid of Example 1 is higher than that of the comparative example 1 and the sodium alginate of the control group as time increases. At 36 hours, the moisture retention rate (71%) of the Bletilla striata fermented liquid of Example 1 is still higher than that of the comparative example 1 (59%) and sodium alginate (57%). Similarly, the moisture retention rate (66%) of the Bletilla striata fermented liquid of Example 2 is also higher than that of the comparative example 1 (59%) and sodium alginate (57%).

[0059] It is explained that the Bletilla striata fermented liquid prepared in Example 1 and Example 2 of the present application using Bacillus subtilis as the fermentation bacteria and dynamically fermented in a constant temperature incubator at 32°C for 15 hours has excellent moisturizing properties, especially the Bletilla striata fermented liquid prepared in Example 1 has the best moisturizing properties.

[0060] The Bletilla striata fermented liquor prepared in Examples 1 and 2 of the present invention not only ensures the increase of antioxidant activity and the decrease of viscosity, but also has a very significant effect on moisture retention.

[0061] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A Bacillus subtilis for preparing a Bletilla striata fermentation broth, characterized in that: Its deposit number is: CGMCC No.29154.

2. A method for preparing a Bletilla striata fermentation broth, characterized in that: The steps include: (a) using Bletilla striata as a raw material, sequentially performing pre-fermentation treatment, sterilization treatment, and cooling to room temperature to obtain an initial fermentation system; (b) inoculating the Bacillus subtilis of claim 1 into the initial fermentation system to obtain an initial fermentation mixture, and fermenting and culturing to obtain a Bletilla striata fermentation liquid; The volume of deionized water in the initial fermentation system was taken as the reference, and the number of viable Bacillus subtilis in the initial fermentation system was 10 8 CFU / mL~10 12 CFU / mL.

3. The preparation method according to claim 2, characterized in that: The pre-fermentation treatment comprises the following steps: (a1) mixing dried and sieved Bletilla striata tuber powder with deionized water to obtain a Bletilla striata raw material liquid; or (a2) subjecting the Bletilla striata raw material solution to extraction treatment to obtain a Bletilla striata extract solution.

4. The preparation method according to claim 3, characterized in that: The bletilla striata tuber powder and deionized water are mixed at a mass volume ratio of 1:10-30; the particle size of the bletilla striata tuber powder is 50-150 meshes, preferably 60 meshes.

5. The preparation method according to claim 3, characterized in that: The extraction method includes: ultrasonic-assisted water extraction, enzyme extraction or physical, chemical and biological combined extraction method.

6. The preparation method according to claim 2, characterized in that: The sterilization temperature is 110° C. to 135° C., the pressure is 0.1 to 0.3 MPa, and the sterilization time is 10 to 35 minutes.

7. The preparation method according to claim 2, characterized in that: The step (b) further comprises the following steps: (b1) fermenting the initial fermentation mixture to obtain a Bletilla striata initial fermentation liquid, sterilizing it, and centrifuging it to obtain a supernatant of the Bletilla striata initial fermentation liquid; (b2) The supernatant of the initial fermentation liquid of Bletilla striata obtained in (b1) is subjected to secondary sterilization to obtain Bletilla striata fermentation liquid, wherein the secondary sterilization adopts a high-temperature water bath sterilization method to reduce residual microorganisms.

8. A Bletilla striata fermented liquid, characterized in that: Prepared according to the preparation method according to any one of claims 3 to 7.

9. Use of the Bletilla striata fermented liquid according to claim 8 for preparing cosmetics.

10. A cosmetic, characterized in that: Calculated by mass percentage, the bletilla striata fermented liquid according to claim 8 comprises 1wt% to 100wt%.

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